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.     

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.     

Design of a complement mannose-binding lectin pathway-specific activation system applicable at low serum dilutions

Recently we showed that alternative pathway (AP) amplification was responsible for more than 80% of specific classical pathway-induced terminal pathway activation under physiological conditions. The present study aimed to design a system for specific lectin pathway (LP) activation applicable at low serum dilutions with a fully functional AP. Comparison between activation of normal human serum (NHS), a mannose-binding lectin (MBL) homozygous D/D-deficient serum, and sera deficient in C1q and C2, all diluted 1 : 2, was essential to document optimal conditions for LP specificity. Mannan on the solid phase of enzyme-linked immunosorbent assay (ELISA) plates was used for activation, showing 0.5 microg mannan/well to give optimal conditions because at this concentration a good signal was preserved for C4 and TCC deposition in NHS, whereas the C3 deposition observed in C2-deficient serum at higher mannan concentrations reached nadir at 0.5 microg/well, indicating a lack of direct AP activation under these conditions. Pooled NHS and C1q-deficient serum gave the same degree of C4 and terminal complement complex (TCC) deposition, whereas deposition of these products was not obtained with MBL-deficient serum. Reconstitution with purified MBL, however, restored the depositions. A blocking anti-MBL monoclonal antibody (mAb) completely abolished the complement deposition, in contrast to a non-inhibiting anti-MBL mAb. Activation of C2-deficient serum induced C4 deposition similar to NHS, but negligible deposition of C3 and TCC, confirming the lack of direct activation of AP. Thus, this assay is unique in being LP-specific at low serum dilution and thus particularly suitable to study LP activation mechanisms and the role of AP amplification under physiological conditions.     

Use of serum or buffer-changed EDTA-plasma in a rapid, inexpensive, and easy-to-perform hemolytic complement assay for differential diagnosis of systemic lupus erythematosus and monitoring of patients with the disease

We previously described a simplified quantitative hemolytic assay for classical pathway (CP) hemolytic function in serum that has been shown to correlate with the 50% hemolytic complement (CH50) assay. In the present study, we used this assay to compare CP functions; plasma levels of C3, C4, and C3dg; and ratios of C3dg to C3 in healthy individuals and patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) with different degrees of complement activation. A significant depression in CP function and levels of C4 and C3 and increased C3dg levels and C3dg/C3 ratios were observed in the SLE patients. In patients with RA, CP function was normal, whereas C3, C4, and C3dg levels and the C3dg/C3 ratio were elevated. The SLE results are compatible with systemic complement consumption, whereas the RA data suggest an acute-phase reaction with a normal C3 catabolic rate. To facilitate the handling of patient samples, we also developed a method to restore the hemolytic function of EDTA-plasma by transferring it to Veronal-buffered saline containing the thrombin inhibitor lepirudin. This process inhibits coagulation and enables complement activation, allowing a longer time lag between sample harvesting and testing. These results, combined with previous correlation studies, suggest that the CP hemolytic assay can effectively replace the CH50 assay for routine SLE differential diagnosis and monitoring of disease activity.     

Functional characterization of the lectin pathway of complement in human serum

Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum.Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms.We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.     

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.     

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.     

Level of complement activity and components C1, C4, C2, and C3 in complement response to bacterial challenge in malnourished rats.

In experimentally induced malnutrition in rats, there was no significant difference between the measured level of complement activity of the classical pathway (50% hemolytic complement [CH50]) and that of the alternative pathway (ACH50), although the levels of complement components C1, C4, C2, and C3 were depressed significantly. The complement activity showed a temporary elevation with a peak at 2 or 3 days after bacterial challenge with Staphylococcus aureus in rats, and we call this the complement response. After 3 days, CH50 and C3 in the malnourished rats and ACH50, CH50, and C3 in the well-nourished rats showed a significant increase, and C1, C4, and C2 in both groups tended to elevate. On the basis of these observations, the significance of the elevation of C3 in the complement response to bacterial infection showed a strong influence by enhancing the activation of both the classical and the alternative pathways, since C3 is known to be the junction of both complement pathways. In this way, C3 responded to an earlier stage than did the other components and may contribute to maintaining the body defense system against infection.     

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.     

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.     

Studies on the complement system from capybara (Hidrochoerus hidrochaeris hidrochaeris) serum

