Terminal Complement Components Research Articles

Complement activation in Puumala hantavirus infection correlates with disease severity

Introduction. Hantaviruses are important human pathogens that cause clinical diseases characterized by renal and cardiopulmonary manifestations. Their pathogenesis is currently poorly understood. We have studied the role of the complement system in the pathogenesis of Puumala (PUUV) hantavirus infection.Material and methods. We studied the activation of complement by measuring the terminal complement complex SC5b-9 and complement component C3 and C4 levels in patients with acute PUUV infection. Several laboratory parameters and clinical findings reflecting the severity of PUUV-HFRS were evaluated with regard to complement activation. Results.The levels of SC5b-9 were significantly increased and C3 decreased in the acute stage as compared to the levels at full recovery (P < 0.001). We found that SC5b-9 levels were higher in patients with chest X-ray abnormalities than in patients with a normal X-ray during the acute stage (P = 0.028). Furthermore, SC5b-9 and C3 levels showed significant correlation with several clinical and laboratory parameters that reflect the severity of the acute PUUV infection.Conclusions. We showed that the complement system becomes activated via the alternative pathway in the acute stage of PUUV infection and the level of activation correlates with disease severity. The results further suggest that complement activation may contribute to the pathogenesis of acute PUUV infection.

Recurrent meningococcal sepsis in a presumptive immunocompetent host shown to be complement C5 deficient-a case report

Invasive meningococcal disease is a world wide challenge. Most cases occur in immunocompetent children and young adults, but some immunodeficiencies are linked to a greater risk of invasive neisserial infections. One of these is complement component deficiencies, particularly deficiency of properdin and the terminal complement components. We describe a case of recurrent meningococcal sepsis in a young man who was later diagnosed with complete lack of complement component C5. This case report emphasizes the need of having complement deficiencies in mind when being introduced to patients with invasive Neisseria-infections.

Cloning of the sixth complement component and, spatial and temporal expression profile of MAC structural and regulatory genes in chicken

Humoral cytotoxicity results from the assembly of terminal components of complement, called membrane attack complex (MAC), which lead to the formation of pores on pathogen membranes. The complement components involved in MAC formation are C5b, C6, C7, C8alpha, C8beta, C8gamma and C9. Among them, C6 protein interacts with C5b through a metastable binding site to form a soluble C5b-6 dimer in the vicinity of the activating cell. Formation of the MAC is controlled by complement regulatory molecules, such as CD59, vitronectin and clusterin. Here, we report the molecular characterization of the C6 complement component, as well as the spatial and temporal expression profile of MAC structural (C6, C7, C8alpha, C8beta, C8gamma) and regulatory (CD59, vitronectin and clusterin) genes in chicken (Gallus gallus). The deduced polypeptide sequence of chicken C6 consists of 935 amino acid residues and exhibits 81%, 58%, 56% and 44% identity with zebra finch, human, frog and trout orthologs, respectively. The 'domain' architecture of chicken C6 resembles that of mammalian counterparts and the cysteine backbone is also conserved. MAC structural and regulatory genes are expressed in a wide range of adult chicken tissues, with the liver being the major source of their produced transcripts. The developmental expression profile of chicken MAC structural genes shows that their transcripts initially appear in the 12th embryonic day in the liver, exhibiting a pick in the 17th, while no expression was detected in the early whole embryo (day 4 and 6), as well as in the 2-day old neonate chicken liver. On the other hand, MAC regulatory genes are expressed in all the developmental stages investigated.

