Ethnic differences in complement system biomarkers and their association with metabolic health in men of Black African and White European ethnicity.

Inflammation plays a fundamental role in the development of several metabolic diseases, including obesity and type 2 diabetes (T2D); the complement system has been implicated in their development. People of Black African (BA) ethnicity are disproportionately affected by T2D and other metabolic diseases but the impact of ethnicity on the complement system has not been explored. We investigated ethnic differences in complement biomarkers and activation status between men of BA and White European (WE) ethnicity and explored their association with parameters of metabolic health. We measured a panel of 15 complement components, regulators, and activation products in fasting plasma from 89 BA and 96 WE men. Ethnic differences were statistically validated. Association of complement biomarkers with metabolic health indices (BMI, waist circumference, insulin resistance, and HbA1c) were assessed in the groups. Plasma levels of the key complement components C3 and C4, the regulators clusterin and properdin and the activation marker iC3b were significantly higher in BA compared to WE men after age adjustment, while FD levels were significantly lower. C3 and C4 levels positively correlated with some or all markers of metabolic dysfunction in both ethnic groups while FD was inversely associated with HbA1c in both groups, and clusterin and properdin were inversely associated with some markers of metabolic dysfunction only in the WE group. Our findings of increased levels of complement components and activation products in BA compared to WE men suggest differences in complement regulation that may impact susceptibility to poor metabolic health.

The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade.

Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts, it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MACs) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerize and a small increase in its ability to induce hemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerization and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.

Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex.

INTRODUCTION: Coagulation and complement systems are simultaneously activated at sites of tissue injury, leading to thrombin generation and opsonisation with C3b. Thrombomodulin (TM) is a cell-bound regulator of thrombin activation, but can also enhance the regulatory activity of complement factor H (FH), thus accelerating the degradation of C3b into inactive iC3b. OBJECTIVES: This study sought to determine the biophysical interaction affinities of two recombinant TM analogs with thrombin, FH and C3b in order to analyze their ability to regulate serum complement activity. METHODS: Surface plasmon resonance (SPR) analysis was used to determine binding affinities of TM analogs with FH and C3b, and compared to thrombin as positive control. The capacity of the two recombinant TM analogs to regulate complement in serum was tested in standard complement hemolytic activity assays. RESULTS: SPR analysis showed that both TM analogs bind FH and C3b-Factor H with nanomolar and C3b with micromolar affinity; binding affinity for its natural ligand thrombin was several fold higher than for FH. At a physiological relevant concentration, TM inhibits complement hemolytic activity in serum via FH dependent and independent mechanisms. CONCLUSIONS: TM exhibits significant binding affinity for complement protein FH and C3b-FH complex and its soluble form is capable at physiologically relevant concentrations of inhibiting complement activation in serum.

Complement activation and expression during chronic relapsing experimental autoimmune encephalomyelitis in the Biozzi ABH mouse.

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE) in mice recapitulates many of the clinical and histopathological features of human multiple sclerosis (MS), making it a preferred model for the disease. In both, adaptive immunity and anti-myelin T cells responses are thought to be important, while in MS a role for innate immunity and complement has emerged. Here we sought to test whether complement is activated in crEAE and important for disease. Disease was induced in Biozzi ABH mice that were terminated at different stages of the disease to assess complement activation and local complement expression in the central nervous system. Complement activation products were abundant in all spinal cord areas examined in acute disease during relapse and in the progressive phase, but were absent in early disease remission, despite significant residual clinical disease. Local expression of C1q and C3 was increased at all stages of disease, while C9 expression was increased only in acute disease; expression of the complement regulators CD55, complement receptor 1-related gene/protein y (Crry) and CD59a was reduced at all stages of the disease compared to naive controls. These data show that complement is activated in the central nervous system in the model and suggest that it is a suitable candidate for exploring whether anti-complement agents might be of benefit in MS.

Deletion of Crry, the murine ortholog of the sporadic Alzheimer's disease risk gene CR1, impacts tau phosphorylation and brain CFH.

