In vivo intracellular cytokine production by leukocytes during haemodialysis

Several chronic inflammatory changes undergone during chronic haemodialysis are associated with increased pro-inflammatory cytokine production. Although generation of anaphylatoxins has been incriminated in the untoward effects of haemodialysis, it is still debated whether anaphylatoxins stimulate monocyte secretion of TNF-alpha and IL-1. We demonstrate that peripheral mononuclear cells isolated from healthy controls and cultured with complement-activated autologous serum or recombinant C5a induced high levels of IL-1, IL-1ra, IL-8 and MCP-1, low levels of TNFalpha and sTNFRII but no IL-10 and MIP-1alpha. Cytokine production by leukocytes was investigated by FACS analysis in six patients dialysed consecutively with three equivalent low permeability membranes known to activate the complement to different degrees: polysulfone (F6HPS), cellulose acetate (CA) and cuprophane (CP). Percentage of leukocytes expressing IL-1, IL-1ra, TNF-alpha and IL-8 is increased in patients dialysed with CP. Moreover, we show for the first time that haemodialysis is associated with the production of cytokines by circulating neutrophils. Predialysis plasma levels of MCP-1 and TNFRII did not increase during the dialysis session at the time when anaphylatoxin generation was highest. Dialysis with membranes that activate the complement to a high extent induce activation of leukocytes which may explain chronic complications associated with dialysing with CP.     

Hydrolysis of myelin basic protein in human myelin by terminal complement complexes

The participation of terminal complement complexes (TCC) in demyelination has been shown in rodent cerebellar cultures. Since TCC modulates activities of various membrane-associated enzymes and increases the level of cellular Ca2+ we investigated whether TCC could activate Ca2+-dependent neutral proteases in myelin that would lead to hydrolysis of myelin basic protein (BP). Addition of antibody and C7-deficient serum plus C7 to sealed myelin vesicles of two to six bilayers caused significant BP hydrolysis compared to the hydrolysis caused by antibody and C7-deficient serum. Significant hydrolysis occurred at the stage of C5b6,7 assembly, which increased in magnitude at the C5b6-8 stage. C5b6-9 formation did not enhance the effect of C5b6-8. BP hydrolysis by C5b6,7 did not require Ca2+ whereas the effect of C5b6-8/C5b6-9 was, in part, Ca2+-dependent. We postulated that TCC formation in myelin membranes causes activation of myelin-associated neutral proteases with subsequent hydrolysis of BP as a consequence of complement peptide insertion and channel formation. Such processes may alter the structure of myelin and augment the action of other inflammatory cells and their products in demyelinating diseases that could ultimately lead to the loss of myelin.     

Complement activation induces the expression of decay-accelerating factor on human mesangial cells.

In the present study we evaluated the effect of complement activation by immune complexes (IC) on the expression of decay-accelerating factor (DAF) on human mesangial cells (MC). MC in culture were incubated with an Ag (DNP-Gelatin) that binds to fibronectin present in the MC matrix. Subsequently, MC were incubated with anti-DNP antibodies in the presence of human serum. By immunoperoxidase staining we showed that these incubations resulted in IC formation and deposition of human C3 and terminal complement components (C5b-9) on the mesangial matrix and on the surface of MC. By immunoperoxidase staining and by RIA we showed that IC formation and complement activation significantly increased DAF expression on the MC plasma membrane. The induction of DAF expression was a consequence of deposition of terminal complement components on the MC because, zymosan-activated serum and IC formation in the presence of C5- or C8-deficient serum failed to increase MC DAF expression. Furthermore, the observed increased DAF expression was the consequence of increased DAF synthesis by MC. Thus, both cycloheximide and actinomycin D blocked the increase on MC DAF observed after incubation with IC and serum. MC DAF had biophysical and functional characteristics similar to DAF in other cells. Thus, 1) MC DAF was resistant to trypsin but was removed from the MC membrane by pronase; 2) phosphatidylinositol-specific phospholipase C removed 48 +/- 4% of MC DAF indicating that MC DAF is anchored in the cell membrane by phosphatidylinositol groups; 3) DAF isolated from MC-inhibited complement-mediated hemolysis and demonstrated a molecular mass of 83 kDa. In conclusion, deposition of terminal complement components on human MC trigger new synthesis and membrane expression of DAF. Because DAF protects cells against complement-mediated lysis, we postulate that DAF may protect glomerular cells during IC and complement-mediated glomerulonephritis.     

