Assay of classical and alternative pathway activities of murine complement using antibody-sensitized rabbit erythrocytes

Methods for measurement of classical complement pathway activity (CH50) and alternative complement pathway activity (ACH50) in mouse serum using rabbit erythrocytes sensitized with guinea pig anti-rabbit erythrocyte antibody have been established. The assays measured CH50 values in mouse sera that could hardly be determined by the conventional method using antibody-sensitized sheep red blood cells. Mouse serum ACH50 values determined by the method were also 5-7 times higher than those obtained in conventional assays with rabbit erythrocytes. Both the CH50 and ACH50 values varied with the strain among the 25 different strains of mice studied. BALB/c (nu/nu, male), LT/SuJ and Jcl-ICR27 strains exhibited higher CH50 values, and NIH (nu/+), ICR (nu/nu), NOD (male) and AKR strains showed lower values. The ACH50 was higher in C3H/HeN (male), C57BL/6J (male), Jcl-ICR27 and BALB/c (nu/nu, male) mice, and lower in ICR (nu/nu), NOD (female) and AKR mice. Sera from 16 out of the 25 mouse strains showed ACH50 values comparable to or higher than those in man. As for CH50, however, even the highest value seen in BALB/c (nu/nu, male) mice corresponded to about three-fifths of an average value in man. It is concluded that the complement system of mice, especially the alternative pathway of complement activation, functions as actively as that in man. It was also found that male mice have higher CH50 and ACH50 values than female mice. The differences in these parameters between males and females were only slight at the age of 4 weeks and became conspicuous after 6 weeks at which time both the CH50 and ACH50 virtually reached their respective peak levels of activity.     

Relationships between the haemolytic activities of the human complement system and complement components.

The relationships between the haemolytic activities of complement and its components were studied. The activities studied included CH50 (classical pathway), AP50 (alternative pathway), CV50 (early part of alternative pathway) and C(3--9)H50 ((the late part of both pathways). The components included C3, C4, C5, C9, B and D. There was a good correlation between CH50 and AP50. AP50 had a good correlation with B and CV50. There was no correlation between AP50 and C(3--9)H50, and none between C(3--9)H50 and C5 or C9. AP50 may primarily represent changes in the early part of the alternative pathway. C(3--9)H50 is not influenced by respective changes in the amounts of C5 or C9. Since cell lesion is now considered to be caused by a unit of C5b to C9, a change in each component of C5 to C9 may not influence haemolytic activity.     

Effect of adsorbent of Riposorber, a cellulose microparticle with immobilized dextran sulfate, on the serum complement system

Abstract-Apheresis. using columns of cellulose microparticles with immobilized dextran sulfate, Riposorber, has been applied to treatment of patients with various diseases, such as hypercholesterolemia and systemic lupus erythematosus. Unfortunately, it has been reported that the apheresis activates the complement system. It might exert unpleasant side effects on patients during lifelong treatment. In this study, the interaction of the serum complement system with cellulose microparticles with immobilized dextran sulfate and its components, nontreated cellulose microparticles and dextran sulfate, were examined in vitro to get some ideas for development of an extracorporeal apparatus which does not give any serious damage to patient blood. The cellulose microparticles with immobilized dextran sulfate reduces both the CH50 and the ACH50. Decrease in CH50 is not due to the classical pathway activation, but to adsorption of C2 or Cl components including Clq. The alternative pathway was not activated by the addition of the dextran sulfate alone to serum, but the addition of non-treated cellulose microparticles to serum activated complement. Form these, decrease in ACH50 is not caused by dextran sulfate on the microparticles, but by the hydroxyl groups of the cellulose microparticles that is the core of the column. For prevention of complement activation, hydroxyl groups remaining after dextran sulfate immobilization should be blocked by further treatment with a reagent that reacts with them, or else dextran sulfate might be immobilized on particles without nucleophiles such as hydroxyl or amino groups.     

The role of Staphylococcus aureus cell-wall peptidoglycan, teichoic acid and protein A in the processes of complement activation and opsonization.

