C3 Glomerulopathy and Related Disorders in Children: Etiology-Phenotype Correlation and Outcomes.

BACKGROUND AND OBJECTIVES: Membranoproliferative GN and C3 glomerulopathy are rare and overlapping disorders associated with dysregulation of the alternative complement pathway. Specific etiologic data for pediatric membranoproliferative GN/C3 glomerulopathy are lacking, and outcome data are based on retrospective studies without etiologic data. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 80 prevalent pediatric patients with membranoproliferative GN/C3 glomerulopathy underwent detailed phenotyping and long-term follow-up within the National Registry of Rare Kidney Diseases (RaDaR). Risk factors for kidney survival were determined using a Cox proportional hazards model. Kidney and transplant graft survival was determined using the Kaplan-Meier method. RESULTS: Central histology review determined 39 patients with C3 glomerulopathy, 31 with immune-complex membranoproliferative GN, and ten with immune-complex GN. Patients were aged 2-15 (median, 9; interquartile range, 7-11) years. Median complement C3 and C4 levels were 0.31 g/L and 0.14 g/L, respectively; acquired (anticomplement autoantibodies) or genetic alternative pathway abnormalities were detected in 46% and 9% of patients, respectively, across all groups, including those with immune-complex GN. Median follow-up was 5.18 (interquartile range, 2.13-8.08) years. Eleven patients (14%) progressed to kidney failure, with nine transplants performed in eight patients, two of which failed due to recurrent disease. Presence of >50% crescents on the initial biopsy specimen was the sole variable associated with kidney failure in multivariable analysis (hazard ratio, 6.2; 95% confidence interval, 1.05 to 36.6; P<0.05). Three distinct C3 glomerulopathy prognostic groups were identified according to presenting eGFR and >50% crescents on the initial biopsy specimen. CONCLUSIONS: Crescentic disease was a key risk factor associated with kidney failure in a national cohort of pediatric patients with membranoproliferative GN/C3 glomerulopathy and immune-complex GN. Presenting eGFR and crescentic disease help define prognostic groups in pediatric C3 glomerulopathy. Acquired abnormalities of the alternative pathway were commonly identified but not a risk factor for kidney failure.

Rare Genetic Variants in Complement Factor I Lead to Low FI Plasma Levels Resulting in Increased Risk of Age-Related Macular Degeneration.

Purpose: Rare genetic variants in complement factor I (CFI) that cause low systemic levels of the protein (FI) have been reported as a strong risk factor for advanced age-related macular degeneration (AMD). This study set out to replicate these findings. Methods: FI levels were measured by sandwich ELISA in an independent cohort of 276 patients with AMD and 205 elderly controls. Single-nucleotide polymorphism genotyping and Sanger sequencing were used to assess genetic variability. Results: The median FI level was significantly lower in those individuals with AMD and a rare CFI variant (28.3 µg/mL) compared to those with AMD without a rare CFI variant (38.8 µg/mL, P = 0.004) or the control population with (41.7 µg/mL, P = 0.0085) or without (41.5 µg/mL, P < 0.0001) a rare CFI variant. Thirty-six percent of patients with AMD with a rare CFI variant had levels below the fifth percentile, compared to 6% in controls with CFI variants. Multiple regression analyses revealed a decreased FI level associated with a rare CFI variant was a risk factor for AMD (early or late AMD: odds ratio [OR] 12.05, P = 0.03; early AMD: OR 30.3, P = 0.02; late AMD: OR 10.64, P < 0.01). Additionally, measurement of FI in aqueous humor revealed a large FI concentration gradient between systemic circulation and the eye (∼286-fold). Conclusions: Rare genetic variants in CFI causing low systemic FI levels are strongly associated with AMD. The impermeability of the Bruch's membrane to FI will have implications for therapeutic replacement of FI in individuals with CFI variants and low FI levels at risk of AMD.

Complement, a target for therapy in inflammatory and degenerative diseases.