Large-scale genome-wide SNP association studies have identified an association between variants of CR1, the gene encoding complement component receptor 1, and the sporadic form of Alzheimer's disease. The role of CR1 and the complement system in Alzheimer's disease remains far from clear. In rodents the closest ortholog of CR1 is the Crry gene (Cr1-related protein Y). To begin to explore its role in Alzheimer's disease we examined hippocampal lysates from Crry(-/-) mice and age matched controls by immunoblotting. We measured complement factor H, a component of the complement system and biomarker for Alzheimer's disease progression, and tau phosphorylation at the serine 235 site, hyperphosphorylated forms of tau being a defining neuropathological hallmark of the disease. We found that levels of CFH and of tau phosphorylation at serine 235 were strongly and significantly reduced in Crry(-/-) samples. These observations provide a starting point for further attempts to determine the role of CR1 in the neuropathological process driving Alzheimer's disease.

Systemic complement profiling in multiple sclerosis as a biomarker of disease state.

BACKGROUND: There is increasing evidence of significant and dynamic systemic activation and upregulation of complement in multiple sclerosis (MS), which may contribute to disease pathogenesis. OBJECTIVE: We aimed to investigate the pathological role of complement in MS and the potential role for complement profiling as a biomarker of MS disease state. METHODS: Key components of the classical, alternative and terminal pathways of complement were measured in plasma and cerebrospinal fluid (CSF) of patients with MS in different clinical phases of disease and in matched controls. RESULTS: Increased plasma levels of C3 (p<0.003), C4 (p<0.001), C4a (p<0.001), C1 inhibitor (p<0.001), and factor H (p<0.001), and reduced levels of C9 (p<0.001) were observed in MS patients compared with controls. Combined profiling of these analytes produced a statistical model with a predictive value of 97% for MS and 73% for clinical relapse when combined with selected demographic data. CSF-plasma correlations suggested that source of synthesis of these components was both systemic and central. CONCLUSION: These data provide further evidence of alterations in both local and systemic expression and activation of complement in MS and suggest that complement profiling may be informative as a biomarker of MS disease, although further work is needed to determine its use in distinguishing MS from its differential.

Variation in complement component C1 inhibitor in age-related macular degeneration.

This study assessed variation in plasma levels of the complement regulatorC1 inhibitor (C1inh) in patients with age related macular degeneration (AMD) and controls. Plasma from391 AMD cases and 370 controls was assayed by rate nephelometry to determine C1inh protein levels. Protein levels were analysed for relationships with age, gender, smoking, AMD disease status and genetic variation in the SERPING1 gene, which encodes C1inh, using a multivariate analysis. t-Tests show a significant difference in C1inh levels in AMD cases compared with controls (p=2.340E-6), smokers compared to non-smokers (p=1.022E-4) and females compared to males (p=1.661E-7). Multivariate analysis shows that after accounting for gender and smoking AMD status remained significant. Age was included in the model but was not significant. Including genetic variation in the model shows that one significant SNP (rs2649663) 5' of the SERPING1 gene is associated with C1inh levels though this SNP is not associated with AMD. This suggests that genetic variation in the promoter region of the SERPING1 gene may influence expression of the gene.

Characterization of a large genomic deletion in four Irish families with C7 deficiency.

Inherited deficiency of the seventh complement component (C7) is associated with increased susceptibility to Neisseria meningitidis infections. The disease is rare in most Western countries. Here we report new investigations of a large, but incompletely characterized genomic deletion of exons 8 and 9 [c.739-?_1093+?del], previously identified in three unrelated Irish families with C7 deficiency. We have analysed DNA from one individual, who is homozygous for the deletion, by PCR using primers progressively proximal to the deleted exons. Thus we were able to map the deletion boundaries. Amplification across the breakpoint and sequencing revealed an indel mutation that included a 6.4kb deletion together with an insertion of a novel 8bp sequence [c.739+1262_1270-2387delinsGCAGGCCA]. We demonstrated the same defect in the C7 deficient patients from each family and developed a duplex PCR method to enable the detection of alleles containing the deletion in heterozygotes. A member of a fourth family was found to be homozygous for the deletion defect. Thus, the deletion defect may be a more commonly distributed cause of C7 deficiency in Ireland.

Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular degeneration.