The role of cell-wall peptidoglycan, teichoic acid and protein A in the processes of Staphylococcus aureus complement activation and opsonization was investigated. CH50 consumption studies reveal that, although all cell-surface fractions were capable of activating the classical C pathway, only peptidoglycan consumed C via the alternative pathway. Using a quantitative immunofluorescence assay, peptidoglycan was shown to bind C3 molecules via the classical as well as via the alternative C pathway and in the absence of IgG and IgA class antibodies. C activation via the classical and the alternative pathway could be distinguished by kinetic analysis. By comparing the rates of staphylococcal C consumption, C3 fixation and opsonization it was found that the CH50 consumption assay is a relatively insensitive method and may yield results that do not necessarily reflect the process of bacterial opsonization.     

Involvement of the lectin pathway of complement activation in antimicrobial immune defense during experimental septic peritonitis

A critical first line of defense against infection is constituted by the binding of natural antibodies to microbial surfaces, activating the complement system via the classical complement activation pathway. In this function, the classical activation pathway is supported and amplified by two antibody-independent complement activation routes, i.e., the lectin pathway and the alternative pathway. We studied the contribution of the different complement activation pathways in the host defense against experimental polymicrobial peritonitis induced by cecal ligation and puncture by using mice deficient in either C1q or factors B and C2. The C1q-deficient mice lack the classical complement activation pathway. While infection-induced mortality of wild-type mice was 27%, mortality of C1q-deficient mice was increased to 60%. Mice with a deficiency of both factors B and C2 lack complement activation via the classical, the alternative, and the lectin pathways and exhibit a mortality of 92%, indicating a significant contribution of the lectin and alternative pathways of complement activation to survival. For 14 days after infection, mannan-binding lectin (MBL)-dependent activation of C4 was compromised. Serum MBL-A and MBL-C levels were significantly reduced for 1 week, possibly due to consumption. mRNA expression profiles did not lend support for either of the two MBL genes to respond as typical acute-phase genes. Our results demonstrate a long-lasting depletion of MBL-A and MBL-C from serum during microbial infection and underline the importance of both the lectin and the alternative pathways for antimicrobial immune defense.     

Pneumolysin-induced complement depletion during experimental pneumococcal bacteremia

To quantify complement depletion by pneumolysin during Streptococcus pneumoniae bacteremia, cirrhotic and control rats were infected intravenously with one of three isogenic mutant strains of S. pneumoniae expressing different forms of pneumolysin. Outcome measures included clearance of the organisms from the bloodstream, alterations in 50% serum hemolytic complement (CH(50)) activity and complement C3 levels during infection, and serum opsonic capacity at 18 h postinfection. Cirrhotic rats had significantly lower CH(50) and C3 levels than control rats, both before and after infection. However, initial complement levels did not predict bacterial load after 18 h of infection. Changes in CH(50) and C3 levels over the 18-h period correlated with numbers of H+C+ but not H+C- or PLY- organisms in the bloodstream at 18 h postinfection. The sera of cirrhotic rats infected with the H+C+ strain had significantly decreased levels of C3 and showed significantly lower opsonizing activity for S. pneumoniae than sera from H+C+-infected control rats. These studies suggest that under limiting concentrations of complement, the expression of pneumolysin by pneumococci has a significant, negative effect on serum complement levels and reduces serum opsonic activity.     

Interaction of complex polysaccharides with the complement system: effect of calcium depletion on terminal component consumption.

Complex polysaccharides and lipopolysaccharides can activate the terminal components of complement by either the classical (antibody, C1, C4, and C2) or alternative complement pathways, but the relative importance of either pathway for terminal component consumption in normal serum is poorly understood. Since classical complement pathway function requires both calcium and magnesium ions, whereas the alternative pathway requires only magnesium ions, selective chelation of calcium ions in serum can be used to block the classical complement pathway while leaving the alternative pathway intact. In these studies, ethyleneglycol-bis-(beta-aminoethyl ether)N, N-tetraacetic acid, a potent chelator or calcium, was used to block the classical complement pathway in normal guinea pig serum.Consumption of the terminal complement components by endotoxin, inulin, and zymosan in such serum was strikingly depressed when compared to serum containing an intact classical complement pathway. These studies demonstrate that in normal serum, both the classical and alternative complement pathways participate in the consumption of the terminal complement components by complex polysaccharides and lipopolysaccharides.     

