The role of complement membrane attack complex in dry and wet AMD - From hypothesis to clinical trials.

Data from human dry and wet age-related macular degeneration (AMD) eyes support the hypothesis that constant 'tickover' of the alternative complement pathway results in chronic deposition of the complement membrane attack complex (MAC) on the choriocapillaris and the retinal pigment epithelium (RPE). Sub-lytic levels of MAC lead to cell signaling associated with tissue remodeling and the production of cytokines and inflammatory molecules. Lytic levels of MAC lead to cell death. CD59 is a naturally occurring inhibitor of the assembly of MAC. CD59 may thus be therapeutically efficacious against the pathophysiology of dry and wet AMD. The first gene therapy clinical trial for geographic atrophy - the advanced form of dry AMD has recently completed recruitment. This trial is studying the safety and tolerability of expressing CD59 from an adeno-associated virus (AAV) vector injected once into the vitreous. A second clinical trial assessing the efficacy of CD59 in wet AMD patients is also under way. Herein, the evidence for the role of MAC in the pathophysiology of dry as well as wet AMD and the scientific rationale underlying the use of AAV- delivered CD59 for the treatment of dry and wet AMD is discussed.

The role of MACPF proteins in the biology of malaria and other apicomplexan parasites.

Apicomplexans are eukaryotic parasites of major medical and veterinary importance. They have complex life cycles through frequently more than one host, interact with many cell types in their hosts, and can breach host cell membranes during parasite traversal of, or egress from, host cells. Some of these parasites make a strikingly heavy use of the pore-forming MACPF domain, and encode up to 10 different MACPF domain-containing proteins. In this chapter, we focus on the two most studied and medically important apicomplexans, Plasmodium and Toxoplasma, and describe the known functions of their MACPF polypeptide arsenal. Apicomplexan MACPF proteins appear to be involved in a variety of membrane-damaging events, making them an attractive model to dissect the structure-function relationships of the MACPF domain.

Chlamydial MACPF protein CT153.

Chlamydiae are obligate intracellular bacterial parasites that infect a wide range of metazoan hosts. Some Chlamydia species are important causes of chronic inflammatory diseases of the ocular, genital and respiratory tracts in humans. Genes located in a variable region of chlamydial genomes termed the plasticity zone are known to be key determinants of pathogenic diversity. The plasticity zone protein CT153, present only in select species, contains a membrane attack complex/perforin (MACPF) domain, which may mediate chlamydial interactions with the host cell. CT153 is present throughout the C. trachomatis developmental cycle and is processed into polypeptides that interact with membranes differently than does the parent protein. Chlamydiae interact extensively with membranes from the time of invasion until they eventually exit host cells, so numerous roles for a MACPF protein in pathogenesis of these pathogens are conceivable. Here, we present an overview of what is known about CT153 and highlight potential roles of a MACPF family protein in a group of pathogens whose intracellular development is marked by a series of interactions with host cell membranes and organelles. Finally, we identify new strategies for identifying CT153 functions made feasible by the recent development of a basic toolset for genetic manipulation of chlamydiae.

The making of a macromolecular machine: assembly of the membrane attack complex.

The complement terminal pathway clears pathogens by generating cytotoxic membrane attack complex (MAC) pores on target cells. For more than 40 years, biochemical and cellular assays have been used to characterize the lytic nature of the MAC and to define its protein composition. Although models for pore formation have been inferred from structures of bacterial cytolysins, it was only recently that we were able to visualize how complement components come together during MAC assembly. This review highlights structural analyses of terminal pathway complexes to explore molecular mechanisms underlying MAC formation.

Sublytic C5b-9 induces IL-6 and TGF-ß1 production by glomerular mesangial cells in rat Thy-1 nephritis through p300-mediated C/EBPß acetylation.

CCAAT/enhancer-binding protein (C/EBPß)-enhanced IL-6 and TGF-ß1 promoter activity and p300-mediated C/EBPß acetylation were involved in up-regulation of IL-6 and TGF-ß1 expression in GMCs attacked by sublytic C5b-9. In detail, the elements of C/EBPß binding to rat IL-6 and TGF-ß1 promoter and 3 acetylated sites of rat C/EBPß protein were first revealed. Furthermore, silencing the p300 or C/EBPß gene in rat kidney significantly reduced the production of IL-6 and TGF-ß1 and renal lesions in Thy-1N rats. Together, these data indicate that the mechanism of IL-6 and TGF-ß1 production in renal tissue of Thy-1N rats is associated with sublytic C5b-9 up-regulated p300 and p300-mediated C/EBPß acetylation as well as C/EBPß-activated IL-6 and TGF-ß1 genes.

Role of C5b-9 complement complex and response gene to complement-32 (RGC-32) in cancer.

Complement system activation plays an important role in both innate and acquired immunity, with the activation of complement and the subsequent formation of C5b-9 terminal complement complex on cell membranes inducing target cell death. Recognition of this role for C5b-9 leads to the assumption that C5b-9 might play an antitumor role. However, sublytic C5b-9 induces cell cycle progression by activating signal transduction pathways and transcription factors in cancer cells, indicating a role in tumor promotion for this complement complex. The induction of the cell cycle by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 also induces response gene to complement (RGC)-32, a gene that plays a role in cell cycle promotion through activation of Akt and the CDC2 kinase. RGC-32 is expressed by tumor cells and plays a dual role in cancers, in that it has both a tumor suppressor role and tumor-promoting activity. Thus, through the activation of tumor cells, the C5b-9-mediated induction of the cell cycle plays an important role in tumor proliferation and oncogenesis.

A role for the NF-κB pathway in cell protection from complement-dependent cytotoxicity.

