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.

Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein.

The glycosylation of the circulating immunoglobulin-gamma (IgG) antibody molecules changes in rheumatoid arthritis. The extent of the changes correlates with the disease severity and reverses in remission. We demonstrate here that the alteration in glycosylation associated with rheumatoid arthritis can create a new mode for the interaction of IgG with complement through binding to the collagenous lectin mannose-binding protein (MBP). Rheumatoid arthritis is associated with a marked increases in IgG glycoforms that lack galactose (referred to as G0 glycoforms) in the Fc region of the molecule and that terminate in N-acetyl glucosamine (GlcNAc). We show, using nuclear magnetic resonance (NMR) and X-ray data, that these terminal GlcNAc residues become accessible for MBP binding. We further demonstrate that multiple presentation of IgG-G0 glycoforms to MBP results in activation of the complement. This suggests that a contribution to the chronic inflammation of the synovial membrane could arise from the localization of the IgG-G0 glycoforms in the affected joint and from resulting activation of complement.

Human leukocyte C1q receptor binds other soluble proteins with collagen domains.

A receptor binding to the C1q subcomponent of complement has been reported by many workers. In this paper we report for the first time that C1q receptor binds not only to C1q, but also to three other structurally similar ligands, namely mannan binding protein (MBP), conglutinin, and lung surfactant protein (SP-A). All these ligands have been reported to enhance removal of species bound to their globular domain from blood (MBP, conglutinin, C1q) or lung (SP-A) through phagocytosis. One of the possible roles for ligand-receptor binding may be initiation of phagocytosis.

Local opsonization by secreted macrophage complement components. Role of receptors for complement in uptake of zymosan.

We have examined the role of macrophage (M phi plasma membrane receptors for the cleaved third complement component (iC3b; CR3) and mannosyl, fucosyl terminated glycoproteins (MFR) in uptake of unopsonized zymosan. Monoclonal antibodies against CR3, M1/70 (Mac-1) and MO1, each inhibited approximately 50% of uptake of 125I-zymosan by murine and human M phi, respectively. Yeast mannan inhibited 0-50% of zymosan uptake in various M phi, in parallel with their expression of MFR. We demonstrated that M phi were the source of C3 in our assay and that the activity of other components of the complement system, namely a C3 convertase, factor I, and a factor I cofactor were also present in serum-free cultures of human monocytes. Macrophage C3 was deposited rapidly, within 10 min, on the zymosan particles and mediated binding, ingestion, and stimulation of superoxide release in BCG-activated and thioglycollate-elicited peritoneal M phi via CR3. Local secretion of complement proteins by M phi themselves can therefore opsonize pathogens and cells able to activate the alternative pathway and effect their destruction.

Interferon gamma induces synthesis of complement alternative pathway proteins by human endothelial cells in culture.

Human umbilical vein endothelial cells grown in vitro under standard conditions contain a high level of mRNA specific for the complement regulatory factors H and I. An additional 1.8-kb mRNA encoding a truncated form of factor H is also present. IFN-gamma stimulation of the cells causes a 6-7 fold increase in both factor H mRNA species, and a greater than 10-fold increase in factor I mRNA. IL-1 and LPS slightly suppressed factor H mRNA, while TNF had no effect. mRNA for factor B is also detectable in IFN-gamma-stimulated cells, but messengers for C1q, C4bp, and CR3 beta chain were not found. Secretion of factor H protein was also stimulated by IFN-gamma. The presence of mRNA for factors H, B, and I, together with C3 secretion, demonstrated by others, suggests that endothelial cells can assemble the complete alternative complement pathway. Endothelial cell complement may be involved in leukocyte-endothelium interactions mediated by leukocyte C3 receptors.

Macrophage complement and lectin-like receptors bind Leishmania in the absence of serum.