Complement factor 7 gene mutations in relation to meningococcal infection and clinical recurrence of meningococcal disease

Meningococcal disease is caused by Neisseria meningitidis which is associated with high morbidity and mortality. Recurrences of meningococcal infection have been observed in patients with terminal complement component defects, because of the inefficient formation of the lytic membrane attack complex (MAC), C5b-9. Complement component C7 is one of the five plasma proteins to form the MAC. The gene C7 may carry mutations that cause functional abnormalities or the mere absence of the C7 protein. More than 200 patients were screened for aberrant C7 protein by isoelectric focusing (C7 IEF). These were compared with patients in whom recurrent meningococcal infection had resulted in the diagnosis of complete C7 absence (C7Q0). A higher proportion of C7 IEF variants were found in meningitis cases compared to controls ( p = 0.03). In contrast to C7Q0 patients, recurrent meningococcal infection was never observed in C7 IEF cases. Whereas C7Q0 sera were defective in meningococcal serogroup B and W135 killing assays, the sera of patients with C7 IEF variants were only defective in complement-mediated killing when classical pathway activation by (endogenous) anti-meningococcal antibodies was blocked. Upon sequence analysis we characterized the genetic background of the C7*6 and C7*8 IEF pattern and identified three novel C7 gene mutations in 13 C7Q0 patients. In conclusion, C7 IEF variants can determine meningococcal killing in the early stage of infection when antibody-independent killing prevails. The results endorse the lack of clinical recurrences once antibodies are present, whereas in C7Q0 patients the anti-meningococcal antibodies may not suffice to protect from recurrent meningococcal infection.

Complement modulates pathogenesis and antibody-dependent neutralization of West Nile virus infection through a C5-independent mechanism

Although the interactions of complement and viruses have been widely studied, the function of C5 and the membrane attack complex in the context of viral infection or antibody-mediated neutralization remains controversial. Using C5-depleted or -deficient human or mouse sera, we show that C5 does not contribute to the antibody-dependent or -independent neutralization of West Nile virus (WNV) in cell culture. Consistent with this, C5 neither contributed to protection against WNV pathogenesis nor augmented the neutralizing efficacy of complement-fixing anti-WNV neutralizing antibodies in mice. Although previous studies established that activation of the classical, lectin, and alternative complement pathways restricts WNV infection, our results show little effect of C5 and by inference the terminal lytic complement components. Overall, these results enhance our mechanistic understanding of how complement controls flavivirus infections.

Molecular cloning of the terminal complement components C6 and C8β of cartilaginous fish

The terminal complement components (TCCs) of mammals, C6, C7, C8α, C8β, 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.

Novel C3 mutation associated with MPGN2/DDD in a multiple-affected pedigree

The IgG fraction of serum from patients with membranoproliferative glomerulonephritis (MPGN) types I and II or partial lipodystrophy (PLD) was found to contain C3 nephritic factor (C3NeF) which reacts with the alternative pathway C3 convertase C3bBb and stabilizes it. Two types of C3NeF were detected, of which one was heat sensitive (56°C for 30 min) and properdin dependent (C3NeF:P) but the other was heat stable and properdin independent (C3NeF:nP). C3NeF:P was found in sera from patients with MPGN types I and II and it displayed the properties of properdin and IgG. C3NeF:P was observed in patients with reduced serum concentrations of C3 and terminal complement components (TCC), and the generation of SC5b-9 complex was increased in mixtures with normal human serum. C3NeF:nP was found in sera from patients with MPGN type II and PLD, whose sera revealed a selective decrease in C3 concentrations.

A Novel Mutation in a Patient with a Deficiency of the Eighth Component of Complement Associated with Recurrent Meningococcal Meningitis

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. 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. 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. This is the first report of a duplication mutation in C8beta deficiency and extends the molecular heterogeneity of the disorder.

Cloning and characterization of the trout perforin

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout ( Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.

Hypoxia increases susceptibility of non-small cell lung cancer cells to complement attack.

