Mechanism Of Complement Activation Research Articles

Complement and Alcoholic Liver Disease: Role of C1q in the Pathogenesis of Ethanol-Induced Liver Injury in Mice

Complement is involved in the development of alcoholic liver disease in mice; however, the mechanisms for complement activation during ethanol exposure have not been identified. C1q, the recognition subunit of the first complement component, binds to apoptotic cells, thereby activating the classical complement pathway. Because ethanol exposure increases hepatocellular apoptosis, we hypothesized that ethanol-induced apoptosis would lead to activation of complement via the classical pathway. Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets or pair-fed control diets for 4 or 25 days. Ethanol feeding for 4 days increased apoptosis of Kupffer cells in both wild-type and C1qa-/- mice. Ethanol-induced deposition of C1q and C3b/iC3b/C3c was colocalized with apoptotic Kupffer cells in wild-type, but not C1qa-/-, mice. Furthermore, ethanol-induced increases in tumor necrosis factor-alpha and interleukin-6 expression at this early time point were suppressed in C1q-deficient mice. Chronic ethanol feeding (25 days) increased steatosis, hepatocyte apoptosis, and activity of serum alanine and aspartate aminotransferases in wild-type mice. These markers of hepatocyte injury were attenuated in C1qa-/- mice. In contrast, chronic ethanol (25 days)-induced increases in cytochrome P450 2E1 expression and oxidative stress did not differ between wild-type and C1qa-/- mice. For the first time, these data indicate that ethanol activates the classical complement pathway via C1q binding to apoptotic cells in the liver and that C1q contributes to the pathogenesis of ethanol-induced liver injury.

Complement activation in emergency department patients with severe sepsis.

This study assessed the extent and mechanism of complement activation in community-acquired sepsis at presentation to the emergency department (ED) and following 24 hours of quantitative resuscitation. A prospective pilot study of patients with severe sepsis and healthy controls was conducted among individuals presenting to a tertiary care ED. Resuscitation, including antibiotics and therapies to normalize central venous and mean arterial pressure (MAP) and central venous oxygenation, was performed on all patients. Serum levels of Factor Bb (alternative pathway), C4d (classical and mannose-binding lectin [MBL] pathway), C3, C3a, and C5a were determined at presentation and 24 hours later among patients. Twenty patients and 10 healthy volunteer controls were enrolled. Compared to volunteers, all proteins measured were abnormally higher among septic patients (C4d 3.5-fold; Factor Bb 6.1-fold; C3 0.8-fold; C3a 11.6-fold; C5a 1.8-fold). Elevations in C5a were most strongly correlated with alternative pathway activation. Surprisingly, a slight but significant inverse relationship between illness severity (by sequential organ failure assessment [SOFA] score) and C5a levels at presentation was noted. Twenty-four hours of structured resuscitation did not, on average, affect any of the mediators studied. Patients with community-acquired sepsis have extensive complement activation, particularly of the alternative pathway, at the time of presentation that was not significantly reversed by 24 hours of aggressive resuscitation.

Membranoproliferative glomerulonephritis

Membranoproliferative glomerulonephritis is an uncommon kidney disorder characterized by mesangial cell proliferation and structural changes in glomerular capillary walls. It can be subdivided into idiopathic and secondary forms, which are differentially diagnosed by a review of clinical features, laboratory data, and renal histopathology. Three types—I, II, and III—have been defined by pathologic features. All three types are associated with hypocomplementemia, but they manifest somewhat different mechanisms of complement activation. Type II, also known as “dense deposit disease”, is associated with the presence of C3-nephritic factor. Membranoproliferative glomerulonephritis primarily affects children and young adults, with patients presenting with nephrotic or nephritic syndrome or with asymptomatic renal disease. This type of glomerulonephritis often progresses slowly to end-stage renal disease, and it tends to recur after renal transplantation, especially type II. The efficacy of various forms of treatment remains controversial; however, long-term steroid treatment seems to be effective in children with nephrotic-range proteinuria. Improvement in renal outcomes largely relies on the evaluation of more selective agents in carefully controlled studies.