Experimental bovine trypanosomiasis. Changes in serum immunoglobulins,complement and complement components in infected animals.

In three calves experimentally infected with Trypanosoma congolense the amounts of IgG1 and IgG2 were little changed and similar to those of normal animals. IgM increased in amount early in the infection and the amount of the increase appeared related to the parasite burden. The amounts of IgA and IgE were both much decreased and this also appeared related to the numbers of parasites in the blood. There was a decrease in the amounts of total haemolytic complement and complement components C1, C1q and C3 in the infected calves. Furthermore the amounts of properdin fluctuated with the cyclical changes in numbers of T. congolense parasites in the individual calves. No significant change in the amount of C8 was observed. It is considered that activation of both the alternative and the classical complement pathways occurs in trypanosome infected animals but that neither pathway goes to its terminal stages.     

Structure-activity relationships within the N-terminal short consensus repeats (SCR) of human CR1 (C3b/C4b receptor, CD35): SCR 3 plays a critical role in inhibition of the classical and alternative pathways of complement activation.

Genes coding for between one and four short consensus repeats (SCR) of the N-terminal region of human complement receptor 1 (CR1) were synthesized from oligonucleotides and those encoding SCR(1-2), SCR(1-3), SCR(1-4), SCR3 and SCR(3-4) were expressed as inclusion bodies in Escherichia coli. Following solubilization in urea, the proteins were partially purified and refolded and the activity of each protein was assessed in both classical and alternative pathway complement assays. All fragments showed a varying degree of activity with the general order being SCR(1-3) = SCR(1-4) > SCR(1-2). Addition of SCR3 to SCR(1-2) significantly improved potency, whereas the addition of SCR4 conferred no additional benefit. This observation, coupled with the ability of the single-domain SCR3 to inhibit classical pathway mediated lysis with an IH50% (inhibition of hemolysis by 50%) of 4.8 microM, demonstrates that SCR3 provides key binding interactions with activated complement components. SCR(1-3) was able to inhibit both classical and alternative pathways of complement activation, showing that the N-terminal SCR of CR1 retain the ability to interact with C3b. Assays for CR1-like cofactor activity for factor I using C4b-like C4 or C3b-like C3 as substrates showed that SCR(1-3) possessed such cofactor activity and that C4b-like C4 was a better substrate. When compared to full-length (30 SCR) soluble CR1 (sCR1), SCR(1-3) was significantly less potent in accord with a model involving multi-valent binding of C3b/C4b to CR1.     

Study on CH50 levels in factor D-depleted serum

It is generally accepted that levels of serum whole complement activity (CH50) reflect the activities of complement (C) components of the classical C pathway (CP), since CH50 is assayed by use of sensitized sheep erythrocytes (EA). However, the alternative C pathway (AP) is considered to be also activated simultaneously in the process of activation of serum CP by EA. Thus, serum CH50 levels may possibly reflect not only CP but also AP activation in CH50 assay. We studied on the influence of AP activation during CH50 assay on CH50 levels, by comparison of CH50 levels in serum samples before and after treatment of factor D depletion. Polystyrene beads carrying polyanion, poly (2-acrylamide 2-methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation for factor D-depleted serum. After treatment of pooled normal human serum (NHS) with PAMPS-beads (2.5 mg/ml of serum), serum ACH50 level decreased to be undetectable, indicating that AP activation is prohibited in PAMPS-beads-treated serum. When isolated factor D was added to this PAMPS-beads-treated serum, ACH50 level recovered to that of before treatment. Immunoblot analysis revealed that factor D band observed in NHS disappeared completely after PAMPS-beads treatment. From these results, it is clear that factor-D deficient serum is prepared by PAMPS-beads treatment. Besides, since serum CH50 level was not decreased by PAMPS-beads treatment, it may be concluded that CH50 level is not affected by AP activation during CH50 assay.     

Normal human serum depleted of C1q, factor D and properdin: its use in studies of complement activation.

