Expression of C5a-like biological activities by the fifth component of human complement (C5) upon limited digestion with noncomplement enzymes without release of polypeptide fragments.

Experimental conditions required for the expression of maximum C5 activation upon limited trypsin hydrolysis were determined to be 0.008 mol of trypsin/mol C5 in a reaction mixture containing 1 mg C5/ml veronal-buffered saline incubated at 37 degrees C for 30 min. Employing these optimal incubation conditions, the primary or preferred site of trypsin hydrolysis of the C5 alpha-chain resulted in the production of C5 alpha 1 (molecular weight, 90,000) and C5 alpha 5 (molecular weight, 25,000) fragments that remained disulfide bonded to the modified C5 molecule (C5'try). Detailed structural-functional analyses clearly indicated the trypsin-mediated conversion of the C5 alpha-chain to C5 alpha 1 and C5 alpha 5 was responsible for the acquisition of neutrophil lysosomal enzyme-releasing and chemotactic activities. Gel filtration column chromatography under physiological ionic strength, pH 7.4, or in the presence of 0.2% SDS further demonstrated that at least 90% of the total recoverable C5a-like biological activity was mediated by the 210,000 molecular weight forms of trypsin-modified C5. Other physiologically relevant, noncomplement protease enzymes (alpha-thrombin, plasmin, and elastase) also activated C5 to express C5a-like reactivities. Analysis of alpha-thrombin-induced, C5 alpha-chain cleavage events by SDS-polyacrylamide slab gel electrophoresis indicated that the mechanism of alpha-thrombin-activation of C5 is similar to that described for trypsin. Reconciliation of this novel mechanism of C5 activation by trypsin with previously published results, and a discussion of the biological significance of noncomplement enzyme-mediated activation of C5 as it might relate to inflammatory processes in vivo, was presented.

The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex.

Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.

The membrane attack mechanism of complement. Verification of a stable C5-9 complex in free solution.

The membrane attack mechanism of complement, C5 to C9, has previously been postulated to associate on the target cell surface to a stable decamolecular complex with a calculated mol wt of 995,000. A soluble and stable complex consisting of C5, C6, C7, C8, and C9 has now been demonstrated to arise as a consequence of complement activation by the classical or alternate pathway. It has a sedimentation coefficient of 22.5S and a mol wt of 1 million daltons, and it migrates on electrophoresis at pH 8.6 as an alpha-globulin. The stable and soluble C5b-9 complex cannot bind to erythrocytes and has no demonstrable cytolytic activity. However, due to partially unsaturated binding sites for C9, it can bind additional C9 and thus function as an inhibitor of lysis of EAC1-8 by C9. These results support the concept according to which the membrane-bound attack system of complement represents a stable, decamolecular assembly of C5b-9. Unlike its analogue in free solution, the membrane-bound complex is cytolytically active.

Complement-dependent proinflammatory properties of the Alzheimer's disease beta-peptide.

Large numbers of neuritic plaques (NP), largely composed of a fibrillar insoluble form of the beta-amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer's disease (AD) patients in association with damaged neuronal processes, increased numbers of activated astrocytes and microglia, and several proteins including the components of the proinflammatory complement system. These studies address the hypothesis that the activated complement system mediates the cellular changes that surround fibrillar Abeta deposits in NP. We report that Abeta peptides directly and independently activate the alternative complement pathway as well as the classical complement pathway; trigger the formation of covalent, ester-linked complexes of Abeta with activation products of the third complement component (C3); generate the cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory C5b-9 membrane attack complex, in functionally active form able to insert into and permeabilize the membrane of neuronal precursor cells. These findings provide inflammation-based mechanisms to account for the presence of complement components in NP in association with damaged neurons and increased numbers of activated glial cells, and they have potential implications for the therapy of AD.

Molecular analysis of the membrane attack mechanism of complement.