The complement system is a key innate immune defence against infection and an important driver of inflammation; however, these very properties can also cause harm. Inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease. Complement provides numerous options for drug development as it is a proteolytic cascade that involves nine specific proteases, unique multimolecular activation and lytic complexes, an arsenal of natural inhibitors, and numerous receptors that bind to activation fragments. Drug design is facilitated by the increasingly detailed structural understanding of the molecules involved in the complement system. Only two anti-complement drugs are currently on the market, but many more are being developed for diseases that include infectious, inflammatory, degenerative, traumatic and neoplastic disorders. In this Review, we describe the history, current landscape and future directions for anti-complement therapies.

Functional analysis of a complement polymorphism (rs17611) associated with rheumatoid arthritis.

Complement is implicated in the pathogenesis of rheumatoid arthritis (RA); elevated levels of complement activation products have been measured in plasma, synovial fluid, and synovial tissues of patients. Complement polymorphisms are associated with RA in genome-wide association studies. Coding-region polymorphisms may directly impact protein activity; indeed, we have shown that complement polymorphisms affecting a single amino acid change cause subtle changes in individual component function that in combination have dramatic effects on complement activity and disease risk. In this study, we explore the functional consequences of a single nucleotide polymorphism (SNP) (rs17611) encoding a V802I polymorphism in C5 and propose a mechanism for its link to RA pathology. Plasma levels of C5, C5a, and terminal complement complex were measured in healthy and RA donors and correlated to rs17611 polymorphic status. Impact of the SNP on C5 functionality was assessed. Plasma C5a levels were significantly increased and C5 levels significantly lower with higher copy number of the RA risk allele for rs17611, suggesting increased turnover of C5 V802. Functional assays using purified C5 variants revealed no significant differences in lytic activity, suggesting that increased C5 V802 turnover was not mediated by complement convertase enzymes. C5 is also cleaved in vivo by proteases; the C5 V802 variant was more sensitive to cleavage with elastase and the "C5a" generated was biologically active. We hypothesize that this SNP in C5 alters the rate at which elastase generates active C5a in rheumatoid joints, hence recruiting neutrophils to the site thus maintaining a state of inflammation in arthritic joints.

Expression of membrane complement regulators, CD46, CD55 and CD59, in mesothelial cells of patients on peritoneal dialysis therapy.

We investigated the expression of membrane complement regulators (CRegs), CD46, CD55 and CD59 in human mesothelial cells, and correlated with clinical background and level of complement (C) activation products in peritoneal dialysis (PD) fluids (PDF) to clarify influence of the C activation system in PD patients. Expression of CRegs was assessed on primary cultures of mesothelial cells (HPMC) harvested from PD fluid of 31 PD patients. Because expression of CD55 but not CD46 and CD59 in mesothelial cells was significantly correlated to value of dialysate-to-plasma creatinine concentration ratio (D/P Cre) (p<0.005) as an indicator of peritoneal function, we focused on analysis of CD55 expression of HPMCs in comparison with levels of C activation products in the PDF of the PD patients, and their background factors. When comparing expression of the CRegs between systemic neutrophils and HPMC, no correlation was observed, supporting that change of CRegs' expression in HPMC was independently occurring in the peritoneum. Expression of CD55 protein in HPMC was closely correlated with expression at the mRNA level (p<0.0001) and was inversely correlated with levels of sC5b-9 (p<0.05), but not C3, C4, IL6 and CA125 in the PDF. Complications of diabetes, usage of icodextrin and residual renal function were not correlated with change of CD55 expression in HPMCs. Our data show that the process of PD therapy modifies expression of CD55 on peritoneal mesothelium and triggers local C activation. These findings support efforts to modify PD therapy to limit effects on activation and regulation of the C system.

C3 glomerulopathy: consensus report.

C3 glomerulopathy is a recently introduced pathological entity whose original definition was glomerular pathology characterized by C3 accumulation with absent or scanty immunoglobulin deposition. In August 2012, an invited group of experts (comprising the authors of this document) in renal pathology, nephrology, complement biology, and complement therapeutics met to discuss C3 glomerulopathy in the first C3 Glomerulopathy Meeting. The objectives were to reach a consensus on: the definition of C3 glomerulopathy, appropriate complement investigations that should be performed in these patients, and how complement therapeutics should be explored in the condition. This meeting report represents the current consensus view of the group.

Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Complement-induced protection: an explanation for the limitations of cell-based tumour immunotherapies.