PURPOSE: There is evidence for complement dysfunction in age-related macular degeneration (AMD). Complement activation leads to formation of the membrane attack complex (MAC), known to assemble on retinal pigment epithelial (RPE) cells. Therefore, the effect of sub-lytic MAC on RPE cells was examined with regard to pro-inflammatory or pro-angiogenic mediators relevant in AMD. METHODS: For sub-lytic MAC induction, RPE cells were incubated with an antiserum to complement regulatory protein CD59, followed by normal human serum (NHS) to induce 5% cell death, measured by a viability assay. MAC formation was evaluated by immunofluorescence and FACS analysis. Interleukin (IL)-6, -8, monocytic chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were quantified by enzyme-linked immunosorbent assay (ELISA). Intracellular MCP-1 was analysed by immunofluorescence, vitronectin by western blotting, and gelatinolytic matrix metalloproteinases (MMPs) by zymography. RESULTS: Incubation of RPE cells with the CD59 antiserum followed by 5% NHS induced sub-lytic amounts of MAC, verified by FACS and immunofluorescence. This treatment stimulated the cells to release IL-6, -8, MCP-1, and VEGF. MCP-1 staining, production of vitronectin, and gelatinolytic MMPs were also elevated in response to sub-lytic MAC. CONCLUSIONS: MAC assembly on RPE cells increases the IL-6, -8, and MCP-1 production. Therefore, sub-lytic MAC might have a significant role in generating a pro-inflammatory microenvironment, contributing to the development of AMD. Enhanced vitronectin might be a protective mechanism against MAC deposition. In addition, the increased expression of gelatinolytic MMPs and pro-angiogenic VEGF may be associated with neovascular processes and late AMD.

Impact of compound heterozygous complement factor H mutations on development of atypical hemolytic uremic syndrome-A pedigree revisited.

Atypical hemolytic uremic syndrome (aHUS) is associated with mutations in the gene CFH encoding the complement regulator factor H (CFH). We previously reported a family, in which three individuals had partial CFH deficiency but only one was affected by aHUS. We have investigated this family further to show that the partial CFH deficiency is associated with a heterozygous CFH mutation (c.2768T>G, p.Tyr899Asp). We used the polymorphic CFH variant p.His402Tyr to track expression of p.Tyr899Asp, and found that this mutant was expressed in minimal quantities in serum. In the one affected individual we found a second CFH mutation (c.3581G>A, p.Gly1194Asp) on the other allele which was expressed normally. We showed that this mutant, which has been described previously in aHUS, has impaired regulation of cell surface complement activation. The affected individual in this family is therefore a compound heterozygote for two functionally significant CFH mutations. Two individuals (mother and male sib) in the pedigree carried only c.2768T>G, p.Tyr899Asp and one (father) carried only c.3581G>A, p.Gly1194Asp, and all three were asymptomatic. Thus, further investigation of this family has enabled us to clarify the genotype-phenotype correlation.

Upregulating CD59: a new strategy for protection of neurons from complement-mediated degeneration.

An increasing number of studies have shown a critical role for the membrane attack complex, synthesized on activation of the terminal pathway of the complement system, in causing demyelination and neuronal death in neurodegeneration. The aim of this study was to develop a strategy to increase the resistance of neurons to complement damage by modulating the expression of membrane complement regulatory protein CD59, the only inhibitor of the terminal pathway of the complement cascade. We exploited our recent finding that CD59 expression is regulated by the neural-restrictive silencer factor (REST) and designed a novel REST-derived peptide (REST5) containing the nuclear localization domain of the wild-type protein. The effect of REST5 and the mechanism by which it modulates CD59 expression were modelled in neuroblastoma cells transfected with expression constructs, and then confirmed in human neurons differentiated from neural progenitor cells. REST5 increased the expression of CD59 in neurons by fivefold and protected them from complement-mediated lysis spontaneously triggered by neurons. As a source of complement, we used either human serum or conditioned medium from primary human oligodendroglia. This study brings new insight into immunopharmacological research that may serve to inhibit neuronal death triggered by the terminal pathway of complement activation.

Systemic and local anti-C5 therapy reduces the disease severity in experimental autoimmune uveoretinitis.