Sialic Acid-Containing Lipopolysaccharides of Salmonella O48 Strains—Potential Role in Camouflage and Susceptibility to the Bactericidal Effect of Normal Human Serum

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.

Multiple routes of complement activation by Mycobacterium bovis BCG

Mycobacterium tuberculosis is the leading cause of infectious disease in humans in the world. It evades the host immune system by being phagocytosed by macrophages and residing intracellularly. Complement-dependent opsonisation of extracellular mycobacteria may assist them to enter macrophages. This work examines in detail the mechanisms of complement activation by whole mycobacteria using Mycobacterium bovis BCG as a model organism. M. bovis BCG directly activates the classical, lectin and alternative pathways, resulting in fixation of C3b onto macromolecules of the mycobacterial surface. Investigation into the classical pathway has shown direct binding of human C1q to whole mycobacteria in the absence of antibodies. Most human sera contain IgG and IgM-anti-( M. bovis BCG), and pre-incubation with human immunoglobulin enhances C1q binding to the bacteria. Therefore classical pathway activation is both antibody-independent and dependent. The bacteria also activate the alternative pathway in an antibody-independent manner, but Factor H also binds, suggesting some regulation of amplification by this pathway. For the lectin pathway we have demonstrated direct binding of both MBL and L-ficolin from human serum to whole mycobacteria and subsequent MASP2 activation. H-ficolin binding was not observed. No M. bovis BCG cell surface or secreted protease appears likely to influence complement activation. Together, these data provide a more detailed analysis of the mechanisms by which M. bovis BCG interacts with the complement system.

Mechanisms of complement activation, C4d deposition, and their contribution to the pathogenesis of antibody-mediated rejection.

Complement split products have emerged as useful markers of antibody-mediated rejection in solid organ transplants. One split product, C4d, is now widely accepted as a marker for antibody-mediated rejection in renal and cardiac allografts. This review summarizes the rationale for the use of C4d as a marker of antibody-mediated rejection, along with the clinical evidence supporting its use in the clinical diagnosis of antibody-mediated rejection. Antibody-independent mechanisms by which C4d can be activated by the classical and lectin pathways of complement activation are also identified. Finally, mechanisms by which complement activation stimulates effector cells (neutrophils, monocytes, macrophages, platelets, and B and T lymphocytes) as well as target cells (endothelial cells) are discussed in relation to antibody-mediated allograft rejection.

Amyloid‐β up‐regulates complement factor B in retinal pigment epithelial cells through cytokines released from recruited macrophages/microglia: Another mechanism of complement activation in age‐related macular degeneration

One of the earliest signs of age-related macular degeneration (AMD) is the formation of drusen which are extracellular deposits beneath the retinal pigmented epithelium (RPE). To investigate the relationship between drusen and AMD, we focused on amyloid beta (Abeta), a major component of drusen and also of senile plaques in the brain of Alzheimer's patients. We previously reported that Abeta was accumulated in drusen-like structure in senescent neprilysin gene-disrupted mice. The purpose of this study was to investigate the influence of Abeta on factor B, the main activator of the complement alternative pathway. The results showed that Abeta did not directly modulate factor B expression in RPE cells, but increased the production of monocyte chemoattractant protein-1 (MCP-1). Abeta also increased the production of IL-1beta and TNF-alpha in macrophages/microglia, and exposure of RPE cells to IL-1beta and TNF-alpha significantly up-regulated factor B. Co-cultures of RPE cells and macrophages/microglia in the presence of Abeta significantly increased the expression of factor B in RPE. These findings indicate that cytokines produced by macrophages/microglia that were recruited by MCP-1 produced in RPE cells stimulated by Abeta up-regulate factor B in RPE cells. Thus, a combined mechanism exists for Abeta-induced for the activation of the complement alternative pathway in the subretinal space; cytokine-induced up-regulation of activator factor B and dysfunction of the inhibitor factor I by direct binding to Abeta as suggested in our earlier study.