The molecular arrangement of the membrane attack mechanism of complement was explored. The molar ratios of the components within the C5-9 assembly on the target cell surface were determined using human complement proteins in highly purified and radiolabeled form. With the aid of monospecific complement antisera it was possible to probe the spatial relationships between the components of the assembly. C5 and C6, in the presence of C7, were bound to EAC1-3 in equimolar quantities irrespective of the amounts and the relative proportions of C5, C6, and C7 offered. The amount of C8 bound to EAC1-7 increased with input and at saturation of all C8 binding sites the molar ratio of bound C8/bound C5 approached 1.0. Uptake of C9 by EAC1-8 increased with input and at saturation of all C9 binding sites the molar ratio of bound C9/bound C8 became 6.0. However, calculations suggest that the binding of three C9 molecules to one C8 molecule is sufficient to achieve a full hemolytic effect. Evidence was obtained indicating that binding and hemolytic function of C9 depends upon cooperative interaction of multiple C9 molecules. Binding of C8 to EAC1-7 and the generation of hemolytic C8 sites were inhibited by antibody to either C5, C6, or C7. Uptake of C9 by EAC1-8 and the generation of hemolytic C9 sites were strongly inhibited by anti-C8 and to a lesser degree by anti-C5. Binding of C9 (but not hemolysis) was also reduced by antibody to C6 or C7. The data are consistent with the concept that the fully assembled membrane attack mechanism of complement consists of a decamolecular complex: a trimolecular arrangement composed of C5, C6, and C7 forms the binding site for one C8 molecule which in turn furnishes binding sites for six C9 molecules, saturation of three sites apparently being sufficient for expression of full cytolytic activity of the complex. This work made it possible to design a simple molecular model.

The membrane attack mechanism of complement. Reversible interactions among the five native components in free solution.

Reversible interactions in free solution were demonstrated to occur (a) between C5 and C8, (b) between C5, 6, 7 and C8, and (c) between C8 and C9. No interaction was observed between C8 and C6 or C7 and between C9 and C5, 6, 7. Interactions between C8 and C9 were enhanced at lowered ionic strength (0.05) and a molar excess of C8 over C9. Complex formation was independent of pH over the range of 6.5-8.5. Under optimal conditions the C8, 9 complex had a sedimentation coefficient of 10.2-10.6S, while native C8 and C9 sedimented at 8.5 and 4.8S, respectively. Specificity and reversibility of these interactions were established. In spite of the limited number of interactions observed, all five of the native proteins of the membrane attack mechanism interacted to form an association product that sedimented at 10.8-11.2S. Demonstration of this product in free solution supports the concept that C5-9 on acquisition of cytolytic activity assemble into a stable multimolecular complex.

Human platelet-initiated formation and uptake of the C5-9 complex of human complement.

We have studied the interaction of radiolabeled complement components with normal human platelets, platelets from a patient with paroxysmal nocturnal hemoglobinuria, and rabbit platelets in the absence of known complement activators or in the presence of cobra venom factor (CVF). When unwashed platelets in platelet-rich plasma, or washed platelets suspended in serum or autologous plasma, were incubated for 30 min, C3 and terminal components (C5, C8, and C9) were found to bind to them. The terminal components were shown to be bound as the C5-9 complex, rather than as individual proteins, by eluting them from the platelet membrane and examining their behavior on ultracentrifugation. They cosedimented at a rate characteristic of the stable C5-9 complex (22S). As many as 370-1,380 C5-9 complexes/platelet were calculated to have been bound during the incubation period. The complex so formed did not differ by ultracentrifugational criteria from that binding to rabbit platelets after CVF activation of complement. Though C3 was not included in the complex, it did not appear to be bound by nonspecific absorption. It could not be removed by washing but rather was eluted by the freeze-thaw technique used to elute the C5-9 complex. Incubation of radiolabeled components in platelet-free plasma did not result in C5-9 complex formation, indicating an initiating role for platelets in this reaction. In contrast to platelets, erythrocytes incubated in analogous plasma did not induce detectable C5-9 formation. Neither EDTA, phenylmethylsulfonylfluoride, nor epsilon-amino-N-caproic acid prevented platelet-initiated formation of C5-9, suggesting that the reaction may involve mechanisms of complement activation not previously described.