Three pentraxins C-reactive protein,serum amyloid p component and pentraxin 3 mediate complement activation using Collectin CL-P1

BackgroundPentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition.MethodsWe used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay.ResultsWe demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation.ConclusionOur current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation.General significanceCL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.     

Attachment of human polymorphonuclear leukocytes to herpes simplex virus-infected fibroblasts mediated by antibody-independent complement activation

Herpes simplex virus (HSV)-infected cells can activate the human complement system without interference of specific anti-HSV antibodies. Analysis by flow cytometry showed that C3-like molecules were deposited on the membrane of the infected cell when incubated with human serum without specific antibodies. Depletion of calcium to block the classical pathway of the complement system had no effect on fluorescence intensity. The complement activation could be blocked by chelating both calcium and magnesium or by heating the serum. Furthermore, in the fluid phase C3 was converted to C3b by infected cells and not by uninfected cells. The antibody-independent activation did not lead to lysis of the virus-infected fibroblasts, indicating that the complement cascade is abrogated before formation of the membrane attack complex. This was also confirmed by measurement of the 50% hemolytic complement activities for total and alternative pathways. Polymorphonuclear leukocytes attached to infected fibroblasts after incubation of these fibroblasts with intact complement. This is most probably mediated by complement receptor binding of C3b and C3bi which is deposited on the membrane of the HSV-infected cell. Both type 1 and type 2 HSVs showed the same characteristics in complement activation and thereby mediated polymorphonuclear leukocyte adherence.     

维生素C和酸应激对中华鳖幼鳖血清补体C3和C4含量的影响

为研究维生素C对中华鳖(Pelodiscus sinensis)血清补体C3和C4的影响及其在酸应激条件下的变化,我们设置了6个实验组,饵料中维生素C的添加量依次为0、250、500、2500、5000和10000mg／kg,喂食4周后取其血清,用透射比浊法测定酸应激前后中华鳖血清补体C3和C4的含量。结果表明,维生素C添加量为250mg／kg时,血清补体C3的含量与对照组间没有明显不同;维生素C添加量为500、2500、5000和10000mg／kg的4组,血清补体C3的含量明显高于对照组和维生素C添加量为250mg／kg组;维生素C添加量为500mg／kg的一组,血清补体CA含量明显高于其它5组;维生素C添加量为250mg／kg组明显高于10000mg／kg组。酸应激后,补体C3的含量没有明显下降,将维生素C添加量为0、250和500mg／kg的三组并为一组处理,则应激后有明显下降。维生素C添加量为0、250和500mg／kg的3组,血清补体CA的含量在酸应激后明显下降,而维生素C添加量为2500、5000和10000mg／kg的3组,应激后血清补体C4没有明显变化。维生素C和酸应激对中华鳖血清补体C3和CA含量的影响没有交互作用。这说明,维生素C在一定剂量范围内,能提高中华鳖血清补体C3和CA的水平,酸应激能导致其含量降低,而高剂量的维生素C对其下降有颉颃作用[动物学报49(6)：769～774,2003]。     

Phylogenetic Analysis of the Homologous Proteins of the Terminal Complement Complex Supports the Emergence of C6 and C7 Followed by C8 and C9

The plasma complement system comprises several activation pathways that share a common terminal route involving the assembly of the terminal complement complex (TCC), formed by C5b–C9. The order of emergence of the homologous components of TCC (C6, C7, C8α, C8β, and C9) has been determined by phylogenetic analyses of their amino acid sequences. Using all the sequence data available for C6–C9 proteins, as well as for perforins, the results suggested that these TCC components originated from a single ancestral gene and that C6 and C7 were the earliest to emerge. Our evidence supports the notion that the ancestral gene had a complex modular composition. A series of gene duplications in combination with a tendency to lose modules resulted in successive complement proteins with decreasing modular complexity. C9 and perforin apparently are the result of different selective conditions to acquire pore-forming function. Thus C9 and perforin are examples of evolutionary parallelism. Received: 16 August 1998 / Accepted: 12 March 1999     

Cold activation of complement i. presence of coagulation-related activator.