Mechanisms of complement activation and effects of C1-inhibitor on the meconium-induced inflammatory reaction in human cord blood

BackgroundMeconium aspiration syndrome has a complex pathophysiology. Meconium activates the complement system and meconium-induced cytokine formation is differentially mediated by complement and CD14. C1-inhibitor (C1-INH) regulates complement and contact-system activation mainly by protease inhibition, but may reduce inflammation by other mechanisms as well. ObjectiveThe aim of the study was to investigate the initial mechanisms of meconium-induced complement activation and to study the effect of C1-INH on the meconium-induced inflammatory reaction. MethodsHuman serum from five donors was preincubated with an anti-MBL monoclonal antibody and then incubated with meconium for 30min at 37°C. Human cord whole blood, anticoagulated with lepirudin, from six donors was preincubated with C1-INH and then incubated with meconium for 30min and 4h at 37°C. Complement activation products specific for the different pathways were measured by ELISAs: classical pathway C1rs/C1-INH complexes, classical and lectin pathway C4d, alternative pathway C3bBbP, and terminal pathway sC5b-9 complex (TCC). A Bio-Plex Array Reader was used to measure 27 inflammatory mediators. ResultsThe anti-MBL monoclonal antibody significantly reduced meconium-induced formation of C4d by 63% (p=0.0159) and TCC by 27% (p=0.0079). C1-INH dose-dependently inhibited meconium-induced formation of C1rs/C1-INH complexes, C4d, C3bBbP, and TCC compared to albumin (p<0.002 for all). C1-INH induced a dose-dependent and substantial inhibition of meconium-induced formation of the proinflammatory cytokines TNFα, IL-1β, IL-6 and IFN-γ (p<0.01 for all), the chemokines IL-8, MCP-1, MIP-1α, MIP-1β, and eotaxin (p<0.02 for all), the growth factors G-CSF, GM-CSF, basic FGF, and PDGFbb (p<0.05 for all), and the anti-inflammatory cytokine IL-1ra (p<0.001). ConclusionsMeconium activated the lectin complement pathway as well as the alternative pathway. C1-INH efficiently reduced a broad spectrum of inflammatory mediators even at the lowest concentration. Administration of C1-INH may thus reduce the inflammatory response in MAS.

Mechanisms of complement activation and effects of C1-inhibitor on the meconium-induced inflammatory reaction in cord blood

Hereditary angioedema (HAE) is a rare, but potentially life-threatening disorder, characterized by acute, recurring, and self-limiting edematous episodes of the face, extremities, trunk, genitals, upper airways, or the gastrointestinal tract. HAE may be caused by the deficiency of C1-inhibitor (C1-INH-HAE) but another type of the disease, hereditary angioedema with normal C1-INH function (nC1-INH-HAE) was also described. The patient population is quite heterogeneous as regards the location, frequency, and severity of edematous attacks, presenting large intra- and inter-individual variation. Here, we review the role of the complement system in the pathomechanism of HAE and also present an overview on the complement parameters having an importance in the diagnosis or in predicting the severity of HAE.

Complement activation by tubular cells is mediated by properdin binding

Activation of filtered complement products on the brush border of the tubular epithelium is thought to be a key factor underlying proteinuria-induced tubulointerstitial injury. However, the mechanism of tubular complement activation is still unclear. Recent studies on mechanisms of complement activation indicate a key role for properdin in the initiation of an alternative pathway. We hypothesized that properdin serves as a focal point for complement activation on the tubulus. We observed a strong staining for properdin on the luminal surface of the tubules in kidney biopsies from patients with proteinuric renal disease. In vitro experiments revealed dose-dependent binding of properdin to proximal tubular epithelial cells (PTEC), whereas no significant binding to endothelial cells was detected. Exposure of PTEC with normal human serum as a source of complement resulted in complement activation with deposition of C3 and generation of C5b-9. These effects were virtually absent with properdin-deficient serum. Preincubation of PTEC with properdin before addition of properdin-depleted serum fully restored complement activation on the cells, strongly suggesting a key role for properdin in the activation of complement at the tubular surface. In proteinuric renal disease, filtered properdin may bind to PTEC and act as a focal point for alternative pathway activation. We propose that this contribution of properdin is pivotal in tubular complement activation and subsequent damage. Interference with properdin binding to tubular cells may provide an option for the treatment of proteinuric renal disease.