We have examined the relative roles of the macrophage (M phi) plasma membrane receptor for the cleaved third complement component (iC3b, CR3) and of the mannosyl/fucosyl receptor (MFR) in binding and ingestion of Leishmania donovani. In the absence of exogenous complement, the binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, were inhibited by the anti-CR3 monoclonal antibody M1/70, by the Fab portion of an anti-C3 antibody, or by the nucleophile, sodium salicyl hydroxamate, an inhibitor of C3 fixation. This provides strong evidence that M phi-derived, cleaved C3 (iC3b) present on the promastigote surface mediates binding to CR3. Equivalent inhibition of promastigote binding and ingestion was also observed using the soluble inhibitors of MFR activity, mannan or ribonuclease B. No additive effect for blocking the two M phi receptors simultaneously was observed. For amastigotes, which are poor activators of the alternative pathway, a lesser but nevertheless equivalent effect was observed for the three soluble inhibitors of CR3-mediated binding vs. the two soluble inhibitors of MFR-mediated binding. Modulation experiments in which either CR3 or MFR had been rendered inaccessible demonstrated that both receptors must be present on the segment of M phi membrane to which the parasite binds. The combined function of these two distinct M phi receptors may provide a general mechanism for recognition and ingestion of other pathogenic protozoa known to activate the alternative pathway.

Resistance of the Echinococcus granulosus cyst wall to complement activation: analysis of the role of InsP6 deposits.

The larva of the cestode Echinococcus granulosus (hydatid cyst) is protected by the acellular laminated layer (LL). The mechanisms that make this thick coat a poor activator of host complement are incompletely understood. The structure binds, through unknown motifs, the host regulator of the alternative complement pathway (ACP), factor H. A second potential mechanism of ACP regulation, the inhibition of factor B activation, was detected in assays employing purified components (Immunopharmacology 42 : 91). The inhibitor was subsequently identified as myo-inositol hexakisphosphate (InsP(6)), which in the form of nano-deposits is a major component of the LL (Biochem J 362 : 297; J Cell Biochem 93 : 1272; FEBS J 273 : 3192). In this report we show that colloidal InsP(6 )solids inhibit factor B activation, through adsorption and associated impairment of C3b binding. However, this interaction is not relevant in the presence of serum proteins. In serum, InsP(6) deposits instead bind C1q, and initiate complement activation. This activation is curtailed through efficient C3b inactivation, previously shown to be entirely factor H-dependent, and now observed to be independent of the InsP(6) deposits. Therefore the complement resistance of the LL must be based on functional factor H binding sites present on the mucin-based meshwork that is its other major constituent.

Autoantibody facilitated cleavage of C1-inhibitor in autoimmune angioedema.

C1-inhibitor (C1-Inh) is an important inhibitor of the inflammatory response and deficiency of this inhibitor, which may be hereditary or acquired, is associated with recurrent episodes of edema. Recently, an autoimmune form of angioedema has been described that is associated with functional deficiency of C1-Inh and an autoantibody that impedes C1-Inh function. In this report we describe the isolation of C1-Inh from the monocytes and plasma of a patient with autoimmune angioedema and demonstrate that the patient's monocytes secrete structurally and functionally normal C1-Inh, but show that this protein circulates in the patient's plasma in an inactive, structurally altered form. Furthermore, using analytic gel electrophoresis techniques it is demonstrated that the patient's autoantibody facilitates cleavage of normal C1-Inh, by its target proteases, to the same species of C1-Inh that is found circulating in the patient's plasma. This autoantibody facilitated cleavage of normal C1-Inh is apparently a consequence of destabilization of protease/inhibitor complexes. These findings contribute to our understanding of protease/C1-Inh interactions and document important observations on pathogenic mechanisms in autoimmune disease.

Interaction of human monocytes, macrophages, and polymorphonuclear leukocytes with zymosan in vitro. Role of type 3 complement receptors and macrophage-derived complement.

