Alpha-synuclein activates the classical complement pathway and mediates complement-dependent cell toxicity.

BACKGROUND: Synucleinopathies are characterized by neurodegeneration and deposition of the presynaptic protein α-synuclein in pathological protein inclusions. Growing evidence suggests the complement system not only has physiological functions in the central nervous system, but also is involved in mediating the pathological loss of synapses in Alzheimer's disease. However, it is not established whether the complement system has a similar role in the diseases Parkinson's disease, Dementia with Lewy bodies, and multiple system atrophy (MSA) that are associated with α-synuclein aggregate pathology. METHODS: To investigate if the complement system has a pathological role in synucleinopathies, we assessed the effect of the complement system on the viability of an α-synuclein expressing cell model and examined direct activation of the complement system by α-synuclein in a plate-based activation assay. Finally, we investigated the levels of the initiator of the classical pathway, C1q, in postmortem brain samples from MSA patients. RESULTS: We demonstrate that α-synuclein activates the classical complement pathway and mediates complement-dependent toxicity in α-synuclein expressing SH-SY5Y cells. The α-synuclein-dependent cellular toxicity was rescued by the complement inhibitors RaCI (inhibiting C5) and Cp20 (inhibiting C3). Furthermore, we observed a trend for higher levels of C1q in the putamen of MSA subjects than that of controls. CONCLUSION: α-Synuclein can activate the classical complement pathway, and the complement system is involved in α-synuclein-dependent cellular cytotoxicity suggesting the system could play a prodegenerative role in synucleinopathies.

Development and optimization of a personalized fibrin membrane derived from the plasma rich in growth factors technology.

PURPOSE: To develop and characterize a new type of plasma rich in growth factors (PRGF) membrane for patients in which immune system is involved in the disease etiology. METHODS: Blood from three healthy donors was collected to obtain the different fibrin membranes by PRGF technology. PRGF obtained volumes were activated and divided into two groups: PRGF membrane (mPRGF) obtained after incubation at 37 °C for 30 min (control); and is-mPRGF: mPRGF obtained after incubation for 30 min at 56 °C. The concentration of several growth factors, proteins, immunoglobulin E and the complement activity was determined in the different mPRGF. The proliferative potential of heat-inactivated mPRGF were assayed on keratocytes (HK) and conjunctival fibroblasts (HConF). In addition, morphological and physical features of the inactivated mPRGF were evaluated in contrast to the control mPRGF. RESULTS: Heat-inactivation of the mPRGF preserves the content of most of the growth factors involved in the ocular wound healing while reducing drastically the content of IgE and the complement activity. The heat-inactivated mPRGF conserve the morphological and physical characteristics of the fibrin meshwork in comparison with the control mPRGF. Furthermore, no significant differences were found in the biological activity of the control mPRGF regarding the heat-inactivated mPRGF (is-mPRGF) in any of both ocular cell types evaluated. CONCLUSIONS: The heat-inactivation of the PRGF membranes (is-mPRGF) reduces drastically the content of IgE and complement activity while preserving the content of most of the proteins and morphogens involved in ocular wound healing. Furthermore, the morphological and physical features of the immunosafe mPRGF were also preserved after heat-inactivation.

Microglia in Brain Development, Homeostasis, and Neurodegeneration.

Advances in human genetics have implicated a growing number of genes in neurodegenerative diseases, providing insight into pathological processes. For Alzheimer disease in particular, genome-wide association studies and gene expression studies have emphasized the pathogenic contributions from microglial cells and motivated studies of microglial function/dysfunction. Here, we summarize recent genetic evidence for microglial involvement in neurodegenerative disease with a focus on Alzheimer disease, for which the evidence is most compelling. To provide context for these genetic discoveries, we discuss how microglia influence brain development and homeostasis, how microglial characteristics change in disease, and which microglial activities likely influence the course of neurodegeneration. In all, we aim to synthesize varied aspects of microglial biology and highlight microglia as possible targets for therapeutic interventions in neurodegenerative disease.