Immunoadsorbent affinity purification of the fifth component (C5) of human complement and development of a highly sensitive hemolytic assay.

The fifth component of complement (C5) has been isolated from human serum in fully hemolytically active form by immunoadsorbent and anion exchange column chromatography. The immunoadsorbent column was prepared by the covalent coupling of the purified IgG fraction obtained from monospecific goat anti-human C5 antiserum to CNBr activated Sepharose 4B. Establishment of appropriate conditions for the dissociation and elution of functionally active C5 from the immunoadsorbent column was of central importance in the development of this purification procedure. The C5 preparations exhibited final yields of 20--50% with 570--710-fold purification factors based on recovery of specific hemolytic activity. These preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide slab gel electrophoretic and immunochemical criteria. A C5-depleted reagent (C5D) was generated from the non-adsorbed protein containing fractions obtained subsequent to the passage of freshly drawn NHS plus 10 mM EDTA through the monospecific anti-C5 Sepharose 4B column. Upon reconstitution of C5D with Ca2+, Mg2+, and C1q, this reagent was utilized for the detection and quantitation of C5 hemolytic activity. The purified C5 preparations contained 1.5--2.5 x 10(12) effective molecules/mg protein and NHS expressed 0.5--2.0 x 10(11) effective molecules/ml.

Biological effects of short-term, high-concentration exposure to methyl isocyanate. VI. In vitro and in vivo complement activation studies.

The ability of MIC to induce complement activation in vitro and in vivo was investigated. For the in vitro studies, both human and guinea pig serum or EDTA-plasma samples were exposed to 1167 to 1260 ppm MIC vapor for 15 min at room temperature. The human serum samples exposed to MIC showed significant reductions in Factor B, C2, C4, C3, C5, and total hemolytic complement CH50 activity levels. C6 functional activity was unaffected. The C3, C5, and CH50 functional activities in guinea pig serum (the only functional tests conducted on these samples) were more sensitive to MIC-mediated reduction than the corresponding activity reductions observed in the human serum samples. The human and single guinea pig EDTA-plasma samples exposed to MIC vapor showed no evidence of C3 consumption but did show significant reductions in CH50 levels. Thus, MIC vapor was able to activate, and thereby reduce serum complement C3 activity in vitro by a complement-dependent process. However, the data suggest at least one complement component other than C3 was inactivated in EDTA-plasma by a complement-independent mechanism. For the in vivo studies, five pairs of guinea pigs were exposed to 644 to 702 ppm MIC vapor until one of the pair died (11-15 min). MIC exposure was then discontinued, the surviving guinea pig was sacrificed, and EDTA-plasma was obtained from both animals and analyzed for complement consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of serum complement by contrast media.

Evidence is presented for the activation of serum complement by contrast media, in vitro and in vivo. Activation as a function of concentration was measured and the increasing order of effectiveness was found to be metrizamide, iothalamate, diatrizoate, acetrizoate, iodipamide and iopanoate. This order is the same as for protein binding and enzyme inhibition. The activation mechanism for iodipamide, and by inference for the other compounds, does not involve gamma-globulin aggregation. Serial daily injections in normal dogs resulted in substantial declines in serum complement over several days. Guinea pigs which were depleted of serum complement with cobra venom factor were found to be no less sensitive to lethal doses of iodipamide than those with normal complement. Implications of these findings are discussed.

Assembly of the functional membrane attack complex of human complement: formation of disulfide-linked C9 dimers.