Efficacy of HIV-specific and 'antibody-independent' mechanisms for complement activation by HIV-infected cells.

Previous studies in this laboratory have shown that efficient activation of complement (C) on HIV isolates and HIV-infected cells requires the binding of specific anti-HIV antibodies, while other investigators have observed 'antibody-independent' C activation. In an attempt to clarify these disparate findings, we investigated the effect of several variables on C activation by HIV-infected cells using flow cytometric analysis of C3 deposition. Antibody-mediated C activation using pooled sera from infected persons or human MoAbs directed against the V3 region of gp120 was always substantially higher than activation without antibody. Normal human serum (NHS) from a subset of HIV antibody-negative donors did, however, induce low levels of C3 deposition. Differences in C3 activation between the various NHS did not correlate with total haemolytic C levels or mannose-binding protein (MBP) levels. IgM isolated from NHS that induced high levels of C activation was at least partly responsible for the 'antibody-independent' C activation. Although there appeared to be a correlation between NHS that induced C activation and the presence of anti-blood type B IgM, absorption of anti-B did not abrogate the C3 deposition. Additionally, MoAb to the B antigen did not induce C3 deposition. These studies show that IgM in sera from HIV-uninfected donors can induce C3 deposition on HIV-infected cells, but that specific antibody-dependent C activation is substantially more efficient. Therefore, 'antibody-independent' C activation on HIV-infected cells may, in some cases, be more accurately described as HIV-cross-reactive antibody-dependent C activation.

Immunohistochemical detection of terminal complement complex and S protein in normal and pre-eclamptic placentae

Terminal complement complex and S protein were searched for in term placentae obtained from 13 women with normal pregnancy and 15 patients with moderate or severe form of pre-eclampsia. Terminal complement complex was found to localize in the fibrinoid material of the decidua of the basal plate, in the stroma of the chorionic villi and in the vessel walls, as subendothelial deposits. S protein had a quite different distribution, being detected in the syncytiotrophoblast located both in the chorionic villi and in the decidua of the basal plate (DBP) and also on the endothelial cells of fetal stem vessels. Mild deposits of C3 were found in the decidua of the basal plate and also in the stroma and on the basal membranes of the villi. Reactivity for C9 neoantigen was also observed in the cytoplasm of some cells, which were recognized to be macrophages by the presence in their cytoplasm of acid phosphatase and by their reaction with a monoclonal antibody specific for macrophages. Differences in complement deposition in normal and pre-eclamptic placentae were essentially quantitative. Possible mechanisms of complement activation are discussed.

Early complement activation follows eversion carotid endarterectomy and correlates with the time of clamping of the carotid artery

Background Complement activation plays an important role in ischemia/reperfusion (I/R) injury. The objective of the present study was to detect the presence and mechanism of complement activation in patients who underwent carotid endarterectomy (CEA). Methods Complement activation products C1rsC1-inhibitor, C4d, C3a and SC5b-9 and concentrations of C-reactive protein (CRP) were measured in samples serially taken from 16 patients with eversion CEA and 10 with carotid artery stenting (CAS) in the first 24 h post-surgery/intervention. MBL2 genotypes were also determined. Results In patients with CEA an intense increase in C3a levels were observed immediately after surgery ( p < 0.001), accompanied by a slight elevation in SC5b-9 levels ( p < 0.05). C3a levels remained elevated until 4 h post-surgery, compared with the baseline values and with CAS patients. Peak C3a levels correlated with the time of carotid clamping ( r = 0.5921, p = 0.02). No significant changes were detected in C1rsC1-inhibitor or C4d levels following CEA, and we found no association between the generation of C3a and MBL2 genotypes or CRP levels. Complement activation was not present in patients with CAS. Conclusions Early complement activation follows CEA and correlates with the time of I/R injury. The lack of C4d generation suggests the role of the alternative and not the lectin pathway in the process.