The complement system can be specifically targeted to tumor cells due to molecular changes on their surfaces that are recognized by complement directly or via naturally occurring antibodies. However, tumor cells often overexpress membrane-bound complement inhibitors protecting them from complement attack. We have previously shown that non-small cell lung cancer (NSCLC) cells, additionally to membrane-bound inhibitors, produce substantial amounts of soluble regulators such as factor I (FI) and factor H (FH). Since low oxygen concentration is associated with rapidly growing solid tumors, we studied how NSCLC cells protect themselves from complement attack under hypoxic conditions. Unexpectedly, mRNA levels and secretion of both FI and FH were significantly decreased already after 24 h exposure to hypoxia while cell viability measured by XTT assay and annexin V/7-AAD staining was affected only marginally. Furthermore, we observed decrease of mRNA level and loss of membrane-bound complement inhibitor CD46 and increased deposition of early (C3b) and terminal (C9) complement components on hypoxic NSCLC cells. All three complement pathways (classical, lectin and alternative) were employed to deposit C3b on cell surface. Taken together, our results imply that under hypoxic conditions NSCLC give up some of their available defense mechanisms and become more prone to complement attack.

Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59

The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42–59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

Binding of Complement Inhibitor C4b-Binding Protein Contributes to Serum Resistance of Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.

Evolutionary origin of the vertebrate blood complement and coagulation systems inferred from liver EST analysis of lamprey

The complement and coagulation systems in mammalian blood are composed of multiple components with unique domain structures, and are believed to be established by exon-shufflings and following gene duplications. To elucidate their origin in vertebrates, liver EST and 5′- and 3′-rapid amplification of cDNA ends (RACE) analyses were performed in lamprey, Lethenteron japonicum. For the complement system, the factor I cDNA was cloned for the first time outside of the jawed vertebrates. Evidence for the C3/ C4/ C5, fB/ C2 and MASP-1/ MASP-2/ C1r/ C1s gene duplications was not found, suggesting that these duplications occurred in the jawed vertebrate lineage. In contrast, the coagulation factors VII and X, prothrombin and protein C-like cDNAs were identified, indicating that duplications among them predated the cyclostome-jawed vertebrate divergence. The genes for terminal complement components, coagulation factors XI and XII, or prekallikrein were not found, suggesting that the complement and coagulation systems of an ancestral vertebrate were simpler compared to their mammalian counterparts.

C4d deposition and cellular infiltrates as markers of acute rejection in rat models of orthotopic lung transplantation.

C4d is a useful marker of antibody-mediated rejection in cardiac and renal transplants, but clinical studies examining correlations between circulating alloantibodies, C4d deposition, and rejection in lung transplants have yielded conflicting results. We studied circulating alloantibody levels and C4d deposition in two rat models of lung transplantation: Brown Norway (BN) to Wistar-Kyoto (WKY) and PVG.R8 to PVG.1U lung allografts. The availability of C6 deficient (C6-) and C6 sufficient (C6+) PVG 1U rats allowed evaluation of the effects of the terminal complement components on graft injury and C4d deposition. The lung allografts had histologic features resembling human posttransplant capillaritis, characterized by neutrophilic infiltration of alveoli, edema, and hemorrhage. Immunoperoxidase stains on cross sections of allografts showed intense, diffuse, C4d deposition in a continuous linear pattern on the vascular endothelium. C4d deposits were found in both BN to WKY and PVG R8 to 1U allografts, whereas no staining was detectable in WKY to WKY isografts or native lungs. Complement deposition was associated with vascular disruption in C6+, but not in C6- recipients. The presence of circulating donor-specific alloantibodies was verified by flow cytometry. Cell-specific staining revealed perivascular accumulation of macrophages and T lymphocytes whereas neutrophils were sequestered in the intravascular and alveolar capillary compartments. The deposition of C4d on vascular endothelium as well as the coincident presence of alloantibodies is consistent with previous findings in antibody-mediated rejection of renal and cardiac transplants. Furthermore, the histological features of our allografts support the concept that posttransplant capillaritis is a form of humoral rejection.