The 158,000 Mr protein, previously designated C5c, present in fully assembled complement (C) membrane attack complexes (MC5b-9) has been identified as a disulfide-bonded dimer of C9. This conclusion was based on the observations that: (i) a portion of the 125I-radiolabeled precursor C9 incorporated into MC5b-9 complexes comigrated with the 158,000 Mr protein band in NaDodSO4/polyacrylamide slab gels; (ii) monospecific antisera produced against native C9 and the 158,000 Mr protein immunologically crossreacted with monomeric native C9 by double immunodiffusion and with monomeric C9 and the 158,000 Mr protein on immunoreplication procedures; and (iii) two-dimensional NaDodSO4/polyacrylamide slab gel electrophoresis, in which the second dimension was conducted under reducing conditions, revealed that the 158,000 Mr protein contained two identical 71,000 Mr subunits which comigrated with monomeric C9. Molar ratio estimates indicated that 1 mol of C5b, C9 dimer, C6, C7, and C8 and 3-4 mol of C9 monomer were present per MC5b-9 complex. Each fully assembled membrane-bound MC5b-9 complex would therefore have a calculated Mr of 982,000. The presence of C9 dimers in the hemolytically active 29S dimeric form of the MC5b-9 complex and the absence of C9 dimers in the hemolytically inactive 23S monomeric form of the fluid phase SC5b-9 complex strongly suggest an important role for C9 dimer formation in MC5b-9 complex structure and function. The most probable function of C9 dimers would be the formation of intercomplex disulfide crosslinks which would provide a mechanism to stabilize the assembly of MC5b-9 into aggregates of increasing size on the target membrane surface which would thus be responsible for the observed pore size heterogeneity of functional C lesions.

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement.

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.

The effect of vitamin C nutriture on complement component C1q concentrations in guinea pig plasma.

This study shows that guinea pigs fed 100 times the amount of vitamin C needed for growth and for prevention of scurvy have elevated levels of complement component C1q. C1q is a plasma protein rich in hydroxyproline, an amino acid whose biosynthesis requires ascorbate. C1q is essential for host defense against pathogens, both as a component of the classical complement pathway and as an opsonin in the phagocytosis process. We measured C1q in vitamin C-depleted guinea pigs that had been repleted for 4 wks with the following daily doses of ascorbate (mg/100 g body wt): 0.50 (suboptimal), 2.0 (adequate), 10 (ample) and 50 (tissue saturating). We measured C1q in three ways: indirectly by quantifying protein-bound hydroxyproline and directly by hemolytic assay and by immunodiffusion against anti-C1q. Regardless of the method, plasma C1q was 30-50% higher in animals fed tissue-saturating ascorbate than in those fed adequate or suboptimal amounts of the vitamin (p less than 0.05, one-way analysis of variance, least significant difference test). These data confirm and significantly extend earlier work that provided indirect evidence for a relationship between C1q and ascorbate nutriture in the guinea pig. They are consistent with a possible relationship between ascorbate nutriture and host defense.

Ba and Bb fragments of factor B activation: fragment production, biological activities, neoepitope expression and quantitation in clinical samples.

Factor B is a centrally important component of the alternative complement pathway. Alternative pathway activation results in factor B cleavage and production of the amino-terminal Ba and the carboxyl-terminal Bb fragments which have molecular weights of approximately 30,000 and 63,000 daltons, respectively. Both Ba and Bb fragments have been reported to express a variety of biological activities in vitro. Thus, binding of Ba and Bb fragments to specific B lymphocyte surface receptors modulates proliferation of prestimulated B cells. In addition, the enzymatically active Bb fragment induces activation and spreading of human and murine macrophages and monocytes as well as regulates C5a des Arg chemotactic activity. The fractional catabolic rate and metabolism of factor B in vivo is similar to that of C3, C4 and C5 complement proteins, which are among the most metabolically active plasma proteins in the circulatory system. Factor B hyperconsumption and increased catabolism, concomitant with factor B fragment production, occurs in a wide variety of diseases, including gram-negative sepsis, autoimmune diseases and burns. Measurement of alternative pathway activation in vivo has been attempted utilized a number of different techniques to quantitate factor B fragments in biological fluids. However, the recent development of enzyme immunoassays (EIA) employing monoclonal antibodies (MoAbs) reactive with factor B fragment neoepitopes provides the best approach currently available for the quantitation of factor B activation fragments. Results obtained using these new MoAb-based EIAs have indicated that factor B fragment concentrations were elevated, as compared with normal donor levels, in EDTA plasma samples obtained from patients with rheumatoid arthritis and systemic lupus erythematosus (SLE). Plasma concentrations of factor B fragments, especially Ba fragment levels, in these patients showed a positive correlation with disease activity scores. One of the highest disease activity correlations was obtained with Ba fragment measurements in SLE plasma samples. In fact, the results strongly suggested that quantitation of Ba fragment levels in SLE plasma samples more accurately reflected disease activity and was a more sensitive predictor of impending flare in these patients than any other test(s) currently available.