Properdin and Complement Activation: A Fresh Perspective

The C3 convertases are the major proteases of the complement cascade and are assembled at the site of complement activation via several different pathways. Properdin's functional role in stabilizing the alternative pathway convertase has been long established; however, new evidence demonstrates that properdin can also bind to certain microbial surfaces, and provide a platform for de novo convertase assembly. Therefore, properdin participates in two distinct mechanisms for complement activation: the alternative pathway and a properdin-directed pathway. Previous work had implicated the alternative pathway in the initiation and/or progression of several autoimmune diseases and in the host defense against certain bacterial pathogens. Those conclusions were based on evidence that cannot distinguish effects of the alternative pathway from effects of the properdin-directed pathway. With the identification of the new role for properdin in C3 convertase assembly there became a pressing need to reassess the mechanisms of complement activation, determine the specific role of properdin in each of these pathways, and explore the new therapeutic avenues that could arise.

Complement driven by conformational changes

Complement in mammalian plasma recognizes pathogenic, immunogenic and apoptotic cell surfaces, promotes inflammatory responses and marks particles for cell lysis, phagocytosis and B-cell stimulation. At the heart of the complement system are two large proteins, complement component C3 and protease factor B. These two proteins are pivotal for amplification of the complement response and for labelling of the target particles, steps that are required for effective clearance of the target. Here we review the molecular mechanisms of complement activation, in which proteolysis and complex formation result in large conformational changes that underlie the key offensive step of complement executed by C3 and factor B. Insights into the mechanisms of complement amplification are crucial for understanding host defence and pathogen immune evasion, and for the development of complement-immune therapies.

ROLE OF THE ALTERNATIVE PATHWAY IN THE EARLY COMPLEMENT ACTIVATION FOLLOWING MAJOR TRAUMA

Complement activation has been reported after major trauma. However, little is known about the clinical relevance and the mechanisms of complement activation early after trauma. Therefore, the aim of this study was to measure complement activation, to identify the roles of injury severity and hypoperfusion, to determine the predominant activated pathway, and to identify the clinical significance of early complement activation in trauma patients. A total of 208 adult trauma patients were enrolled in this prospective single-center cohort study of major trauma patients. Blood samples were obtained within 30 min after injury before any significant fluid resuscitation. Complement (C5b-9) was activated early after trauma, correlated with injury severity and tissue hypoperfusion, and was associated with increased mortality rate and with the development of organ failure such as acute lung injury and acute renal failure. The alternative pathway seems to be the predominant activated complement pathway early after trauma. However, the classical and/or the lectin pathway initiated complement activation because of the correlation between plasma levels of C4d and C3a/C5b-9. Finally, in patients with low C3a levels, C5b-9 levels correlated with plasma levels of prothrombin fragments 1 + 2, a marker of thrombin generation, suggesting additional C3-independent complement activation by thrombin after severe trauma. In summary, complement activation via its amplification by the alternative pathway is observed early after trauma and correlates with injury severity, tissue hypoperfusion, and worse clinical outcomes. Besides complement activation by the classical and/or lectin pathways, there is an independent association between thrombin generation and complement activation.

The mechanisms of activation of normal human serum complement byEscherichia coli strains with K1 surface antigen

Ten E. coli K1 strains isolated from the urine of children with urinary tract infections were sensitive to the bactericidal action of normal human serum (NHS). The role of the particular mechanisms of complement activation was determined in the process of killing these strains, showing variable sensitivity to the bactericidal action of NHS; three mechanisms of activation of human complement were observed. Important role of alternative pathway activation in the bactericidal action of NHS against E. coli K1 strains independent of the classical and lectin pathways was not established.

Activation of complement-a mechanism for the inflammation in acne

An immunofluorescence investigation of thirty-six non-inflamed and thirty-four inflamed acne lesions has been carried out. Fluorescence for C3, unaccompanied by fluorescence for immunoglobulins, was found in the walls of dermal blood vessels or at the basement membrane zone of the comedo or at both these sites in fifty-seven lesions. Fluorescence for C3 accompanied by fluorescence for immunoglobulins was found in nine lesions. In two papules and three nodules the comedo was surrounded or replaced by a mononuclear cell infiltrate. These findings have been taken to indicate that cellular immune mechanisms are a late event and activation of complement is an early event in the pathogenesis of inflammation in acne. The mechanism of complement activation remains uncertain.