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

INTRODUCTION: Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. RESULTS AND CONCLUSIONS: In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

An Essential Role for the Transcription Factor Runx1 in T Cell Maturation.

The transcription factor Runx1 has essential roles throughout hematopoiesis. Here, we demonstrate that Runx1 is critical for T cell maturation. Peripheral naïve CD4(+) T cells from CD4-cre Runx1 cKO mice are phenotypically and functionally immature as shown by decreased production of TNF-α upon TCR stimulation. The loss of peripheral CD4(+) T cells in CD4-cre Runx1 cKO mice is not due to defects in homeostasis or decreased expression of IL-7Rα, as transgenic expression of IL-7Rα does not rescue the loss of CD4(+) T cells. Rather, immature Runx1-deficient CD4(+) T cells are eliminated in the periphery by the activation and fixation of the classical complement pathway. In the thymus, there is a severe block in all aspects of intrathymic T cell maturation, although both positive and negative selection are unaltered. Thus, loss of Runx1 leads to the earliest characterized block in post-positive selection intrathymic maturation of CD4 T cells.

The role of complement in tumor growth.

Complement is a central part of the immune system that has developed as a first defense against non-self cells. Neoplastic transformation is accompanied by an increased capacity of the malignant cells to activate complement. In fact, clinical data demonstrate complement activation in cancer patients. On the basis of the use of protective mechanisms by malignant cells, complement activation has traditionally been considered part of the body's immunosurveillance against cancer. Inhibitory mechanisms of complement activation allow cancer cells to escape from complement-mediated elimination and hamper the clinical efficacy of monoclonal antibody-based cancer immunotherapies. To overcome this limitation, many strategies have been developed with the goal of improving complement-mediated effector mechanisms. However, significant work in recent years has identified new and surprising roles for complement activation within the tumor microenvironment. Recent reports suggest that complement elements can promote tumor growth in the context of chronic inflammation. This chapter reviews the data describing the role of complement activation in cancer immunity, which offers insights that may aid the development of more effective therapeutic approaches to control cancer.

The alternative and terminal pathways of complement mediate post-traumatic spinal cord inflammation and injury.

Complement is implicated in the inflammatory response and the secondary neuronal damage that occurs after traumatic spinal cord injury (SCI). Complement can be activated by the classical, lectin, or alternative pathways, all of which share a common terminal pathway that culminates in formation of the cytolytic membrane attack complex (MAC). Here, we investigated the role of the alternative and terminal complement pathways in SCI. Mice deficient in the alternative pathway protein factor B (fB) were protected from traumatic SCI in terms of reduced tissue damage and demyelination, reduced inflammatory cell infiltrate, and improved functional recovery. In a clinically relevant paradigm, treatment of mice with an anti-fB mAb resulted in similarly improved outcomes. These improvements were associated with decreased C3 and fB deposition. On the other hand, deficiency of CD59, an inhibitor of the membrane attack complex, resulted in significantly increased injury and impaired functional recovery compared to wild-type mice. Increased injury in CD59-deficient mice was associated with increased MAC deposition, while levels of C3 and fB were unaffected. These data indicate key roles for the alternative and terminal complement pathways in the pathophysiology of SCI. Considering a previous study demonstrating an important role for the classical pathway in promoting SCI, it is likely that the alternative pathway plays a critical role in amplifying classical pathway initiated complement activation.

Complement classical pathway components are all important in clearance of apoptotic and secondary necrotic cells.