Fibrotic Mechanisms in Idiopathic Rapidly Progressive Glomerulonephritis: The Role of TGF-β1 and C5b-9

Background. Idiopathic IRPGN is a form of renal vasculitis in which a high chronicity index is present despite minimal impairment of renal function. The present study investigated the mechanisms underlining the relatively early appearance of fibrosis. Methods. In all, 34 patients (17 males) with biopsy proven idiopathic RPGN were included. On light microscopy, the percentage and evolution stage of crescents, the presence of glomerular necrosis, the degree or severity of arteriosclerosis, as well as the extent of tubulointerstitial (TIN) infiltration, interstial fibrosis, and tubular atrophy were assessed. Monoclonal antibodies were used to identify infiltrating macrophages, HLA-DR (+), α-SMA (+), and PCNA (+) cells, the expression of the adhesion molecule ICAM-1, the growth factor TGF-β1, and the terminal complement component C5b-9. Results. The presence of glomerular necrosis correlated positively with the number of SMA (+) cells in TIN (p = 0.036). Glomerular TGF-β1 expression had positive correlation with tubular C5b-9 expression. The tubulointerstitial TGF-β1 expression correlated with tubular C5b-9 expression (p = 0.001) and TGF-β1 expression (p = 0.009). Independent factors predicting the severity of renal function impairment were the CRP levels (p = 0.002) and the degree of arteriosclerosis (p = 0.01). CRP levels correlated with the severity of interstitial infiltration and fibrosis (p = 0.02), the expression of TGF-β1 in the glomeruli (p = 0.009) and the interstitial space (p = 0.001), and the intensity of tubular ICAM-1 and C5b-9 expression (p = 0.023, p = 0.002, respectively). The severity of proteinuria showed a significant correlation with the expression of TGF-β1 in the glomeruli (p = 0.033) and the tubulointerstitium (p = 0.019). Conclusions. The activation of interstitial fibroblasts seems to be an early phenomenon that is related to the extent of glomerular necrosis. Glomerular TGF-β1 may induce tubular expression of C5b-9. Increased tubular C5b-9 expression may result in interstitial fibrosis through increased TGF-β1 production.

Inhibition of Terminal Complement Components in Presensitized Transplant Recipients Prevents Antibody-Mediated Rejection Leading to Long-Term Graft Survival and Accommodation

Ab-mediated rejection (AMR) remains the primary obstacle in presensitized patients following organ transplantation, as it is refractory to anti-T cell therapy and can lead to early graft loss. Complement plays an important role in the process of AMR. In the present study, a murine model was designed to mimic AMR in presensitized patients. This model was used to evaluate the effect of blocking the fifth complement component (C5) with an anti-C5 mAb on prevention of graft rejection. BALB/c recipients were presensitized with C3H donor skin grafts 7 days before heart transplantation from the same donor strain. Heart grafts, transplanted when circulating anti-donor IgG Abs were at peak levels, were rejected in 3 days. Graft rejection was characterized by microvascular thrombosis and extensive deposition of Ab and complement in the grafts, consistent with AMR. Anti-C5 administration completely blocked terminal complement activity and local C5 deposition, and in combination with cyclosporine and short-term cyclophosphamide treatment, it effectively prevented heart graft rejection. These recipients achieved permanent graft survival for >100 days with normal histology despite the presence of systemic and intragraft anti-donor Abs and complement, suggesting ongoing accommodation. Furthermore, double-transplant experiments demonstrated that immunological alterations in both the graft and the recipient were required for successful graft accommodation to occur. These data suggest that terminal complement blockade with a functionally blocking Ab represents a promising therapeutic approach to prevent AMR in presensitized recipients.

Complement Induction in Spinal Cord Microglia Results in Anaphylatoxin C5a-Mediated Pain Hypersensitivity

Microarray expression profiles reveal substantial changes in gene expression in the ipsilateral dorsal horn of the spinal cord in response to three peripheral nerve injury models of neuropathic pain. However, only 54 of the 612 regulated genes are commonly expressed across all the neuropathic pain models. Many of the commonly regulated transcripts are immune related and include the complement components C1q, C3, and C4, which we find are expressed only by microglia. C1q and C4 are, moreover, the most strongly regulated of all 612 regulated genes. In addition, we find that the terminal complement component C5 and the C5a receptor (C5aR) are upregulated in spinal microglia after peripheral nerve injury. Mice null for C5 had reduced neuropathic pain sensitivity, excluding C3a as a pain effector. C6-deficient rats, which cannot form the membrane attack complex, have a normal neuropathic pain phenotype. However, C5a applied intrathecally produces a dose-dependent, slow-onset cold pain in naive animals. Furthermore, a C5aR peptide antagonist reduces cold allodynia in neuropathic pain models. We conclude that induction of the complement cascade in spinal cord microglia after peripheral nerve injury contributes to neuropathic pain through the release and action of the C5a anaphylatoxin peptide.