The presence of the fifth component of complement (C5) in rabbit uterine flushings in relation to reproductive state.

The fifth component of complement (C5) has been detected in rabbit uterine flushings. The C5 activity was evaluated using a hemolytic assay which requires the use of a C5-depleted reagent (C5D) prepared by affinity chromatography of normal human serum. In the absence of C5D, there was no hemolysis of antibody-sensitized erythrocytes by rabbit uterine flushings, whereas the presence of the C5D reagent resulted in substantial hemolysis. The amount of hemolysis was correlated with the reproductive state of the rabbits, with higher amounts of hemolysis (expressed per mg uterine flushing protein) evident in estrous rabbits. In addition, the amounts of immunoglobulin G (IgG), albumin, and total protein were also determined in the uterine flushings. The amounts of total protein and IgG were increased in day-6 pregnant animals compared to estrus while the amount of albumin per ml uterine flushing was not significantly changed.

Nonimmunologic complement activation in normal human serum induced by radiographic contrast media.

Two different radiographic contrast media (RCM), iothalamate and iodipamide, induced the activation of several complement (C) components in normal, genetically C2-deficient and agammaglobulinemic human sera in vitro. This activation was dose dependent and demonstrable by a reduction in whole C as well as C4, C2, C3, and C5 hemolytic activities. C6, C8, and C9 hemolytic activities were unaffected. Concommitant with the loss of C3 hemolytic activity was the appearance of C3 proteolytic cleavage products that were identified by immunoelectrophoresis. Both the loss of C3 hemolytic activity and the production of C3 fragments occurred in the presence of 10 mM EDTA, indicating RCM-induced C3 cleavage occurred without participation of the multicomponent C3/C5 convertases of either the classical or alternative C pathways. Furthermore, loss of C3 hemolytic activity was not due to the direct alteration of the C3 molecule by RCM because purified C3 was unaffected upon incubation with RCM at a concentration that induced 80% reduction in the C3 hemolytic activity in normal human serum. Serum samples obtained from 40 patients, before and 30 min after undergoing i.v. pyelography, revealed no significant change in total hemolytic C activity; 34 patients received sodium and methylglucamine diatrizoate and six received sodium iothalamate. Hemolytic C3 levels were also determined for the six patients before and 30 min after administration of sodium iothalamate and no significant change in activity was detectable.

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.

C1Q receptors on cultured human gingival fibroblasts: analysis of binding properties.

The interaction of the purified C1q component of human C with synchronized, quiescent human gingival fibroblasts was investigated, and the presence of a specific binding site was demonstrated. Quantitative binding studies with radioiodinated C1q showed that binding was specific, saturable, and reversible upon addition of unlabeled C1q or by increasing the salt concentration. Scatchard plot analysis of the data yielded an affinity constant of 2 X 10(7) M-1 for all cell strains examined. The capacity for C1q binding varied among the eight cell strains examined. The number of binding sites per cell ranged from 2.6 to 17.7 X 10(6) with an average of 8.4 X 10(6). The receptor was insensitive to trypsin digestion, and it bound the collagen-like portion of the C1q molecule. Specific immunofluorescence staining showed that virtually all the viable cultured fibroblasts were able to bind added C1q. Flow cytometric analysis demonstrated a spectrum of fluorescence intensity among the cell strains, and there was a positive correlation between fluorescence intensity and the number of binding sites detected by using radiolabeled C1q.