Determination of the complement titer in the serum and plasm of 120 patients with chronic liver diseases showed that in eight (7%) patients with cirrhosis of the liver, chronic active or chronic inactive hepatitis complement in the serum was less than half in the plasma. The dissociation of complement serum and plasma was due to cold activation of the classical pathway of complement in vitro since serum drawn from these patients at 37 degrees C lost hemolytic activity in 4 hours when transferred to a cold environment. Neither HB antigen nor cryoglobulin participated in this phenomenon. The activation of complement in the cold could be prevented by increasing the ionic strength, or by adding vitamin E or, to a lesser extent its vehicle HCO-60, while heparin, Trasylol, soybean trypsin inhibitor, or hirudin had no effect. Trans-AMCHA prevented activation in one case. It is speculated that a factor appearing as a result of blood clotting is able to activate the classical pathway of complement in the cold; it is probably not related to Hageman factor (factor XII), factor VII, thrombin, kallikrein.     

Activation of complement at plasma-air or serum-air interface of rabbits

The possibility of the air-plasma interface giving rise to complement activation is investigated. After incubation of the plasma of a group of rabbits with zymosan and measurement of the degree of autologous polymorphonuclear leukocyte aggregation that follows the injection of a sample of the incubated plasma into a leukocyte suspension, it is found that the rabbits can be divided into two groups, sensitive and insensitive, depending on the degree of leukocytes aggregation. For the sensitive group it is found that both the plasma-air interface and the serum-air interface give rise to significant leukocyte aggregation. If the animal is decomplemented before the plasma is incubated in the presence of the air interface, there is no longer any significant leukocyte aggregation. It would appear that the complement system is activated by the presence of the air interface in plasma, but that fibrinogen does not play a pivotal role in the process.     

The release of C5a in complement-activated serum does not require C6

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.     

Membrane attack complex formation on yeast as trigger of selective release of terminal complement proteins from human polymorphonuclear leukocytes

It has recently been shown that measurable amounts of complement proteins, C6 and in particular C7, are released from human polymorphonuclear leukocytes (PMNs). The aim of the present study was to investigate the impact of opsonized Candida albicans on this release. Stimulation with opsonized C. albicans led to a rapid and sustained increase of C6 and C7 in the cell culture supernatant beginning within 5 min of placing in co-culture, whereas co-culture with unopsonized C. albicans or C. albicans mock-opsonized with inactivated human serum did not affect the release. In contrast, even after stimulation employing opsonized C. albicans, no release of the complement component C8 and only trace amounts of C9 were detected. The presence of the membrane attack complex (MAC) on C. albicans after opsonization was demonstrated by indirect immunofluorescence. Opsonization of C. albicans with human serum deficient in or depleted of a terminal complement component resulted in only minor stimulation of C6 and C7 release, although C3 deposition on the surface of C. albicans was not affected as determined by direct immunofluorescence. Detailed analyses with inactivated or deficient sera showed that detection of C6 and C7 was not due to insufficient washing of the opsonized yeast prior to co-culture and suggest that only a small proportion of these proteins was derived from the membrane bound and then cleaved off MAC. Thus, these findings imply that MAC on the fungal surface may represent an additional trigger for the release of C6 and C7 from PMNs, suggesting a new role for the terminal complement complex (TCC) on target membranes as modulator of PMN functions locally at the site of inflammation.     