Inherited deficiencies in components of the classical complement pathway are strong disease susceptibility factors for the development of systemic lupus erythematosus (SLE) and there is a hierarchy among deficiency states, the strongest association being with C1q deficiency. We investigated the relative importance of the different complement pathways regarding clearance of apoptotic cells. Phagocytosis of labelled apoptotic Jurkat cells by monocyte-derived macrophages in the presence of sera from individuals with complement deficiencies was studied, as well as C3 deposition on apoptotic cells using flow cytometry. Sera from individuals deficient in C1q, C4, C2 or C3 all showed decreased phagocytosis. Mannose binding lectin (MBL) and the alternative pathway did not influence phagocytosis. Notably, the components of the complement classical pathway, including C1q, were equally important in clearance of apoptotic cells. This indicates that deposition of C3 fragments is of major significance; we therefore studied C3 deposition on apoptotic cells. Experiments with MBL-deficient serum depleted of C1q or factor D confirmed the predominance of the classical pathway. At low dilution, sera deficient of C1q, C4 or C2 supported C3 fragment deposition demonstrating alternative pathway activation. In conclusion, we have found that complement-mediated opsonization and phagocytosis of apoptotic cells, particularly those undergoing secondary necrosis, are dependent mainly upon an intact classical pathway. The alternative pathway is less important, but may play a role in some conditions. C1q was not more important than other classical pathway components, suggesting a role in additional pathogenetic processes in SLE other than clearance of apoptotic cells.

Early complement activation and decreased levels of glycosylphosphatidylinositol-anchored complement inhibitors in human and experimental diabetic retinopathy.

Diabetic retinal microangiopathy is characterized by increased permeability, leukostasis, microthrombosis, and apoptosis of capillary cells, all of which could be caused or compounded by activation of complement. In this study, we observed deposition of C5b-9, the terminal product of complement activation, in the wall of retinal vessels of human eye donors with 9 +/- 3 years of type 2 diabetes, but not in the vessels of age-matched nondiabetic donors. C5b-9 often colocalized with von Willebrand factor in luminal endothelium. C1q and C4, the complement components unique to the classical pathway, were not detected in the diabetic retinas, suggesting that C5b-9 was generated via the alternative pathway, the spontaneous activation of which is regulated by complement inhibitors. The diabetic donors showed a prominent reduction in the retinal levels of CD55 and CD59, the two complement inhibitors linked to the plasma membrane by glycosylphosphatidylinositol anchors, but not in the levels of transmembrane CD46. Similar complement activation in retinal vessels and selective reduction in the levels of retinal CD55 and CD59 were observed in rats with a 10-week duration of streptozotocin-induced diabetes. Thus, diabetes causes defective regulation of complement inhibitors and complement activation that precede most other manifestations of diabetic retinal microangiopathy. These are novel clues for probing how diabetes affects and damages vascular cells.

Recent progress in the understanding of the structure-function relationships of the globular head regions of C1q.

The first step in the activation of the classical pathway of complement cascade by immune complexes involves the binding of the C-terminal globular head regions of C1q to the Fc regions of IgG or IgM, each globular head being composed of the C-terminal halves of one A-, one B- and one C-chain. Recent studies using recombinant forms of globular region appear to suggest that each globular head of C1q may be composed of three, structurally and functionally, independent domains/modules. The heterotrimeric organisation thus could offer functional flexibility and versatility to the whole C1q molecule. The crystal structure of an adipocyte-specific serum protein, Acrp-30, has revealed the existence of a structural fold shared by members of a new C1q/tumor necrosis factor (TNF) superfamily, characterized by a distinctive globular domain. The protein members seem to be active as self-assembling noncovalent trimers, whose individual chains fold as compact 'jellyroll' beta sandwiches. The recognition of a C1q/TNF superfamily, which has wide-ranging functions, highlights the possibility that the globular regions of C1q may fulfill more binding functions than previously envisaged.

Increased susceptibility of the left compared to the right ventricle to remote ischemia/reperfusion injury in human C1-inhibitor-overexpressing transgene mice.

