Gamma subunit of complement component 8 is a neuroinflammation inhibitor.

The complement system is part of the innate immune system that comprises several small proteins activated by sequential cleavages. The majority of these complement components, such as components 3a (C3a) and C5a, are chemotactic and pro-inflammatory. However, in this study, we revealed an inhibitory role of complement component 8 gamma (C8G) in neuroinflammation. In patients with Alzheimer's disease, who exhibit strong neuroinflammation, we found higher C8G levels in brain tissue, CSF, and plasma. Our novel findings also showed that the expression level of C8G increases in the inflamed mouse brain, and that C8G is mainly localized to brain astrocytes. Experiments using recombinant C8G protein and shRNA-mediated knockdown showed that C8G inhibits glial hyperactivation, neuroinflammation, and cognitive decline in acute and chronic animal models of Alzheimer's disease. Additionally, we identified sphingosine-1-phosphate receptor 2 (S1PR2) as a novel interaction protein of C8G and demonstrated that astrocyte-derived C8G interacts with S1PR2 to antagonize the pro-inflammatory action of S1P in microglia. Taken together, our results reveal the previously unrecognized role of C8G as a neuroinflammation inhibitor. Our findings pave the way towards therapeutic containment of neuroinflammation in Alzheimer's disease and related neurological diseases.

Membrane attack complex-associated molecules from redlip mullet (Liza haematocheila): Molecular characterization and transcriptional evidence of C6, C7, C8ß, and C9 in innate immunity.

The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were detected in the TCC components, but the CCP and FIMAC domains were absent in MuC8ß and MuC9. Expression analysis of four TCC genes from healthy redlip mullets showed the highest expression levels in the liver, whereas limited expression was observed in other tissues; immune-induced expression in the head kidney and spleen revealed significant responses against Lactococcus garvieae and poly I:C injection, suggesting their involvement in MAC formation in response to harmful pathogenic infections. Furthermore, the response to poly I:C may suggest the role of TCC components in the breakdown of the membrane of enveloped viruses. These findings may help to elucidate the mechanisms behind the complement system of the teleosts innate immunity.

Identification and expression of complement component C8α, C8ß and C8γ gene in half-smooth tongue sole (Cynoglossus semilaevis) and C8α recombinant protein antibacterial activity analysis.

Complement component C8, which mediates membrane attack complex formation and bacterial lysis, plays important roles in the complement system. The cDNA sequences of the C8α, C8ß and C8γ genes were cloned from half-smooth tongue sole (Cynoglossus semilaevis). Full-length cDNA of CsC8α (C8α of C. semilaevis), CsC8ß and CsC8γ was 1990, 2219 and 886 bp, respectively, which contained open reading frames of 1797, 1749 and 666 bp, encoding 598, 582 and 221 amino acids, respectively. The deduced proteins of CsC8α, CsC8ß and CsC8γ showed the closest amino acid similarity to C8α (73%) of Siniperca chuatsi, C8ß (76%) of Oryzias latipes and C8γ (72%) of Takifugu rubripes, respectively. The highest expression level of CsC8α, CsC8ß and CsC8γ among the 13 normal tissues was observed in liver tissue, followed by much lower levels in other tissues. After infection with Vibrio anguillarum, CsC8α, CsC8ß and CsC8γ were significantly up-regulated in all of the detected tissues, including the intestine, liver, gill, head kidney, blood and spleen. Then, a recombinant expression plasmid was constructed, and the recombinant CsC8α protein was expressed in GS115 pichia pastoris yeast. Furthermore, to investigate the biological functions of recombinant CsC8α, an antibacterial assay was performed, and the results showed that recombinant CsC8α obviously inhibited growth of V. anguillarum, Edwardsiella tarda and Vibrio parahaemolyticus. Taken together, these results suggest that CsC8α, CsC8ß and CsC8γ may play important roles in the immune defense of C. semilaevis.

Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation.

BACKGROUND: Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host's immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated. METHODS AND FINDINGS: The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration. CONCLUSION: Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9.

Membrane pore formation by human complement: functional importance of the transmembrane ß-hairpin (TMH) segments of C8α and C9.

Human C8 and C9 have a key role in forming the pore-like "membrane attack complex" (MAC) of complement on bacterial cells. A possible mechanism for membrane insertion of these proteins was suggested when studies revealed a structural similarity between the MACPF domains of the C8α and C8ß subunits and the pore-forming bacterial cholesterol-dependent cytolysins (CDCs). This similarity includes a pair of α-helical bundles that in the CDCs refold during pore formation to produce two transmembrane ß-hairpins (TMH1 and TMH2). C9 is the major pore-forming component of the MAC and is also likely to contain two TMH segments because of its homology to C8α and C8ß. To determine their potential for membrane insertion, the TMH sequences in C8α and those predicted to be in C9 were substituted for the TMH sequences in perfringolysin O (PFO), a well-characterized CDC. Only chimeric proteins containing TMH2 from C8α (PFO/αT2) or C9 (PFO/C9T2) could be expressed in soluble, active form. The PFO/αT2 and PFO/C9T2 chimeras retained significant hemolytic activity, formed pore-like structures on membranes, and could combine with PFO to form hemolytically active mixed complexes that were functionally similar to PFO alone. These results provide experimental evidence in support of the hypothesis that TMH segments in C8α and those predicted to be in C9 have a direct role in MAC membrane penetration and pore formation.

Genomic characterization and expression analysis of complement component 8α and 8ß in rock bream (Oplegnathus fasciatus).

The complement component 8α and 8ß are glycoproteins that mediate formation of the membrane attack complex (MAC) on the surface of target cells. Full-length complement C8α (Rb-C8α) and C8ß (Rb-C8ß) sequences were identified from a cDNA library of rock bream (Oplegnathus fasciatus), and their genomic sequences were obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The Rb-C8α gene contains 64bp of 5'-UTR, open reading frame (ORF) of 1794bp, which encodes a polypeptide of 598 amino acids, 212bp of 3'-UTR. The Rb-C8ß gene contains 5'-UTR of 27bp, open reading frame (ORF) of 1761bp, which encodes a polypeptide of 587 amino acids, 3'-UTR of 164bp. Rb-C8α consists of 11 exons interrupted by 10 introns and Rb-C8ß consists of 12 exons interrupted by 11 introns. Sequence analysis revealed that both Rb-C8α and Rb-C8ß contain thrombospondin type-1, a low-density lipoprotein receptor domain class A, membrane attack complex/perforin (MACPF) domain and epidermal growth factor like domain. The promoter regions of both genes contain important putative transcription factor binding sites including those for NF-κB, SP-1, C/EBP, AP-1, and OCT-1. Rb-C8α and Rb-C8ß showed the highest amino acid identity of 62% and 83% to rainbow trout C8α and Japanese flounder C8ß respectively. Quantitative real-time PCR analysis confirmed that Rb-C8α and Rb-C8ß were constitutively expressed in all examined tissues, isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus led to up regulation of Rb-C8α and Rb-C8ß in liver. Positive regulations upon bacterial and viral challenges, and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C8α and Rb-C8ß important immune genes, likely involved in the complement system lytic pathway of rock bream.

Trichinella spiralis paramyosin binds to C8 and C9 and protects the tissue-dwelling nematode from being attacked by host complement.