Activation of the Classical Pathway of Complement by Resting and Shear Stress-Stimulated Human Endothelial Cells.

Complement activation is associated with a variety of inflammatory conditions including atherosclerosis, but the mechanism of complement activation in these settings is poorly understood. Endothelial cells (EC) play an important role in vascular pathology and express a variety of complement receptors, including gC1qR/p33, recognizing the globular domain of the complement component C1q. In preliminary studies, purified recombinant gC1qR/p33 was found to support C1q-dependent C4 activation in vitro, comprising 19.5% ± 8.3% (mean ± S.D., n=5) of that produced by aggregated IgG. In contrast, a truncated form of gC1qR/p33, lacking the C1q binding domain, failed to support C4 activation. Additional studies were performed with immortalized bone marrow microvascular EC to investigate classical complement pathway activation and deposition. EC were exposed to anticoagulated (0.32 % sodium citrate) human plasma, diluted (1/10) in 0.01 M HEPES buffered modified Tyrode's solution, pH 7.5, containing 2 mM Mg Cl2 and 1 mM CaCl2, for 60 min, 37°C. A solid phase ELISA approach was used to detect EC-associated C1q and C4 activation (C4d). Statistically significant deposition of C4d (0.72 ± 0.3. OD units (ODU), n=4)(p=0.04) and C1q (0.57 ± 0.19. ODU, n=4) (p=0.002) was observed on EC that had been immobilized on poly-L-lysine coated microtiter wells. Consistent with classical complement pathway activation, C4d deposition remained at baseline (0.23 ± 0.13, ODU, n=4) in the presence of 10 mM EDTA, but C1q deposition was unaffected. Moreover, no significant C1q or C4d deposition occurred when endothelial cells were exposed to C1q depleted serum. Similar studies were performed using EC grown to confluence on Type I collagen to examine the effect of shear stress (12 dynes/cm2 for 1 hour in a cone-and-plate shearing device), simulating flow conditions in coronary arteries, on classical complement pathway activation and deposition. Compared to static conditions, shear stress resulted in an approximately 50% increase in C1q and C4d deposition on EC. This was accompanied by an approximately 2-fold increase in EC binding of a monoclonal antibody, 60.11, recognizing the N-terminal C1q binding domain of gC1qR/p33. Taken together, these data present evidence for a potential paradigm shift, illustrating immune complex independent classical complement pathway activation by gC1qR/p33, and deposition of activated classical complement components on EC. The generation and deposition of active complement components on EC is likely to contribute directly to vascular inflammation and atherosclerotic changes.

Complement consumption by Photobacterium damselae subsp. piscicida in seabream, red porgy and seabass normal and immune serum. Effect of the capsule on the bactericidal effect

A virulent strain of Photobacterium damselae subsp. piscicida ( Pdp) was grown without (C form) or with (C+ form) glucose supplementation, the latter to enhance capsule formation. Both forms were resistant to killing by normal serum of seabream, red porgy and seabass. However, the C form was killed by immune serum of all three fish species while the C+ form was killed only by seabream and red porgy sera and to a lesser extent than the C form. Both C and C+ forms consumed complement in normal serum and this consumption was enhanced by precoating the bacteria in specific fish antibody. Complement consumption was greatest in seabass serum, especially with antibody-coated C+ form yet in this case the bacteria were not killed. The killing of the C form in immune serum of all three fish species was completely inhibited by EGTA/Mg 2+, indicating that the mechanism of complement activation leading to killing of the bacteria was by the classical pathway. The results suggest that immune serum killing by the classical complement pathway may provide some degree of protection against pasteurellosis, but enhanced expression of the capsule by Pdp in vivo may restrict complement-mediated killing, especially in immunised seabass.