Complement is involved in the inflammatory response and clearance of infected or altered cells. It is therefore unexpected that complement-deficient animals are less susceptible to carcinogen-induced tumours and more readily control growth of injected tumour cell lines than their wild-type counterparts, implying that complement promotes tumour development and progression. Conversely, natural killer (NK) and CD8(+) T cells are known to limit progression of the same tumours. Previous studies indicate that sublytic levels of the complement membrane attack complex protect cells against further attack by lytic doses of complement and other pore-formers such as perforin. We hypothesise that inefficient attack by complement in vivo allows tumour cells to avoid lysis by both NK cells and antigen-specific cytotoxic T cells, thereby promoting tumour outgrowth. Complement could thus be limiting the efficacy of NK and T cell-targeted cancer therapies, and the inclusion of complement inhibitors could optimise these immunotherapeutic regimes.

Combined yeast {beta}-glucan and antitumor monoclonal antibody therapy requires C5a-mediated neutrophil chemotaxis via regulation of decay-accelerating factor CD55.

Administration of a combination of yeast-derived beta-glucan with antitumor monoclonal antibodies (mAb) has significant therapeutic efficacy in a variety of syngeneic murine tumor models. We have now tested this strategy using human carcinomas implanted in immunocompromised severe combined immunodeficient mice. Combined immunotherapy was therapeutically effective in vivo against NCI-H23 human non-small-cell lung carcinomas, but this modality was surprisingly ineffective against SKOV-3 human ovarian carcinomas. Whereas NCI-H23 tumors responded to this combination therapy with increased intratumoral neutrophil infiltration and C5a production, these responses were lacking in treated SKOV-3 tumors. Further results suggested that SKOV-3 tumors were protected by up-regulation of the membrane complement regulatory protein CD55 (decay-accelerating factor). Blockade of CD55 in vitro led to enhanced deposition of C activation product C3b and increased cytotoxicity mediated by beta-glucan-primed neutrophils. In vivo, administration of anti-CD55 mAb along with beta-glucan and anti-Her-2/neu mAb caused tumor regression and greatly improved long-term survival in animals bearing the previously resistant SKOV-3 tumors. This was accompanied by increased intratumoral neutrophil accumulation and C5a production. We conclude that CD55 suppresses tumor killing by antitumor mAb plus beta-glucan therapy (and, perhaps, in other circumstances). These results suggest a critical role for CD55 to regulate iC3b and C5a release and in turn to influence the recruitment of beta-glucan-primed neutrophils eliciting killing activity.

In vivo characterization and therapeutic efficacy of a C5-specific inhibitor from the soft tick Ornithodoros moubata.

The involvement of complement (C) in inflammatory diseases has driven the search for agents capable of inhibiting dysregulated complement activation. Many of these reagents inhibit the C3 convertases during the early stages of the cascade. However, a drawback of total systemic C inhibition, particularly in longterm treatment of chronic disease, is potentiation of infection and immune complex disease due to an inability to opsonize complexes and foreign cells and to lyse pathogens. Recent identification of a C5-binding protein in the salivary gland of the soft tick Ornithodoros moubata has enabled development of a terminal pathway-specific reagent, OmCI, with potential to ameliorate disease while leaving key physiological processes unaffected. Here we demonstrated that OmCI has broad cross-species activity. When given intravenously to rodents, OmCI totally ablated complement hemolytic activity, which gradually restored as C5 was resynthesized. The circulating half-life of OmCI was 30 h, demonstrating a much slower clearance than other small, biological agents. Using C5-sufficient and C5-deficient mice we showed that prolonged half-life was due to binding to plasma C5. Surface plasmon resonance analysis of C5 binding to OmCI confirmed a high binding affinity with a slow dissociation rate. OmCI was effective in preventing experimental autoimmune myasthenia gravis induced by passive transfer in normal Lewis rats. OmCI ablated clinical disease, reduced C3 and C9 deposition at the neuro-muscular junction, and effected a marked reduction in cellular infiltration at this site. These data offer exciting prospects for targeted treatment of complement-mediated diseases without the detrimental inhibition of the opsonic roles of complement.

Decay-accelerating factor (CD55) is expressed by neurons in response to chronic but not acute autoimmune central nervous system inflammation associated with complement activation.