Normal human sera were depleted of C1q, factor D (D) and properdin (P) by a simple and reproducible procedure providing reagents for analysis of complement-dependent functions. Classical pathway activity was restored with purified C1q, and alternative pathway activity with purified D and P. Since both activation pathways were abolished, antibodies and other components could be removed without loss of complement activity during immunoabsorption procedures. Synergism between the two pathways during haemolysis of rabbit erythrocytes was clearly demonstrated, and was also found on analysis of C3 cleavage in serum incubated with other alternative pathway activators such as zymosan and inulin. Experiments with a Neisseria meningitidis serogroup W-135 strain isolated from a patient with inherited P deficiency showed that both pathways were capable of supporting antibody-dependent killing of the bacteria in serum. The alternative pathway was possibly more efficient than the classical pathway in the assay system. In C1q,D,P-depleted serum with high concentrations of anticapsular IgG antibodies, the addition of D alone resulted in efficient alternative pathway-mediated killing. The alternative pathway was equally efficient in a C1q,D,P-depleted serum with low concentrations of anticapsular antibody, but in this case the reaction required both D and P.     

In vitro C3 deposition on Cryptococcus capsule occurs via multiple complement activation pathways

Complement can be activated via three pathways: classical, alternative, and lectin. Cryptococcus gattii and Cryptococcus neoformans are closely related fungal pathogens possessing a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM), which serves as a site for complement activation and deposition of complement components. We determined C3 deposition on Cryptococcus spp. by flow cytometry and confocal microscopy after incubation with serum from C57BL/6J mice as well as mice deficient in complement components C4, C3, factor B, and mannose binding lectin (MBL). C. gattii and C. neoformans activate complement in EGTA-treated serum indicating that they can activate the alternative pathway. However, complement activation was seen with factor B(-/-) serum suggesting activation could also take place in the absence of a functional alternative pathway. Furthermore, we uncovered a role for C4 in the alternative pathway activation by Cryptococcus spp. We also identified an unexpected and complex role for MBL in complement activation by Cryptococcus spp. No complement activation occurred in the absence of MBL-A and -C proteins although activation took place when the lectin binding activity of MBL was disrupted by calcium chelation. In addition, alternative pathway activation by C. neoformans required both MBL-A and -C, while either MBL-A or -C was sufficient for alternative pathway activation by C. gattii. Thus, complement activation by Cryptococcus spp. can take place through multiple pathways and complement activation via the alternative pathway requires the presence of C4 and MBL proteins.     

Terminal complement complex in plasma from patients with systemic lupus erythematosus and other glomerular diseases

To evaluate terminal complement pathway activation in plasma from patients with systemic lupus erythematosus (SLE) and primary glomerular diseases, we developed an enzyme-linked immunosorbent assay (ELISA) for measuring the terminal complement complexes (TCC). The method is based on a sandwich technique using rabbit antibodies against native human C5, C7 and C9. To avoid interference by native components, we equilibrated plasma specimens with 5% polyethylene glycol buffer. The precipitates were measured by ELISA. TCC was detectable in all 14 normal controls (0.48 +/- 0.06 AU/ml; mean +/- s.e.m.). TCC levels were elevated in 18 of 54 patients with SLE (0.89 +/- 0.07 AU/ml; P less than 0.01) and in eight of 11 patients with membranoproliferative glomerulonephritis (MPGN) (3.15 +/- 0.62 AU/ml; P less than 0.01). However, only one of six patients with membranous nephropathy and none of 13 with mesangial proliferative glomerulonephritis showed high values. In SLE, TCC was correlated with circulating immune complexes and inversely with CH50, C3, C4, C5 and alternative complement pathway activity (AH50), and showed significantly high values even in normal CH50 cases (n = 34; P less than 0.01). In MPGN, TCC was inversely correlated with CH50, AH50, C3, C5 and C9. These results suggest that the classical pathway plays an important role for TCC generation in SLE and that the alternative pathway does in MPGN.     

Assessment of complement activation in clinical samples. Comparison of immunochemical and functional measurements of complement components with quantitation of activation fragments.