BACKGROUND: Paramyosin is a thick myofibrillar protein found exclusively in invertebrates. Evidence suggested that paramyosin from helminths serves not only as a structural protein but also as an immunomodulatory agent. We previously reported that recombinant Trichinella spiralis paramyosin (Ts-Pmy) elicited a partial protective immunity in mice. In this study, the ability of Ts-Pmy to bind host complement components and protect against host complement attack was investigated. METHODS AND FINDINGS: In this study, the transcriptional and protein expression levels of Ts-Pmy were determined in T. spiralis newborn larva (NBL), muscle larva (ML) and adult worm developmental stages by RT-PCR and western blot analysis. Expression of Ts-Pmy at the outer membrane was observed in NBL and adult worms using immunogold electron microscopy and immunofluorescence staining. Functional analysis revealed that recombinant Ts-Pmy(rTs-Pmy) strongly bound to complement components C8 and C9 and inhibited the polymerization of C9 during the formation of the membrane attack complex (MAC). rTs-Pmy also inhibited the lysis of rabbit erythrocytes (E(R)) elicited by an alternative pathway-activated complement from guinea pig serum. Inhibition of native Ts-Pmy on the surface of NBL with a specific antiserum reduced larvae viability when under the attack of complement in vitro. In vivo passive transfer of anti-Ts-Pmy antiserum and complement-treated larvae into mice also significantly reduced the number of larvae that developed to ML. CONCLUSION: These studies suggest that the outer membrane form of T. spiralis paramyosin plays an important role in the evasion of the host complement attack.

Structure of human C8 protein provides mechanistic insight into membrane pore formation by complement.

C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like "membrane attack complex" (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12-18 molecules of C9. C8 is composed of three genetically distinct subunits, C8α, C8ß, and C8γ. The C6, C7, C8α, C8ß, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N- and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8α and C8ß are located on the periphery of C8 and not likely to interact with the target membrane. The C8γ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8α and C8ß are related by a rotation of ∼22° with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane ß-hairpins to form a circular pore.

Molecular cloning of the terminal complement components C6 and C8beta of cartilaginous fish.

The terminal complement components (TCCs) of mammals, C6, C7, C8alpha, C8beta, and C9, are a group of serum proteins involved in the cytolytic killing of microbial pathogens. The mammalian TCCs share a unique core domain structure and were probably generated by the duplication of the ancestral TCC gene and subsequent addition and/or deletion of the N- and C-terminal domains. Proteins and genes for all the TCCs have been identified from bony fish. In contrast, no TCC gene has been identified from cyclostome lamprey using whole-genome shotgun-sequence analysis and liver EST analysis. To clarify the evolutionary origin of TCCs, we performed degenerate RT-PCR and RACE analyses of the cartilaginous fish liver and identified the C6 gene from a shark, Mustelus manazo, and the C8B gene from a chimaera, Chimaera phantasma. The presence of the C6 gene in shark suggests that one of the most crucial steps in the establishment of the cytolytic complement pathway, the addition of the FIM and CCP domains to the primitive TCC, occurred in a common ancestor of the jawed vertebrates. These results also indicate that the gene duplications among TCCs occurred at an early stage of the jawed vertebrate evolution.

A novel mutation in a patient with a deficiency of the eighth component of complement associated with recurrent meningococcal meningitis.

INTRODUCTION: Complement component C8 is one of the five terminal complement components required for the formation of the membrane attack complex. Complete absence of C8 results in increased susceptibility to gram-negative bacteria such as Neisseria species. MATERIALS AND METHODS: Two functionally distinct C8 deficiency states have been described: C8 alpha-gamma deficiency has been predominantly reported amongst Afro-Caribbeans, Hispanics, and Japanese and C8beta mainly in Caucasians. RESULTS: We report a case of functional and immunochemical deficiency of the complement component C8, diagnosed in a Caucasian adult following three episodes of meningitis. Western blotting and hemolytic assay demonstrated absence of C8beta. In genetic studies, the common exon 9 C > T transition responsible for 85% of C8beta deficiencies was not found. Two mutations were identified: a novel duplication mutation, c.1047_1053 dupGGCTGTG in exon 7 that introduces a frame shift, resulting in the addition of seven novel amino acid residues and a premature stop codon, and a previously reported mutation, c.271C > T in exon 3. The parents each expressed one of these mutations, confirming compound heterozygosity. DISCUSSION: This is the first report of a duplication mutation in C8beta deficiency and extends the molecular heterogeneity of the disorder.

Differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus.

The complement system is an important component of the innate immune response to virus infection. The role of human complement pathways in the in vitro neutralization of three closely related paramyxoviruses, Simian Virus 5 (SV5), Mumps virus (MuV) and Human Parainfluenza virus type 2 (HPIV2) was investigated. Sera from ten donors showed high levels of neutralization against HPIV2 that was largely complement-independent, whereas nine of ten donor sera were found to neutralize SV5 and MuV only in the presence of active complement pathways. SV5 and MuV neutralization proceeded through the alternative pathway of the complement cascade. Electron microscopy studies and biochemical analyses showed that treatment of purified SV5 with human serum resulted in C3 deposition on virions and the formation of massive aggregates, but there was relatively little evidence of virion lysis. Treatment of MuV with human serum also resulted in C3 deposition on virions, however in contrast to SV5, MuV particles were lysed by serum complement and there was relatively little aggregation. Assays using serum depleted of complement factors showed that SV5 and MuV neutralization in vitro was absolutely dependent on complement factor C3, but was not dependent on downstream complement factors C5 or C8. Our results indicate that even though antibodies exist that recognize both SV5 and MuV, they are mostly non-neutralizing and viral inactivation in vitro occurs through the alternative pathway of complement. The implications of our work for development of paramyxovirus vectors and vaccines are discussed.

Crystal structure of complement protein C8gamma in complex with a peptide containing the C8gamma binding site on C8alpha: implications for C8gamma ligand binding.

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex. It contains three genetically distinct subunits; C8alpha and C8gamma form a disulfide-linked C8alpha-gamma heterodimer that is noncovalently associated with C8beta. The C8alpha subunit is homologous to C8beta, C6, C7 and C9 and together they form the MAC family of proteins. By contrast, C8gamma is the only lipocalin in the complement system. Like other lipocalins, it has a core beta-barrel structure forming a calyx with a distinct binding pocket for a small and as yet unidentified ligand. The binding site on C8alpha for C8gamma was previously localized to a 19-residue segment which contains an insertion (indel) that is unique to C8alpha. Included in the indel is C8alpha Cys 164 which links to Cys 40 in C8gamma. In the present study, C8gamma containing a C40A substitution was co-crystallized with a synthetic indel peptide containing the equivalent of a C8alpha C164A substitution. The X-ray crystal structure shows that the indel peptide completely fills the upper portion of the putative C8gamma ligand binding pocket and is in contact with all four loops at the calyx entrance. The lower part of the C8gamma cavity is either unoccupied or contains disordered solvent. The validity of the structure is supported by the spatial arrangement of C8alpha Ala 164 in the peptide and C8gamma Ala 40, which are within disulfide-bonding distance of each other. Corresponding studies in solution indicate the C8gamma ligand binding site is also occupied by the indel segment of C8alpha in whole C8. These results suggest a role for C8alpha in regulating access to the putative C8gamma ligand binding site.

Structure of C8alpha-MACPF reveals mechanism of membrane attack in complement immune defense.

Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8alpha at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane beta hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8alpha is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion.

Mapping of the complement C9 binding domain in paramyosin of the blood fluke Schistosoma mansoni.