Group B streptococci inhibit the chemotactic activity of the fifth component of complement

Infection with group B streptococci (GBS) is associated with a poor acute inflammatory response in which neutrophils fail to localize at the site of invasion. In the present studies, we have examined the effects of group B streptococci on C-derived chemotactic activity in human serum. Fresh human serum was activated to form C5a and C5adesarg by incubation with zymosan. The activated serum was then incubated with group B organisms, centrifuged, and the supernatants tested for chemotactic activity for human polymorphonuclear leukocytes. Group B organisms caused a dose-dependent decrease in C-dependent chemotactic activity. The degree of inhibition was profound with 1 X 10(9) bacteria/ml (10% of control). Experiments indicated that significant chemotactic factor inactivation occurred within 2 min of exposure to GBS organisms, while maximal inhibition occurred after 30 min incubation. A number of different strains of GBS of types I, II, and III possessed inhibitory activity. In contrast, group D streptococci, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae failed to inhibit the C-derived chemotactic activity in human serum. Group A streptococci that were M protein positive also inactivated C-dependent chemotactic activity in serum, as previously reported. The inhibitory activity of the GBS strains could be abolished by heat or trypsin treatment but not by neuraminidase, pronase, or pepsin. C5a levels in zymosan-activated serum as measured by RIA were not decreased after incubation with an inhibitory strain suggesting that absorption was not involved. SDS-PAGE analysis revealed that group B streptococci degrade the C5a molecule, increasing its electrophoretic mobility by removing a fragment with a m.w. of approximately 650 Da. Thus, one of the reasons for the poor inflammatory response at the site of GBS infection may reside in the ability of these pathogens to inactivate C-derived inflammatory mediators. The GBS C5a-ase activity probably serves as an additional virulence factor for these organisms contributing to the poor inflammatory response characteristic of group B streptococcal infection.     

Activation of the alternative complement pathway and generation of stimulating factors for granulocytes by glass fibers

Continuous-filament glass fibers coated with organic agents, candidate asbestos substitutes, were assessed for their ability to elicit from normal human serum complement-derived cleavage products which are able to stimulate the chemotaxis and the respiratory burst of polymorphonuclear leukocytes. Glass fibers generated chemoattracting and respiratory stimulating factors for polymorphonuclears from human serum. The effect was dose related for chemotaxis from the serum fiber concentration of 75 g/ml to 1,250 g/ml. The serum chemoattracting activity, as well the respiratory stimulation, were dramatically impaired when serum had been preliminarily absorbed with antiC5 antiserum. Since the impairment of chemotactic activity occurred also in the presence of EDTA, but not in the presence of EGTA, we assumed an activation of the alternative complement pathway.Glass fibers were studied in comparison to a UICC sample of Canadian chrysotile asbestos, which is able to activate in vitro the alternative complement pathway.Glass fibers exhibited less ability than asbestos fibers to generate complement cleavage products with chemotactic activity for polymorphonuclears; however, they produced an activity about equal to 80% of a chemotactic standard stimulus such as zymosan-activated plasma.Abbreviations AF asbestos fibers - antiCS-abs-S NHS absorbed with antiserum against C5 - EDTA-CH-S NHS treated with EDTA - EGTA-Ch-S NHS treated with EGTA - GF continuous filament glass fibers coated with a binder of organic substances - NHP normal human plasma - NHS normal human serum - PMN polymorphonuclear luekocytes - ZAP zymosan-activated plasma     

Multiple signal messengers generated by terminal complement complexes and their role in terminal complement complex elimination

The best established function of C5b-9 is the ability to lyse or kill cells after assembly in the plasma membrane. In addition to this cytolytic function, increasing evidence suggests that C5b-9 also stimulate a variety of cell functions in vitro. Relatively little is known about the C5b-9 signals responsible for cell activation other than a transient increase in cytosolic Ca2+ primarily due to Ca2+ influx that have been determined in a cell population. In this report, signal messenger generation in Ehrlich cells by the sublytic terminal complement complexes (TCC), C5b-9, C5b-8, and C5b-7, was further examined, as well as the role of signal messengers in stimulating elimination of TCC from the cell surface. Changes in cytosolic Ca2+ were monitored in individual cells after a single dose of C5b-9 by digital imaging fluorescence microscopy that revealed oscillations in cytosolic Ca2+ over a period of 10 min. Sublytic C5b-9 substantially increased protein kinase C (PKC) activity at an external Ca2+ concentration of 1.5 mM. C5b-9-mediated PKC activation could be inhibited by 60 to 80% when external Ca2+ was reduced to 0.015 mM. C5b-8, but not C5b-7, activated PKC to a lesser extent. C5b-8 and C5b-7 also stimulated an increase in cAMP. Rapid elimination of TCC known to be stimulated by Ca2+ signal was partially inhibited by protein kinase inhibitors, H-7 and to a lesser extent by HA1004, suggesting a role for PKC in the elimination response. TCC elimination was not accelerated by agents that increase cAMP.     