We have compared functional and immunochemical measurements of complement components with assays measuring the generation of activation fragments, for the assessment of classical pathway activation in vitro and in vivo. The generation of the C3a, C3b/C3bi cleavage fragments of C3, and of the C4d cleavage fragment of C4 measured by ELISA and RIA, was correlated with the decrease in total complement hemolytic activity (CH50) and in functional activity of C3 and C4 in normal human serum in which the classical pathway had been activated with aggregated IgG. The decrease in CH50 in in vitro activated serum was also correlated with the generation of C5a and soluble SC5b-9 complexes. In contrast, no or little increase in the concentration of C3a, C3b/C3bi and C4d was observed in plasma samples from patients with low CH50 and with low levels of immunochemical C3 and C4, indicating that fragment quantitation assays provide no information on the presence and extent of complement activation in vivo. Analysis of samples from patients expressing the four C4 genes and patients having one or two C4 null alleles indicated that a ratio of hemolytic C4 to C2 > or = 1 was indicative of complement activation without C4 deficiency, whereas a ratio below 1 was indicative of C4 deficiency with or without classical pathway consumption. Classical pathway activation and C4 deficiency in clinical samples are best predicted by the concomitant assessment of immunochemical levels of C3 and C4 and hemolytic levels of C4, C2 and C3.     

Role of the classical pathway of complement activation in experimentally induced polymicrobial peritonitis.

The complement system and the natural antibody repertoire provide a critical first-line defense against infection. The binding of natural antibodies to microbial surfaces opsonizes invading microorganisms and activates complement via the classical pathway. Both defense systems cooperate within the innate immune response. We studied the role of the complement system in the host defense against experimental polymicrobial peritonitis using mice lacking either C1q or factor B and C2. The C1q-deficient mice lacked the classical pathway of complement activation. The factor B- and C2-deficient mice were known to lack the classical and alternative pathways, and we demonstrate here that these mice also lacked the lectin pathway of complement activation. Using inoculum doses adjusted to cause 42% mortality in the wild-type strain, none of the mice deficient in the three activation routes of complement (factor B and C2 deficient) survived (mortality of 100%). Mortality in mice deficient only in the classical pathway of complement activation (C1q deficient) was 83%. Application of further dilutions of the polymicrobial inoculum showed a dose-dependent decrease of mortality in wild-type controls, whereas no changes in mortality were observed in the two gene-targeted strains. These results demonstrate that the classical activation pathway is required for an effective antimicrobial immune defense in polymicrobial peritonitis and that, in the infection model used, the remaining antibody-independent complement activation routes (alternative and lectin pathways) provide a supporting line of defense to gain residual protection in classical pathway deficiency.     

New perspectives on mannan-binding lectin-mediated complement activation

The complement system is an important part of the innate immune system, mediating several major effector functions and modulating adaptive immune responses. Three complement activation pathways exist: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). The LP is the most recently discovered, and least characterized. The CP and the LP are generally viewed as working through the generation of the C3 convertase, C4bC2b, and are here referred to as the "standard" pathways. In addition to the standard CP and LP, so-called bypass pathways have also been reported, allowing C3 activation in the absence of components otherwise believed critical. The classical bypass pathways are dependent on C1 and components of the AP. A recent study has shown the existence also of a lectin bypass pathway dependent on mannan-binding lectin (MBL) and AP components. The emerging picture of the complement system is more that of a small "scale-free" network where C3 acts as the main hub, than that of three linear pathways converging in a common terminal pathway.     

ELISA for evaluating the incorporation of plasma derived complement split-products C3b/iC3b into solid-phase immune complexes

An ELISA that measures plasma derived complement (C) split-products C3b/iC3b deposited on solid-phase immune complexes during C activation is described. Plates are coated with BSA, anti-BSA and plasma is added. Deposited C3b/iC3b is then detected by biotinylated anti-C3c-antibodies, avidin-alkaline phosphatase and para-nitrophenylphosphate. A novel feature is that the assay measures residual C activation capacity rather than in vivo generated C activation products. The assay was applied to plasma from 250 healthy blood donors. No difference in activation capacity of either the alternative (AP) or classical pathway (CP) with regard to age or gender was demonstrated. The total coefficient of variation was <5.7%. The ELISA procedure was compared to a standard hemolytic complement CH(50) assay using plasma from 23 out-patients with systemic lupus erythematosus (SLE). There was a weak correlation between the two assays for both C pathways, but neither the ELISA nor the CH(50) assay showed any correlation with the diagnostic ACR-criteria for SLE. However, the capacity of the CP was significantly reduced in SLE out-patients compared to healthy blood donors (P<0.0001).     