The complement system of the serum from capybara (Hidrochoerus hidrochaeris hidrochaeris), the large wild relative of the guinea-pig, was analysed in this study. Capybara serum was efficient in the lysis of sheep erythrocytes sensitised with rabbit antibodies and unsensitised rabbit erythrocytes, being easily measured in assays of classical (CP) and alternative (AP) pathway activities. Lysis or agglutination due to natural antibodies was not observed under the conditions used. The effect of temperature on complement was studied. The lytic activity of CP and AP was stable for 30 min at 37°C. After 30 min at 50°C, CP activity fell to about 65%, and AP was totally inactivated. As expected, both pathways were well preserved on storage at ?70°C. Lyophilisation led to minor losses (about 10%) of CP and AP. CP and AP haemolytic assays were performed in parallel using guinea-pig serum for comparison. When the mean was calculated from individual values, CP and AP showed 73% and 95% of guinea-pig serum activity, respectively, in a typical assay under the same experimental conditions. C3 activation and induction of phagocytosis were also evaluated. Zymosan was opsonised with serum, incubated with rat polymorphonuclear leucocytes (PMN), and luminol-dependent chemiluminescence (CL) was measured. The activity of capybara serum was significantly higher than the activity of guinea-pig serum. In one representative assay, PMN chemiluminescence stimulated with zymosan treated with a pool of capybara serum was about 150% of that obtained when a pool of guinea-pig serum was used. The present data contribute to the understanding of the complement system, which is implicated in defence mechanisms and has not been previously studied in this animal. The importance of capybaras for biology, research and zootechnical exploitation is discussed.     

Effect of body weight and caloric restriction on serum complement proteins, including Factor D/adipsin: studies in anorexia nervosa and obesity

Complement plays important roles in host immune defences, and recent studies suggest that adipose tissue is an important site of production for some complement proteins. Starvation has been associated with low complement levels, but studied populations have usually had concomitant opportunistic infections or other conditions which might affect complement levels. To determine the impact of body weight and changes in body weight on serum complement, we investigated levels of complement proteins in otherwise healthy patients with a wide range of body weights, including patients with anorexia nervosa before and after treatment, obese dieters before and after weight loss, and normal weight controls. We found that complement proteins of the alternative pathway (C3, B, and D), alternative pathway haemolytic activity (AP50) and the inhibitors H and I were low in starving anorectics and normalized with weight gain. C3a levels were comparable in anorectics at low weight and after weight gain, indicating that low serum complement levels were attributable to hypoproduction and not complement cascade activation with consumption. Further, levels of C3, B, AP50, H and I, but not D, were higher than controls in obese patients and decreased toward normal after weight loss. Overall, percentage of ideal body weight, changes in body weight, and serum transferrin were each highly correlated with serum levels of complement proteins. We conclude that levels of alternative pathway complement components are determined in part by factors that influence body weight and by weight changes, possibly due to changes in production in adipose tissue or at other sites.     

Alternative pathway amplification and infections

The alternative pathway (AP) is the phylogenetically oldest arm of the complement system and may have evolved to mark pathogens for elimination by phagocytes. Studies using purified AP proteins or AP-specific serum showed that C3b amplification on bacteria commenced following a lag phase of about 5 min and was highly dependent on the concentration of complement. Most pathogens have evolved several elegant mechanisms to evade complement, including expressing proteases that degrade AP proteins and secreting proteins that block function of C3 convertases. In an example of convergent evolution, many microbes recruit the AP inhibitor factor H (FH) using molecular mechanisms that mimic FH interactions with host cells. In most instances, the AP serves to amplify C3b deposited on microbes by the classical pathway (CP). The role of properdin on microbes appears to be restricted to stabilization of C3 convertases; scant evidence exists for its role as an initiator of the AP on pathogens in the context of serum. Therapeutic complement inhibition carries with it an increased risk of infection. Antibody (Ab)-dependent AP activation may be critical for complement activation by vaccine-elicited Ab when the CP is blocked, and its molecular mechanism is discussed.     

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.     

Interactions of chrysotile asbestos fibres with the complement system.

Type A chrysotile fibres (white asbestos) were tested in vitro for activation of the complement system. Fibres were incubated in normal human serum (NHS), factor B-depleted human serum, and normal and C4-deficient guinea-pig sera; the supernates were assayed for the remaining complement activity. Activation of the alternative pathway (AP) was shown in three ways. First, quantitative measurement of factor B; second, kinetic analysis of rabbit red blood cell lysis in whole alternative pathway (AP) and factor B lytic assays; third, qualitative measurement of C3 and factor B conversion by crossed immunoelectrophoresis. No C3 convertase activity could be demonstrated on the fibres but other possible mechanisms of AP activation are discussed. Magnesium itself is not responsible for this activation because acid-treated fibres retain this property. The early classical pathway is not involved as shown by normal whole complement activity of a factor B-depleted human serum and the absence of decrease of C4 functional activity. Knowing that complement proteins are present in pulmonary alveoli, mainly provided by cell synthesis, we suggest that complement activation in vivo may be relevant to the genesis of the chronic inflammation and fibrosis in the lung.     

Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies

The complement (C)-activating capabilities in human serum of 32 mouse and 10 mouse/human chimeric MoAbs of different isotypes, and their fragments, were tested in vitro. Activation of C via the classical pathway (CP) was performed in 1% factor D-deficient serum in gelatin containing Veronal buffer in the presence of calcium and magnesium (GVB⁺⁺), while activation of the alternative pathway of C (AP) was assessed in 10% Clq-depleted serum in the presence of 5 mm MgCl₂ in GVB⁺⁺. The C-activating ability of MoAbs was expressed relative to the degree of activation of complement by aggregated IgG for the CP and relative to mouse IgG 1 for the AP. All of seven mouse IgG2a MoAbs were potent activators of the CP. The results of CP activation by IgG1, IgG2b and IgG3 isotypes were different for individual MoAbs. Only three (two IgG 1 and one IgG3) of 32 mouse MoAbs were potent activators of the AP. IgG2a and IgG2b were relatively poor AP activators. There were a few MoAbs which activated both the AP and CP. Of 10 chimeric MoAbs, two IgG1, one IgG2 and one IgG4 were poor or non-activators of the CP. On the other hand, IgG2 and IgG4 were good AP activators. IgG3 was the most potent AP activator. Most of the F(ab')₂ fragments were activators of the AP and displayed no activation of the CP. Fc fragments only activated the CP. whereas Fab'did not activate the CP or the AP. These studies suggest that the route of complement activation by class and subclass MoAbs can not always be predicted in advance and based only on their subclass identity.     

Binding of Properdin to Solid-Phase Immune Complexes: Critical Role of the Classical Activation Pathway of Complement

The capacity of serum to support deposition of C3, properdin and factor B was studied by enzyme-linked immunosorbent assay using solid-phase immune complexes (IC) for activation of complement. Deposition of C3 and properdin occurred in fairly dilute normal human serum (NHS), but factor B uptake was hardly detectable. Alternative pathway-mediated deposition of C3 with slow kinetics was demonstrated in C2-deficient serum and in NHS depleted of C1q, factor D and properdin (C1qDP-depleted serum) after reconstitution with factor D and properdin. Efficient uptake of properdin required a functional classical pathway, in the presence of which C3 and properdin were rapidly deposited onto the IC. Judging from findings in C3-deficient serum, factor I-deficient serum, and C1qDPB-depleted serum, the uptake of properdin was strictly C3-dependent, and did not require the presence of factors B and D. Thus, C3b fixed to IC was the principal ligand for properdin in the assay. The findings could have biological implications relating to complement-mediated modification of immune complexes in disease.     

Activation of the alternative and classical complement pathways by Entamoeba histolytica

Entamoeba histolytica HM1 supported the activation of human alternative and classical complement pathways in the absence of ameba-reactive antibodies. Nonimmune serum depleted of C1q and factor D (NHS s C1q + D) and reconstituted with C1q was able to specifically deposit C3b onto trophozoites and produce lysis. This activity was not modified by the absorption of serum on E. histolytica. Serum depleted of factor B allowed C3b binding to amebae. Serum devoid of C4 effected only small amounts of C3 uptake. The kinetics of lysis of E. histolytica by serum in the presence of Mg-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] (lacking classical pathway function) or by NHS s C1q + D and reconstituted with factor D was slow and only produced one-half the amount of lysis produced by NHS s C1q + D supplemented with C1q. These results indicate that the surface of the ameba can promote complement activation by the classical pathway, without the participation of specific antibodies, and that the magnitude of this activation is greater than that induced by the alternative pathway.     

The alternative pathway is required, but not alone sufficient, for retinal pathology in mouse laser-induced choroidal neovascularization

Human genetic studies have demonstrated that polymorphisms in different complement proteins can increase the risk for developing AMD. There are three pathways of complement activation, classical (CP), alternative (AP), and lectin (LP), which all activate a final common pathway. Proteins encoded by the AMD risk genes participate in the AP (CFB), CP/LP (C2), or in the AP and final common pathway (C3). Here we tested which pathway is essential in mouse laser-induced CNV. CNV was analyzed using single complement pathway knockouts (i.e., eliminating one complement pathway at a time), followed by a double knockout in which only the AP is present, and the CP and LP are disabled, using molecular, histological and electrophysiological outcomes. First, single-gene knockouts were analyzed and compared to wild type mice; C1q(-/-) (no CP), MBL(-/-) (no LP), and CFB(-/-) (no AP). Six days after the laser-induced lesion, mice without a functional AP had reduced CNV progression (P<0.001) and preserved ERG amplitudes, whereas those without a functional CP or LP were indistinguishable from the wild type controls (P>0.3). Second, AP-only mice (C1q(-/-)MBL(-/-)) were as protected from developing CNV as the CFB(-/-) mice. The degree of pathology in each strain correlated with protein levels of the angiogenic and anti-angiogenic protein VEGF and PEDF, respectively, as well as levels of terminal pathway activation product C5a, and C9. The analysis of complement activation pathways in mouse laser-induced CNV allows for the following conclusions. Comparing the single pathway knockouts with those having only a functional AP showed: (1) that AP activation is necessary, but not alone sufficient for injury; and (2) that initial complement activation proceeds via both the LP and CP. Thus, these data indicate an important role for the AP in the generation of complement-dependent injury in the RPE and choroid via amplification of CP- and LP-initiated complement activation. Improving our understanding of the local regulation of this pathway in the eye is essential for developing improved treatment approaches for AMD.