Activation of complement occurs during autoimmune retinal and intraocular inflammatory disease as well as neuroretinal degenerative disorders. The cleavage of C5 into fragments C5a and C5b is a critical event during the complement cascade. C5a is a potent proinflammatory anaphylatoxin capable of inducing cell migration, adhesion and cytokine release, while membrane attack complex C5b-9 causes cell lysis. Therapeutic approaches to prevent complement-induced inflammation include the use of blocking monoclonal antibodies (mAb) to prevent C5 cleavage. In these current experiments, the rat anti-mouse C5 mAb (BB5.1) was utilized to investigate the effects of inhibition of C5 cleavage on disease progression and severity in experimental autoimmune uveoretinitis (EAU), a model of organ-specific autoimmunity in the eye characterized by structural retinal damage mediated by infiltrating macrophages. Systemic treatment with BB5.1 results in significantly reduced disease scores compared with control groups, while local administration results in an earlier resolution of disease. In vitro, contemporaneous C5a and interferon-gamma signalling enhanced nitric oxide production, accompanied by down-regulation of the inhibitory myeloid CD200 receptor, contributing to cell activation. These experiments demonstrate that C5 cleavage contributes to the full expression of EAU, and that selective C5 blockade via systemic and local routes of administration can suppress disease. This presents great therapeutic potential to protect against tissue damage during autoimmune responses in the retina or inflammation-induced degenerative disease.

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.

Complement in multiple sclerosis: its role in disease and potential as a biomarker.

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system with a poorly defined and complex immunopathogenesis. Although initiated by reactive T cells, persistent inflammation is evident throughout the disease course. A contribution from complement has long been suspected, based on the results of pathological and functional studies which have demonstrated complement activation products in MS brain and biological fluids. However, the extent and nature of complement activation and its contribution to disease phenotype and long-term outcome remain unclear. Furthermore, functional polymorphisms in components and regulators of the complement system which cause dysregulation, and are known to contribute to other autoimmune inflammatory disorders, have not been investigated to date in MS in any detail. In this paper we review evidence from pathological, animal model and human functional and genetic studies, implicating activation of complement in MS. We also evaluate the potential of complement components and regulators and their polymorphic variants as biomarkers of disease, and suggest appropriate directions for future research.

Complement, roles in renal disease and modulation for therapy.

The complement system, an essential part of the innate immune system, defends the host against invading pathogens, prevents immune complex disease and aids the acquired immune response. Under normal conditions the host is protected from complement attack by an array of complement regulatory proteins. However, in certain contexts inappropriate complement activation can occur associating the C system with a variety of disease pathologies. This review focuses upon the role complement plays in a number of renal pathologies as well as the role of complement in three examples of extrarenal diseases: paroxysmal nocturnal hemoglobinuria, age-related macular degeneration and liver fibrosis. From the evidence discussed it is clear that mutations or polymorphisms in the complement regulators resulting in reduced levels or inefficient action dramatically enhance susceptibility to certain diseases and in particular render the kidney more vulnerable to complement attack. Additionally, deficiency in the complement components can predispose to disease through reduced clearance of apoptotic cells and subsequent generation of complement activating autoantibodies or enhanced formation of convertases resulting in heightened complement activation. As complement has devastating effects, in such disease contexts it has become a therapeutic target. Therapeutic intervention strategies discussed here focus upon the use of recombinant agents, the most promising of which are the anti-C5 antibody-derived reagents. These agents have proved effective in the treatment of paroxysmal nocturnal hemoglobinuria, nephritis and ischemia-reperfusion injuries and will no doubt, along with other reagents currently being developed, prove invaluable in the treatment of renal pathologies.

Complement membrane attack is required for endplate damage and clinical disease in passive experimental myasthenia gravis in Lewis rats.

Myasthenia gravis (MG) is a debilitating and potentially fatal neuromuscular disease characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular endplate with resultant failure of neuromuscular transmission. A role for complement (C) in the pathology of human MG has been suggested based upon identification of C activation products in plasma and deposited at the endplate in MG. In the rat model, experimental autoimmune MG (EAMG), C depletion or inhibition restricts clinical disease, further implicating C in pathology. The mechanisms by which C activation drives pathology in MG and EAMG are unclear. Here we provide further evidence implicating C and specifically the membrane attack complex (MAC) in the Lewis rat passive EAMG model of MG. Rats deficient in C6, an essential component of the MAC, were resistant to disease induction and endplate destruction was reduced markedly compared to C6-sufficient controls. After reconstitution with C6, disease severity and endplate destruction in the C6-deficient rats was equivalent to that in controls. The data confirm the essential role of the MAC in the destruction of the endplate in EAMG and raise the prospect of specific MAC inhibition as an alternative therapy in MG patients resistant to conventional treatments.