Macrophages take up zymosan in the absence of exogenous complement via receptors for iC3b (type 3 complement receptors) acting with or without lectin-like receptors for mannosyl-fucosyl-terminated glycoconjugates. We previously provided evidence that macrophages themselves secrete complement-alternative pathway components able to opsonize zymosan locally (Ezekowitz et al., J. Exp. Med. 1984. 159:244-260). We show here that covalently bound C3 cleavage products C3b and iC3b can be eluted from zymosan particles cultivated with 36-h adherent human monocytes in the absence of serum. The ligand binding site of type 3 complement receptors is involved in macrophage-zymosan interactions as shown by inhibition studies of zymosan binding and uptake with Fab fragments of anti-C3 antibodies and monoclonal antireceptor antibodies M01 and OKM10. In contrast, antibody IB4, which binds to a receptor epitope distinct from the binding site, does not inhibit zymosan uptake. Selective modulation of macrophage receptors onto anticomplement receptor antibody and mannose-rich yeast mannan, respectively, confirms that the complement and lectin-like receptors are distinct. Human polymorphonuclear leukocytes, which express receptors for complement, but are not known to secrete complement proteins, bind and ingest only exogenously opsonized zymosan. Unopsonized zymosan is a poor trigger of respiratory burst activity in neutrophils or 7-d adherent human macrophages, but induces cell aggregation and secretion of large amounts of superoxide anion when these cells are co-cultivated in serum-free medium and challenged with zymosan. Our studies indicate that complement and/or other products synthesized by macrophages at extravascular sites could play an important role in opsonization and lysis of pathogens able to activate the alternative pathway and mediate macrophage-neutrophil collaboration in first-line host defence.

Collectins and host defence.

The collectins are a small family of soluble oligomeric proteins containing collagenous regions and C-type lectin domains. They are related in structure and function to complement protein C1q, and to H-, L- and M-ficolins. In humans, the collectins mannose-binding lectin (MBL) and surfactant proteins A and D (SP-A, SP-D) have important roles in innate immunity. MBL occurs mainly in blood plasma and in the upper respiratory tract. It binds to neutral sugar arrays on microorganisms and acts as an opsonin either directly (by binding to cell-surface calreticulin) or indirectly by activating complement. MBL circulates in complex with any of three proteases, named MBL-associated serine proteases (MASPs)-1, -2 and -3. MBL-MASP-2 complexes activate complement, but the role of MBL-MASP-1 and MBL-MASP-3 complexes is not yet known. MBL deficiency occurs at high frequency, and is associated with susceptibility to infection, particularly in infants. SP-A and SP-D are most abundant in the lungs, and also bind to microorganisms and inhaled particulates, mainly by lectin-sugar interactions. They do not activate complement, but act as opsonins and agglutinators, and have additional effects on cellular regulation. Mice deficient in SP-A or SP-D are susceptible to lung infections, and SP-D-deficient mice develop an emphysema-like condition.

Discrete MBL-MASP complexes show wide inter-individual variability in concentration: data from UK vs Armenian populations.

Mannan-binding lectin (MBL) circulates in plasma in complex with MBL-associated serine proteases (MASP) -1, -2 and -3 and a smaller component, MAp19. When MBL binds to the surface of foreign material (microorganisms), MASP-1, -2, -3 are activated. MASP-2 then activates the complement system. MASP-1 and -3 may activate other (unidentified) systems. MBL levels, MBL-bound MASP-1 and MBL-bound MASP-2 activities have been evaluated in healthy individuals from UK and Armenian populations. MBL-bound MASP-2 activity declines in aging (P<0.04). MBL correlates with smoking (P<0.02). There were significant differences between the two populations in MBL-bound MASP-1 activity and in MBL, but no difference in MBL-bound MASP-2 activity. When MASP activities were normalised to MBL (i.e. MASP-1 activity/MBL, MASP-2 activity/MBL), normalised MASP-2 activity in UK individuals was more than 2 fold higher than in Armenians. The difference in normalised MASP-2 activity level between these two Caucasoid populations, suggests that concentration of the MBL-(MASP-2) complex, and therefore the function of activating complement, depends not only on the quantity of MBL in serum and its oligomeric state, but also on the quantity of MASP-2 in serum. It is likely that in individuals with high MBL concentration there is excess free MBL not occupied by MASPs, particularly not by MASP-2.