Schistosomes are believed to evade complement-mediated damage by expression of complement inhibitory proteins. Our previous results [Deng, J., Gold, D., LoVerde, P.T., Fishelson, Z., 2003. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect. Immun. 71, 6402-6410.] have demonstrated that paramyosin (Pmy) of the blood fluke S. mansoni binds to the human complement proteins C8 and C9, inhibits complement activation at the terminal stage and protects the parasite from complement-mediated damage. In order to locate the Pmy binding site to C8 and C9, various fragments of Pmy cDNA were PCR-cloned into a pET28a bacterial expression vector. Recombinant His-tagged Pmy fragments were expressed in BL21 Escherichia coli and purified over a nickel-nitrilotriacetic acid column. Binding assays by Western blotting with monoclonal anti-His antibody demonstrated that PmyCC (Pmy amino acids (744)Asp-(866)Met) was the only Pmy fragment that bound to human C8 and C9. Functional analyses demonstrated that PmyCC inhibited hemolysis of rabbit erythrocytes and of antibody-sensitized sheep erythrocytes by human complement. Importantly, PmyCC inhibited in vitro killing of trypsin-sensitized schistosomula of S. mansoni by human complement. In the presence of PmyCC, Zn(2+)-induced C9 polymerization was inhibited. Most of the immunodominant B-cell antigenic epitopes of Pmy are present in the PmyCC region, as antibodies collected from mice immunized with recombinant Pmy bound primarily to PmyCC. Taken together, this study has mapped the complement regulatory domain in Pmy, capable of binding to C8 and C9 and preventing polyC9 formation, to its C-terminal region.

The gamma subunit of the eighth complement component (C8) in rainbow trout.

Of the 35 proteins, enzymes, receptors and regulatory components of the complement system, C8gamma is unique in that it is the only lipocalin. C8gamma is a subunit of the C8 molecule, which is one of the five components (C5b, C6, C7, C8 and C9) that interact as a consequence of complement activation to form the membrane attack complex. Until now, C8gamma has been characterized only in mammalian species. In order to elucidate the phylogeny of this molecule, we have cloned the C8gamma subunit in rainbow trout (Oncorhynchus mykiss), a teleost fish representing a critical point in the evolutionary divergence of the complement system. The deduced amino acid sequence of trout C8gamma shows significant identity (37%) to the human C8gamma homolog and much lower to the other known lipocalins. The lipocalin domain is present and all the cysteine residues are conserved. The trout C8gamma gene is probably present as a single copy in the trout genome showing a differential expression pattern among tissues investigated.

Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis.

The membrane attack complex (MAC) of the complement system is causing membrane damage and cell death. For protection, cells have adopted several resistance mechanisms, including removal of the membrane-inserted MAC by vesiculation. To identify proteins involved in MAC vesiculation, extracellular proteins released from K562 cells in response to treatment with sub-lytic complement were separated by acrylamide gel electrophoresis and protein bands were extracted, digested into peptides and the peptides were analyzed by mass spectrometry. A 75-kDa protein that was abundant in the supernatant of complement-treated cells was identified as mortalin/GRP75. Analysis by western blotting demonstrated that as early as 5 min after exposure to sub-lytic doses of complement, mortalin was released from K562 cells. Mortalin was released after complete activation of the complement system and formation of C5b-8, and even more so when C5b-9 was formed. Other pore formers, such as streptolysin O and melittin, did not induce release of mortalin. As shown, mortalin can bind to complement C8 and C9 and is shed in vesicles containing C9 and complement MACs. Anti-mortalin antibodies reduced mortalin release from complement-treated cells and elevated the extent of cell death by complement. Inhibitors of protein kinase C and extracellular signal-regulated protein kinase also prevented mortalin release from complement-activated cells. These results suggest that mortalin/GRP75 promotes the shedding of membrane vesicles loaded with complement MAC and protects cells from complement-mediated lysis.

Hepatocyte nuclear factor 1alpha controls the expression of terminal complement genes.