There is compelling evidence that a unique innate immune response in the CNS plays a critical role in host defense and clearance of toxic cell debris. Although complement has been implicated in neuronal impairment, axonal loss, and demyelination, some preliminary evidence suggests that the initial insult consequently activates surrounding cells to signal neuroprotective activities. Using two different models of experimental autoimmune encephalomyelitis, we herein demonstrate selective C1q complement activation on neuron cell bodies and axons. Interestingly, in brains with chronic but not acute experimental autoimmune encephalomyelitis, C3b opsonization of neuronal cell bodies and axons was consistently associated with robust neuronal expression of one of the most effective complement regulators, decay-accelerating factor (CD55). In contrast, levels of other complement inhibitors, complement receptor 1 (CD35), membrane cofactor protein (CD46), and CD59 were largely unaffected on neurons and reactive glial cells in both conditions. In vitro, we found that proinflammatory stimuli (cytokines and sublytic doses of complement) failed to up-regulate CD55 expression on cultured IMR32 neuronal cells. Interestingly, overexpression of GPI-anchored CD55 on IMR32 was capable of modulating raft-associated protein kinase activities without affecting MAPK activities and neuronal apoptosis. Critically, ectopic expression of decay-accelerating factor conferred strong protection of neurons against complement attack (opsonization and lysis). We conclude that increased CD55 expression by neurons may represent a key protective signaling mechanism mobilized by brain cells to withstand complement activation and to survive within an inflammatory site.

Decay-accelerating factor induction by tumour necrosis factor-alpha, through a phosphatidylinositol-3 kinase and protein kinase C-dependent pathway, protects murine vascular endothelial cells against complement deposition.

We have shown that human endothelial cells (EC) are protected against complement-mediated injury by the inducible expression of decay-accelerating factor (DAF). To understand further the importance of DAF regulation, we characterized EC DAF expression on murine EC in vitro and in vivo using a model of glomerulonephritis. Flow cytometry using the monoclonal antibody (mAb) Riko-3 [binds transmembrane- and glycosylphosphatidylinositol (GPI)-anchored DAF], mAb Riko-4 (binds GPI-anchored DAF) and reverse transcription-polymerase chain reaction (RT-PCR), demonstrated that murine EC DAF is GPI-anchored. Tumour necrosis factor-alpha (TNF-alpha) increased EC DAF expression, detectable at 6 hr and maximal at 24-48 hr poststimulation. DAF upregulation required increased steady-state DAF mRNA and protein synthesis. In contrast, no increased expression of the murine complement receptor-related protein-Y (Crry) was seen with TNF-alpha. DAF upregulation was mediated via a protein kinase C (PKC)alpha, phosphoinositide-3 kinase (PI-3 kinase), p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB)-dependent pathway. The increased DAF was functionally relevant, resulting in a marked reduction in C3 deposition following complement activation. In a nephrotoxic nephritis model, DAF expression on glomerular capillaries was significantly increased 2 hr after the induction of disease. The demonstration of DAF upregulation above constitutive levels suggests that this may be important in the maintenance of vascular integrity during inflammation, when the risk of complement-mediated injury is increased. The mouse represents a suitable model for the study of novel therapeutic approaches by which vascular endothelium may be conditioned against complement-mediated injury.

Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: tissue distribution and functional comparison.

CD59, the sole membrane regulator of the membrane attack complex of complement, is broadly and abundantly expressed in man and other mammals. In mouse, CD59 is encoded by two homologous genes. The expression patterns and functional roles of the proteins encoded by these genes, mCD59a and mCD59b, have not been well characterized. Here we describe the generation of monoclonal and polyclonal antibodies detecting specifically mCD59a and mCD59b. These reagents have been used to study function and to ascertain the cell and tissue distributions of mCD59a and mCD59b. mCD59a was broadly distributed on endothelia, erythrocytes, platelets, and on numerous other cell types in organs, a distribution pattern resembling that of CD59 in other species. In marked contrast, expression of mCD59b was restricted to germ cell elements in the testis and mature spermatozoa. Both mCD59a and CD59b inhibited human and rodent complement with similar efficiency. These findings demonstrate that the broadly distributed mCD59a is the key regulator of the terminal complement pathway in mice whereas CD59b, expressed only in testis and on sperm, probably plays other roles in vivo.