Complement activation by cell-associated immune complexes in contact sensitivity

Lymph node cells collected 4 days after painting the skin with picryl chloride activate the first components of the classical pathway of complement cascade, as shown by consumption of C4 of rabbit complement with total sparing of C5 and factor B activity. In contrast, lymph node cells collected 1 or 6 days after sensitization fail to do so. The ability of “4-day” cells to activate complement is inhibited by treating the cells with specific low-molecular-weight hapten, which is known to dissociate the immune complex present on the cell surface. When mouse serum was used as source of complement, a different behavior in complement activation between CBA/J and B.10.D2-New/SnJ serum was observed: “4-day” cells failed to consume CBA/J serum whereas a normal complement activation was detected when B.10.D2-New/SnJ serum was used. Using these two sera which differ in the level of C4, an inverse relationship between the ability of “4-day” cells to activate complement and their capacity to induce contact sensitivity when injected into the footpad of normal recipients was reported. Experiments performed using sera from C5 genetically deficient mice demonstrate that only the early complement components are involved, suggesting that membrane immune complexes are solubilized as a result of complement activation; on the other hand, membrane bound activated complement components could alter the immunizing potential of “4-day” cells.     

Collectin CL-P1 utilizes C-reactive protein for complement activation

BackgroundC-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown.MethodsWe prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1.ResultsHere, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions.General SignificanceThe interaction of collectin CL-P1 with CFH might be key for preventing attack on “self” as a result of complement activation induced by the CL-P1 and CRP interaction.     

Functional analysis of Ficolin-3 mediated complement activation

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.     

The development of a homologous teleost insulin radioimmunoassay and its use in the study of adrenaline on insulin secretion from isolated pancreatic islet tissue of the rainbow trout, Salmo gairdinerii (R.)

A homologous teleost insulin radioimmunoassay (RIA) employing bonito insulin RIA components is described. The RIA sensitivity and specificity was sufficient to measure endogenous immunoreactive insulin (IRI) levels in trout serum, pancreatic islet tissue extract and in vitro culture medium. Adrenaline at 10(-6)M evoked a marked stimulation of basal insulin release from isolated islets; whereas adrenaline at 10(-10)M evoked an inhibition of basal insulin release.     

C3, C4, and the terminal complement complex differ from C1q by binding predominantly to the antigenic part of solid phase immune complexes

The binding of the C components C1q, C4, C3, the terminal C5b-9 complement complex (TCC) and S protein to immune complexes was studied. The hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) conjugated to BSA was adsorbed to polystyrene plates and reacted with a human IgG3-mouse chimeric anti-NIP antibody. After addition of serum a dose-dependent binding of C1q, C4, C3, and TCC to the immune complexes was found. An increase in the amount of NIP-BSA was associated with an increase in the binding of TCC and a decrease in the binding of S-protein. After addition of soluble NIP only 4 to 6% of the anti-NIP antibody remained bound to the Ag. C1q showed diminished binding after addition of NIP, whereas C4, C3, and TCC quantitatively remained bound to the Ag. Binding of TCC to the immune complexes was also found in an alternative assay, in which the anti-NIP antibody was adsorbed to the solid phase before NIP-BSA and an additional layer of anti-NIP antibody were added. The supernatants from the solid phase assay were tested for C3 activation and formation of the fluid phase TCC (SC5b-9). Activation of the C3 was reflected in the fluid phase by a dose-dependent increase in C3 activation products. This was not seen for TCC despite increased binding to the solid phase.