The terminal components of the complement system contribute to host defense by forming the multiprotein membrane attack complex (MAC) which is responsible for cell lysis and several noncytotoxic effects. Most of the complement proteins are synthesized in the liver, but the mechanisms controlling their tissue-specific expression have not been elucidated. In this study we show that mice lacking the hepatic transcription factor hepatocyte nuclear factor 1alpha (HNF1alpha) fail to transcribe C5 and C8A complement genes. In addition, mRNAs encoding for several other terminal complement components or subunits are expressed at lower levels, including C8beta, C8gamma, and C9. We next used a reconstitution assay involving human sera with selective complement deficiencies to assess mouse complement activity. Sera from HNF1alpha-deficient mice showed negligible hemolytic activity of both C5 and C8alpha-gamma subunits. The activity of C8beta was severely affected despite only a 50% reduction in C8beta mRNA levels in the liver. This is reminiscent of C8alpha-gamma-deficient patients who accumulate extremely low levels of the C8beta subunit. Our results demonstrate that HNF1alpha plays a key role in the expression of C5 and C8A genes, two terminal complement component genes that are essential for the assembly of MAC as a result of complement activation.

CD59 protects rat kidney from complement mediated injury in collaboration with crry.

BACKGROUND: As previously reported, the membrane-bound complement regulator at the C3 level (Crry/p65) is important in maintaining normal integrity of the kidney in rats. However, the role of a complement regulator at the C8/9 level (CD59) is not clear, especially when activation of complement occurs at the C3 level. The aim of this work was to elucidate the in vivo role of CD59 under C3 activating conditions. METHODS: Two monoclonal antibodies, 5I2 and 6D1, were used to suppress the function of Crry and CD59, respectively. In order to activate alternative the pathway of complement, the left kidney was perfused with 5I2 and/or 6D1 and was recirculated. RESULTS: In the kidneys perfused with 5I2 alone, deposition of C3 and membrane attack complex (MAC) was observed in the peritubular capillaries, vasa recta, and tubular basement membranes. Cast formation, tubular dilation and degeneration, and cellular infiltration were observed at days 1 and 4, and they recovered by day 7. Further suppression of CD59 by 6D1 significantly enhanced the deposition of MAC and worsened the already exacerbated tubulointerstitial injury. These effects of 6D1 were dose dependent. Perfusion with 6D1 alone did not induce histologic damage or MAC deposition in the tubulointerstitium. CONCLUSIONS: In rats, CD59 maintains normal integrity of the kidney in collaboration with Crry in rats against complement-mediated damage in vivo.

Selective blockade of membrane attack complex formation during simulated extracorporeal circulation inhibits platelet but not leukocyte activation.

OBJECTIVE: Complement activation is induced by cardiopulmonary bypass, and previous work found that late complement components (C5a, C5b-9) contribute to neutrophil and platelet activation during bypass. In the present study, we blocked C5b-9 formation during extracorporeal recirculation of whole blood to assess whether the membrane attack complex was responsible for both platelet and leukocyte activation. METHODS: In a simulated extracorporeal model that activates complement (C3a and sC5b-9), platelets (CD62P expression, leukocyte-platelet conjugate formation), and leukocytes (increased CD11b expression and neutrophil elastase), we examined an anti-human C8 monoclonal antibody that inhibits C5b-9 generation for its effects on cellular activation. RESULTS: Anti-C8 significantly inhibited sC5b-9 formation but did not block C3a generation. Anti-C8 also significantly inhibited the increase in platelet CD62P and monocyte-platelet conjugate formation seen with control circulation. Moreover, compared with control circulation, in which the number of circulating platelets fell by 45%, addition of anti-C8 completely preserved platelet counts. In contrast to blockade of both C5a and sC5b-9 during simulated extracorporeal circulation, neutrophil activation was not inhibited by anti-C8. However, circulating neutrophil and monocyte counts were preserved by addition of anti-C8 to the extracorporeal circuit. CONCLUSIONS: The membrane attack complex, C5b-9, is the major complement determinant of platelet activation during extracorporeal circulation, whereas C5b-9 blockade has little effect on neutrophil activation. These data also suggest a role for platelet activation or C5b-9 (or both) in the loss of monocytes and neutrophils to the extracorporeal circuit.

Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient.

Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.