Acute myocardial injury has been demonstrated as a remote sequela of severe lower torso ischemia-reperfusion (I/R) due to proinflammatory events. In a model of I/R injury, administration of C1 esterase inhibitor (C1-Inh) reduces myocardial necrosis. We investigated the susceptibility of the left (LV) versus right ventricle (RV) and the protective effect of transgenic C1-Inh-overexpressing mice. Two groups of mice (n = 6) underwent a 2-h lower torso ischemia followed by 3 h of reperfusion: transgenic and wild type with sham-operated controls. Animals were then injected with (125)I bovine albumin. Heart was removed and samples from right and left ventricular free wall were harvested, weighted, and radioactivity was determined. Permeability index for wild-type animals in the RV was 0.22 +/- 0.04, compared to 0.17 +/- 0.07 in controls (NS), and in the LV 0.36 +/- 0.08, compared to 0.21 +/- 0.05 in controls (p <.01). The LV showed a significantly higher value compared to the right (0.22 +/- 0.04 vs. 0.36 +/- 0.08, p <.01). No difference was seen in the RV between transgenic and wild-type mice; however, in the LV the values decreased significantly in transgenic animals (p <.015). Thus, remote myocardial injury after lower torso I/R is present in both ventricles; however, the LV seems to be more susceptible as assessed by albumin permeability. Inhibition of the classic complement cascade may be a promising therapeutic approach for myocardial protection in reperfusion injury.

Activation capacity of the alternative and classic complement pathways in patients operated on for colorectal cancer.

PURPOSE: Tumor cells may suppress activation of the host's complement system, and the functional state of the complement system may be a prognostic marker of outcome in patients with malignancies. Serial plasma samples from patients undergoing intended curative surgery for colorectal cancer were analyzed for complement factor C3 activation capacity. METHODS: Samples were collected from 91 patients with colorectal cancer and 13 with benign colorectal diseases before surgery and 1, 2, and 7 days after surgery, between 8 and 13 days after surgery, and 3, 6, 12, 18, 24, 36, 48, and 60 months after surgery. The samples were analyzed with an enzyme-linked immunosorbent assay that measured C3 activation capacity by the alternative and classic complement pathways. Cancer patients were compared according to Dukes stage, type of surgery performed, transfusion of blood, development of infection, venous thromboembolism, and cancer recurrence. RESULTS: Plasma samples obtained from cancer patients before surgery showed C3 activation capacities corresponding to those of samples from patients with benign disease. For both patient groups, C3 activation capacity decreased after surgery and normalized within seven days. Significant differences in C3 activation capacities were observed between cancer patients that were related to Dukes stage and in patients with and without buffy coat-depleted red cells suspended in saline, adenine, glucose, and mannitol transfusion, infectious events, and deep venous thromboembolism. Measurement of C3 activation capacity was of predictive value in patients who developed infection. CONCLUSION: Serial measurements of C3 activation capacity in plasma from patients who had undergone surgery for colorectal cancer revealed significant differences related to Dukes staging after surgery and to the development of infections but not to cancer recurrence.

Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59.

PURPOSE: To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system. METHODS: Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH(50) and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity. RESULTS: Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH(50)). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an M(r) >/= 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (>/=19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M(r) >/= 3 kDa also had an inhibitory effect on the alternative pathway activity (AH(50)). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid. CONCLUSIONS: Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59.

Therapeutic inhibition of the complement system.

The use of powerful methodologies in molecular biology, biochemistry, and physiology in the last 2 decades had led to impressive progress in our understanding of the mechanisms of complement activation and its role as either a protective or a pathogenic factor in human disease. With respect to disease pathogenesis, the complexity of the complement cascade provides opportunities for several different therapeutic targets within the complement pathways. More than a century after complement was first described, we are about to witness in the near future the availability of a variety of complement inhibitors for specific therapies. Progress in the area of xenotransplantation has been substantial, but formidable obstacles remain to selective inhibition of the factors that block successful clinical xenotransplantation. Bispecific antibodies, designed to enhance rather than inhibit existing complement pathways, hold strong promise for the clearance of viral and bacterial pathogens from the circulation.

Antibody dependent haemolysin, complement and opsonin in sera of a major carp, Cirrhina mrigala and catfish, Clarias batrachus and Heteropneustes fossilis.