Complement research in the 18th-21st centuries: Progress comes with new technology.

The complement system has been studied for about 120 years. Progress in defining this large and complex system has been dependent on the research technologies available, but since the introduction of protein chromatography, electrophoresis, and antibody-based assay methods in the 1950s and 60s, and sequencing of proteins and DNA in the 70s and 80s, there has been very rapid accumulation of data. With more recent improvements in 3D structure determination (nmr and X-ray crystallography), the structures of most of the complement proteins have now been solved. Complement research since 1990 has been greatly stimulated by the discoveries of the multiple proteins in the lectin pathway, the strong association of Factor H, C3, Factor B allelic variants with adult macular degeneration and atypical haemolytic uremic syndrome, and the introduction of the anti-C5 monoclonal antibody as a therapy for paroxysmal nocturnal hemoglobinuria and atypical haemolytic uremic syndrome. Potential new roles for complement in tissue development and the search for novel therapeutics suggest a very active future for complement research.

Collectins and viral infection.

Members of the collectin protein family are beta-inhibitors of influenza virus infectivity. They bind to carbohydrate on the surface of influenza virus and sterically inhibit virus interaction with host cells, and may also act as opsonins. We propose that collectins, by interacting with glycosylated viruses, act as innate inhibitors of viral infection.

Localisation of a group of antigenic sites in complement component C3, and identification of a new fragmentation pattern.

Immunisation of rabbits or goats with denaturated human C3 reproducibly gives rise to an antiserum of restricted specificity which precipitates native C3, C3b and iC3b, but not C3c or C3d isolated from 'aged' serum. The antigenic sites recognised by the antiserum appear to be localised in a small segment of the polypeptide chain located close to the N-terminus of the C3b alpha' chain. Affinity chromatography using antibodies developed by this procedure reveals an unusual pattern of degradation of C3 which occurs on conventional 'ageing' of serum at 37 degrees C.

Complement C4bC2 complex formation: an investigation by surface plasmon resonance.

Complex formation between the human complement proteins C4b and C2 was investigated by surface plasmon resonance. C4b was immobilised and C2 was used in the fluid phase to measure interaction at different ionic strengths (30-830 mM NaCl) and in the absence and presence of MgCl2. Maximum binding was observed at 30 mM NaCl, and was negligible above 300 mM NaCl. Binding was not greatly influenced by variation in Mg(2+) in the range of 2.5-15 mM. C4bC2 affinity (Kd) was determined by steady-state analysis to be 7.2x10(-8) M in physiological conditions (10 mM Hepes, 2.5 mM MgCl2, 0.75 mM CaCl2 and 140 mM NaCl, pH 7.4). For C4(H2O)C2 complex formation, a Kd of 4.0x10(-8) M was calculated. As far as detected by the applied method, complex formation does not involve conformational changes of one of the binding partners. Consistent with previous reports, C4bC2 binding takes place as a multiple-site binding event in the presence of Mg2+. C4bC2 complex formation in 10 mM Hepes, 2.5 mM EDTA and 140 mM NaCl (pH 7.4) was also observed and the interaction showed characteristics of a single-site binding event. Kd was 1.5x10(-8) M. Complement factor B (FB) was also tested for its binding to immobilised C4b. Weak interaction was observed at FB concentrations in the physiological range (500-1000 nM). Kd was 1.2x10(-6) M, indicating possible cross-reactivity between classical and alternative pathways of the activation of the complement system.

Interaction of C1-inhibitor with the C1r and C1s subcomponents in human C1.

1. Insoluble IgG-ovalbumin aggregates were used to bind and activate C1 from human serum. The bound C1 provided a useful reagent for studying the interaction of C1 subcomponents with C1-inhibitor. 2. C1-inhibitor bound to both subcomponents (C1r and C1s in C1 and formed stable complexes of respective apparent molecular weights 197,000 and 185,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The binding reaction proceeded more readily with C1s than with C1r and was correlated with the inhibition of C1s esterase activity. 3. At physiological ionic strength, binding of C1-inhibitor to subcomponents C1r and C1s caused release of these subcomponents from the C1-immune aggregates complex, indicating that C1-inhibitor binding decreased the inter-subcomponent binding forces in C1. At low ionic strength, however, this release did not occur.