The Alternative Pathway of Complement Activation Is Critical for Blister Induction in Experimental Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita is a subepidermal blistering disease associated with tissue-bound and circulating autoantibodies against type VII collagen, a major constituent of the dermal-epidermal junction. The passive transfer of Abs against type VII collagen into mice induces a subepidermal blistering disease dependent upon activation of terminal complement components. To further dissect the role of the different complement activation pathways in this model, we injected C1q-deficient, mannan-binding lectin-deficient, and factor B-deficient mice with rabbit Abs against murine type VII collagen. The development and evolution of blistering had a similar pattern in mannan-binding lectin-deficient and control mice and was initially only marginally less extensive in C1q-deficient mice compared with controls. Importantly, factor B-deficient mice developed a delayed and significantly less severe blistering disease compared with factor B-sufficient mice. A significantly lower neutrophilic infiltration was observed in factor B-deficient mice compared with controls and local reconstitution with granulocytes restored the blistering disease in factor B-deficient mice. Our study provides the first direct evidence for the involvement of the alternative pathway in an autoantibody-induced blistering disease and should facilitate the development of new therapeutic strategies for epidermolysis bullosa acquisita and related autoimmune diseases.

Hereditary complement C7 deficiency in nine families: Subtotal C7 deficiency revisited

Deficiencies in terminal complement components, including the component C7, are uncommon and associated with an increased risk of recurrent systemic neisserial infection. A total of 22 molecular defects have been reported in the C7 gene with both complete (C7Q0) and subtotal (C7SD) C7 deficiencies. In this study we report the molecular basis of nine new cases of C7 deficiencies that were characterized by exon-specific sequence analysis. Seven different C7 gene mutations were identified corresponding to small deletions (n=2), splice site changes (n=1) and single base pair substitutions leading to nonsense (n=1) or missense (n=3) mutations. Altogether, three changes of the C7 gene (G357R, R499S and 5' splice donor site of intron 16) account for half of the molecular defects which emphasize that a restricted number of molecular abnormalities are involved in this deficiency. We identified two patients with combined C7Q0/C7SD(R499S) and established the C7SD(R499S) frequency at about 1% in normal Caucasian population. We demonstrated that C7(R499S) mutant protein is retained in the endoplasmic reticulum whereas the wild-type C7 is located in the Golgi apparatus. Our results provide evidence that R499S represents a loss-of-function polymorphism of C7 due to a defective folding of the protein.

Expression of terminal complement components by human keratinocytes

Human keratinocytes are important constituents of the skin immune system. They produce several cytokines, chemokines as well as some complement proteins. As regards soluble complement proteins, so far keratinocytes have been shown to synthesize only C3, factor B, factor H and factor I. Synthesis and regulation of synthesis of other complement proteins has not yet been studied. Here we studied the synthesis of terminal complement components, C5–C9 by human keratinocytes. We also studied the regulation of terminal complement synthesis in keratinocytes by several cytokines, namely, IL-1α, IL-2, IL-6, TGF-β1, TNF-α, and IFN-γ. Human keratinocytes constitutively expressed C5, C7, C8γ and C9 mRNA but not C6, C8α and C8β mRNA. They released C7 and C9, but not C5, C6 and C8. None of the cytokines tested had any influence on the synthesis of terminal components except TNF-α, which strongly upregulated C9 production. In conclusion, we demonstrate that keratinocytes are capable of synthesizing some of the terminal complement components and that the synthesis of C9 is regulated by TNF-α.