A novel assay for serum complement activity: C42 generation assay.

BACKGROUND: In most clinics, laboratory tests for serum complement are limited to immunochemical determinations of C3 and C4 and are occasionally extended to the hemolytic titration of total complement functional activity (CH50). However, these tests are often not sufficient for the analysis of low CH50 serum. METHODS: A novel assay for serum complement activity, the C42 generation assay, has been developed. The principle of this assay is based on the hemolysis of sensitized sheep erythrocytes (EA) by complement components in two sera: C42 (the classical pathway C3 convertase) is generated on EA by C1, C4 and C2 in the first serum, followed by a second reaction leading to hemolysis by C3-C9 supplied by the addition of the second serum in the presence of EDTA. RESULTS: This methodology permits the evaluation of two distinct serum complement activities of a test serum. The combined activity of C1, C4 and C2, as well as the combined activity of C3-C9, can be estimated from the observed degrees of hemolysis. Information obtained from this assay is helpful for the analysis of test serum determined to have decreased CH50. Several clinical cases are presented in which this assay was utilized. CONCLUSIONS: The C42 generation assay is another functional assay of serum complement which can provide information beyond that obtained from the typical serum CH50 assay. Since intermediate cells or isolated complement components are not necessary, this assay can be employed rapidly and economically in a clinical setting.     

Detection of complement activation by counterimmunoelectrophoresis (CIE)

Counterimmunoelectrophoresis (CIE) was used as a method of detecting activation of the third component of the complement system (C3). Highly purified C3, normal human serum (NHS), EDTA-treated plasma and serum activated with aggregated human immunoglobulin (agg-IgG) or inulin were used as sources of C3 and/or C3 split products. Activation of the alternative pathway of complement was assayed in the presence of EGTA (10 mM) and MgCl₂ (0.3 mM), conditions which block activation of the classical pathway. When purified native C3, fresh NHS and fresh EDTA-plasma were tested in CIE against either antisera to whole C3 or to C3 split products, only one precipitin line was found, which was identified as native C3. However, when serum activated with agg-IgG or inulin were tested against the same reagents, two precipitin lines were seen. The first, with more cathodal mobility was identical to that of native C3. The second line had a more anodal mobility, was distinctly separated from the first and contained C3c and C3d as shown immunochemically with specific antisera. Native C3 and split products of C3 were identified by this CIE method in patients showing evidence of activated complement by having subnormal total complement (CH50) levels. When C3 split products were identified, the C3c-C3d precipitin line could always be distinguished from native C3 by its different electrophoretic mobility, even when C3 concentrations in serum varied from 0.25 mg/ml to 1.5 mg/ml. The sensitivity of CIE was compared to that of CH50 by assaying at different time intervals after agg-IgG was added to fresh NHS. C3c-C3d split products were detected by CIE before any fall in CH50 and at all times when a significant decrease in CH50 was present. This study shows that the CIE technique is a highly sensitive, specific and rapid method for detecting activation of the complement system via classical or alternative pathways in human disease.     

Complement abnormalities during an epidemic of group B meningococcal infection in children

During an epidemic of group B meningococcal infection mean values obtained in 96 consecutively affected children showed a reduction in classical pathway function (CH50), normal alternate pathway function (AP50), C4 and factor B levels, and raised C3 levels. CH50, C3 and Factor B were however significantly lower in those children who had a rapid onset of illness, were in shock, had signs of septicaemia, had extensive skin purpura, or who died. The presence of detectable meningococcal antigen by Counter Immuno Electrophoresis (CIE) and laboratory evidence of Disseminated Intravascular Coagulation (DIC) also correlated with lower complement levels. The significant reduction in CH50 and Factor B in the more severely affected patients suggests that activation of both classical and alternate pathways occurs in group B meningococcal infection.