The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice.

The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end-plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end-plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild-type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end-plates. Cd59a-deficient mice had very mild disease with some muscle inflammation and essentially undamaged end-plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end-plates. Inhibition of MAC assembly abrogated clinical disease in these double-deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti-C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.

Blockade of the C5a receptor fails to protect against experimental autoimmune encephalomyelitis in rats.

Complement activation contributes to inflammation and tissue damage in human demyelinating diseases and in rodent models of demyelination. Inhibitors of complement activation ameliorate disease in the rat model antibody-dependent experimental autoimmune encephalomyelitis and rats unable to generate the membrane attack complex of complement develop inflammation without demyelination. The role of the highly active chemotactic and anaphylactic complement-derived peptide C5a in driving inflammation and pathology in rodent models of demyelination has been little explored. Here we have used a small molecule C5a receptor antagonist, AcF-[OPdChaWR], to examine the effects of C5a receptor blockade in rat models of brain inflammation and demyelination. C5a receptor antagonist therapy completely blocked neutrophil response to C5a in vivo but had no effect on clinical disease or resultant pathology in either inflammatory or demyelinating rat models. We conclude that C5a is not required for disease induction or perpetuation in these strongly complement-dependent disease models.

Deletion of the gene encoding CD59a in mice increases disease severity in a murine model of rheumatoid arthritis.

OBJECTIVE: To investigate the roles of CD59a in the protection of joint tissue in the context of murine antigen-induced arthritis (AIA). METHODS: AIA was triggered in CD59a-deficient (CD59a(-/-)) mice and in CD59a-sufficient (CD59a(+/+)) controls; the course and severity of disease were compared between groups. The effects on arthritis of restoring CD59 to the joint in CD59a(-/-) mice by use of a membrane-targeted recombinant CD59 were also explored. RESULTS: Disease, as assessed clinically by measurement of joint swelling on day 1 (P < 0.0001), day 2 (P < 0.01), and day 7 (P < 0.02) and histologically from indicators of joint damage on day 21 (P < 0.02), was significantly enhanced in CD59a(-/-) mice compared with CD59a(+/+) wild-type controls. Membrane attack complex (MAC) deposition in the arthritic joints of CD59a(-/-) mice was also increased compared with that in the joints of CD59a(+/+) controls. Restitution of CD59 activity in joints of CD59a(-/-) mice was attempted with soluble recombinant rat CD59 (sCD59) or with a novel membrane-targeted rat CD59 derivative (sCD59-APT542). Strong immunohistochemical staining of the synovial membrane and subsynovial tissue was apparent in sCD59-APT542-injected joints, but not in joints injected with untargeted sCD59. Intraarticular administration of sCD59-APT542 markedly ameliorated disease severity in CD59a(-/-) mice, knee swelling was significantly reduced over the time course of the disease, and joint damage, assessed histologically, was significantly milder on day 21 (P < 0.05). CONCLUSION: These data firmly implicate the MAC of complement as a major effector of joint damage in the murine AIA model of rheumatoid arthritis (RA), and they provide a rationale for the inhibition of MAC assembly as a therapeutic strategy for RA.

The possibilities and pitfalls for anti-complement therapies in inflammatory diseases.

The complement system is a key component of innate immunity, acting to protect the host from micro-organisms such as bacteria and other "foreign" threats, including tumor cells. However, excessive activation of complement can injure the host and can even be life threatening. These toxic effects are caused primarily by the excessive production of the anaphylatoxins C3a and C5a during complement activation and excessive formation of membrane attack complex on the host cell membrane. Many inflammatory diseases, including rheumatoid arthritis and glomerulonephritis, are thought to involve excessive activation of complement, both for their development and perpetuation. Uncontrolled complement activation is also implicated in post-ischemic inflammation and tissue damage and in sepsis. Therefore, it is important to regulate the complement system to treat disease. There are still no broadly applicable agents for the therapeutic regulation of excessive complement activation. However, there are now some agents in the development that might provide useful anti-complement therapies in the near future. Current strategies include the use of neutralizing antibodies, small synthetic antagonists, soluble recombinant forms of the natural complement regulators, and gene therapies to control excessive complement activation. Here we describe these new agents, their strengths and weaknesses and progress in testing the agents in relevant animal models.