The present communication is a continuation of our earlier study on the natural serum haemagglutinin/lectins of Cirrhina mrigala, Clarias batrachus and Heteropneustes fossilis. Sera of Cirrhina mrigala, belonging to the major carp family, could not only agglutinate heterologous rabbit erythrocytes, but also lyse them spontaneously. This lysis of rabbit RBC by Cirrhina mrigala sera was calcium ion dependent and heat sensitive, indicating thereby that the haemolysis was mediated by the fish serum complement system via the classical pathway. Quantification of CH50 and APCH50 levels in the sera of Clarias batrachus and Heteropneustes fossilis as well as in the sera of amphibia, aves and mammals showed that lower vertebrates predominantly possessed an alternative pathway of the complement system, while on the other hand, in the higher vertebrates the major pathway of complement activation was classical. Furthermore sera of Clarias batrachus and Heteropneustes fossilis had opsonins, which could stimulate heterologous rat peritoneal macrophages to engulf Staphylococcus aureus with the production of superoxide anion. From this study we concluded that fishes have been armed with various powerful natural humoral defense systems for their protection against environmental pathogens.

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM-Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2-terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway.

Cold target competition analysis of the classical activation pathway of complement-mediated cytotoxicity: a non-interaction model for competing lysis.

A mathematical analysis of cold target competition experiments of complement-mediated lysis is presented, aimed at developing a minimal model of lysis where no interaction between the competing populations of sensitized blood group A and B erythrocytes is presumed. The model is able to predict the extent of lysis from the input values with remarkable accuracy suggesting that under the conditions used no stimulation and/or inhibition of the lysis of the sensitized erythrocytes occurs. The distribution of complement between the competing A and B erythrocyte populations is approximated by the model and found to be proportional to the 5th and 4th power of the ratios of the antibody and target cell concentrations, respectively. In accordance with earlier observations, suggesting that the interaction between the antibody and the C1q molecules is based on polar electrostatic charges, we propose that the sensitizing antibody provides an electrostatic field around the erythrocytes which attracts C1q molecules towards their membranes.

Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways.

Endotoxemia was evoked by bolus injection of Escherichia coli endotoxin (2 ng/kg body weight) in six healthy subjects to investigate the early kinetics of cytokine release in relation to the development of clinical and hematologic abnormalities frequently seen in gram-negative septicemia. The plasma concentration of tumor necrosis factor (TNF) increased markedly after 30 to 45 minutes, and reached a maximal level after 60 to 90 minutes. In each volunteer, the initial increase of plasma interleukin 6 (IL-6) concentrations occurred 15 minutes after the initial TNF increase, and maximal IL-6 concentrations were reached at 120 to 150 minutes. A transient increase in body temperature and pulse rate occurred simultaneously with the initial TNF and IL-6 increases, whereas a significant decrease in blood pressure occurred after 120 minutes. These changes were proportional to the changes in TNF and IL-6 concentrations. Coagulation activation, as assessed by a rise of prothrombin fragments and thrombin-antithrombin III complexes, was noted after 120 minutes, in the absence of activation of the contact system. A two- to sixfold increase in the concentrations of tissue plasminogen activator (t-PA) and von Willebrand factor antigen indicated endothelial cell activation. This increase started at 120 and 90 minutes, respectively. The release of t-PA coincided with activation of the fibrinolytic pathway, as measured by plasmin-alpha 2-antiplasmin complexes. The fibrinolytic activity of t-PA was subsequently offset by release of plasminogen activator inhibitor, observed 150 minutes after the endotoxin injection, and reaching a peak at 240 minutes. No complement activation was detected. These results show that in humans endotoxin induces an early, rapidly counteracted fibrinolytic response, and a more long-lasting activation of thrombin by a mechanism other than contact system activation. In addition, our data suggest that endotoxin-induced leukopenia and endothelial cell activation are mediated by TNF.