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

The secondary structure of the von Willebrand factor type A domain in factor B of human complement by Fourier transform infrared spectroscopy. Its occurrence in collagen types VI, VII, XII and XIV, the integrins and other proteins by averaged structure predictions.

The type A domain of the von Willebrand Factor is found also in the complement proteins factor B (FB), C2, CR3 and CR4, the integrins, collagen types VI, VII, XII and XIV, and other proteins. FB is a component of the alternative pathway of the complement system of immune defence, and is cleaved into the fragments Bb and Ba during complement activation. Bb contains a von Willebrand Factor type A (vWF) domain of unknown secondary structure and a serine proteinase (SP) domain, whereas Ba contains three short consensus repeat/complement control protein (SCR/CCP) domains. Fourier transform infrared (FT-IR) spectroscopy on a recombinant vWF domain and on FB and its Bb and Ba fragments shows a broad amide I band. In H2O buffer, second derivative spectra of the amide I band show subcomponents at 1654 to 1657 cm-1, which is typical of alpha-helix, and at 1676 to 1685 cm-1 and 1636 to 1637 cm-1, which are typical of beta-strand. alpha-Helix was detected in the vWF domain, the Bb fragment and FB, and the proportion of alpha-helix present decreased in that order. This shows that the vWF domain contains appreciable amounts of alpha-helix, while the SP and SCR/CCP domains are almost entirely beta-sheet in their secondary structures. Quantitative integration of the vWF FT-IR spectrum showed that this contained 31% alpha-helix and 36% beta-sheet. In 2H2O buffer, the alpha-helix content in the vWF domain is sensitive to the solvent, while the beta-sheet content is less so. An alignment of 75 vWF type A sequences from 25 proteins was used for averaged secondary structure predictions of the total length of 206 residues by the Robson and Chou-Fasman methods. In support of the FT-IR analysis, a total of at least five well-predicted alpha-helices (35% of residues) and at least five well-predicted beta-strands (21% of residues) were identified by both predictive methods, all of which were interspersed by regions of coil or turn conformations. Eight of the ten predicted alpha-helices and beta-strands form an alternating arrangement with each other. Since the predicted alpha-helices are mostly amphipathic, and since the alpha-helix FT-IR band is sensitive to solvent, the alpha-helices are inferred to be on the protein surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Solution structure of a pair of complement modules by nuclear magnetic resonance.

A portion of human complement factor H spanning the 15th (H15) and 16th (H16) of its 20 modules, has been expressed in a yeast vector and subjected to structure determination in solution using two-dimensional 1H-NMR. The structure of H15 is very similar to that already established for the fifth module of factor H and H16, consistent with the view that all such complement control (C-) modules share a common overall topology. In addition, the tertiary structures of the component modules of the H15-16 pair are very similar to those of the modules when expressed individually, implying that each folds entirely autonomously within intact factor H. Aromatic residues in the third turn of H15 and the second turn of H16, together with a leucine residue from the linker region, contribute to a small intermodular interface. Comparatively few nuclear Overhauser effects were observable between protons on different modules. Consequently, a wide range of angles of "twist" (131 (+/- 146) degrees, mean value (+/- 1 standard deviation)), i.e. rotation about the long axis of one module with respect to the other, exists in the family of structures generated on the basis of the experimental data. However, much smaller variations occur in the two, orthogonal, angles (175 (+/- 12) degrees and 103 (+/- 6) degrees) that describe the "tilt". These observations may suggest upper limits on the relative flexibility of the two modules. Models were built to assess the outcome of applying such restrictions to all the neighbours within a string of 20 C-modules, and the resulting structures compare well with factor H as visualized by electron microscopy.