Nucleated cells are equipped with several mechanisms that support their resistance to complement-dependent cytotoxicity (CDC). The role of the NF-κB pathway in cell protection from CDC was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 subunit of NF-κB or the IκB kinases IKKα or IKKß, and in cells treated with p65 small interfering RNA. Pretreatment with the IKK inhibitor PS-1145 also enhanced CDC of wild-type cells (WT) but not of p65(-/-) cells. Furthermore, reconstitution of p65 into p65(-/-) cells and overexpression of p65 in WT cells lowered their sensitivity to CDC. The postulated effect of p65 on the JNK-mediated death-signaling pathway activated by complement was examined. p65 small interfering RNA enhanced CDC in WT cells but not in cells lacking JNK. JNK phosphorylation induced by complement was more pronounced in p65(-/-) cells than in WT cells. The results indicate that the NF-κB pathway mediates cell resistance to CDC, possibly by suppressing JNK-dependent programmed necrotic cell death.

The protective role of CD59 and pathogenic role of complement in hepatic ischemia and reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI) is a major factor influencing graft outcome in liver transplantation, but its mechanism is not well defined. Although complement, including the membrane attack complex (MAC), a terminal product of complement activation, is thought to be involved in the multiple reactions subsequent to the ischemia-reperfusion (IR) process, the role of MAC in the pathogenesis of hepatic IRI requires further investigation. We used a warm ischemia-reperfusion injury model in mice and a syngeneic orthotopic liver transplantation model in rats to define the role of complement, including MAC, in hepatic IR. CD59-deficient mice had more severe liver dysfunction, evidenced by increased aspartate aminotransferase levels and increased injury of liver parenchymal and nonparenchymal cells than did CD59-sufficient mice during warm hepatic IR. Furthermore, complement depletion in CD59-deficient mice by pretreatment with cobra venom factor (CVF) or the genetic introduction of C3 deficiency partially protected against development of the severe liver dysfunction that occurred in CD59-deficient mice. Severity of liver dysfunction correlated with MAC deposition, apoptotic cells, and increased inflammatory mediators such as tumor necrosis factor α. Moreover, depletion of complement with CVF in orthotopic liver transplantation recipient rats attenuated IRI of the donor livers. Taken together, these results highlight the protective role of CD59 and pathogenic role of complement, including MAC, in the pathogenesis of hepatic IRI.

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.

The soluble terminal complement complex (SC5b-9) up-regulates osteoprotegerin expression and release by endothelial cells: implications in rheumatoid arthritis.

OBJECTIVE: Complement activation products contribute to a large number of inflammatory diseases, including RA. We have investigated whether osteoprotegerin (OPG) may concur with the soluble terminal complement complex (SC5b-9) to the inflammatory cascade characterizing RA. METHODS: Levels of SC5b-9 and OPG in the plasma and SF of patients with active RA were determined by ELISA. The presence of SC5b-9 and OPG in RA synovial lesions was analysed by immunohistochemistry. Cultured endothelial cells were used for in vitro leucocyte/endothelial cell adhesion assays. In addition, endothelial cells were exposed to SC5b-9 in order to evaluate the effects on the production of OPG protein, as well as the activation of the OPG promoter. RESULTS: Patients affected by active RA are characterized by elevated levels of both SC5b-9 and OPG in plasma and/or SF. Of note, we have observed a co-localization of SC5b-9 and OPG in endothelial cells of post-capillary venules of RA synovial lesions. Data on endothelial cell cultures showed that exposure to SC5b-9 induced the up-regulation of OPG expression/release, stimulating the transcriptional activity of the OPG promoter, and synergized with TNF-alpha in up-regulating OPG production. CONCLUSIONS: Our findings demonstrate that SC5b-9 induces OPG production by endothelial cells and we propose that the SC5b-9-mediated up-regulation of OPG may be an important mechanism whereby complement contributes in promoting and/or enhancing the inflammation in RA.

Evaluation of adenovirus-delivered human CD59 as a potential therapy for AMD in a model of human membrane attack complex formation on murine RPE.

PURPOSE: Complement-mediated damage to the retinal pigment epithelium (RPE), Bruch membrane, and choroid has been associated with pathogenesis in age-related macular degeneration (AMD). The terminal step of complement activation involves lysis of cells by the insertion of the membrane attack complex (MAC) in the plasma membrane. The hypothesis that local overexpression of human CD59 (hCD59) delivered by an adenovirus (Ad) vector to primary murine RPE cells in vitro, RPE in vivo, or cornea ex vivo protects those cells from human MAC deposition and lysis was tested. METHODS: A humanized model of MAC deposition on murine cells and murine ocular tissues including RPE and cornea was developed to permit testing of human complement regulators in mice. A recombinant adenovirus-expressing hCD59 was generated, and this virus was injected into the subretinal space of adult mice. Subsequently, eyecups from these mice were exposed to human serum, and the levels of MAC deposition on the RPE were quantified. hCD59 was also expressed on murine cornea ex vivo and in murine hepatocytes, and primary RPE cells in vitro and levels of human MAC deposition and cell lysis were measured. RESULTS: Adenovirus-mediated delivery of hCD59 to the RPE, cornea, or cells in culture protects those cells from human MAC deposition and MAC-mediated damage and vesiculation. CONCLUSIONS: The humanized model of MAC deposition on murine ocular tissues allows testing of human complement regulators that may have potential in the treatment of AMD or other diseases associated with complement activation.

CD59 efficiently protects human NT2-N neurons against complement-mediated damage.

The complement regulatory protein CD59 controls cell survival by the inhibition of C5b-9 formation on the cell membrane. Loss of CD59 increases the susceptibility of cells to complement-mediated damage and lysis. Deposition of IgM can induce complement activation with subsequent cell death. We have previously demonstrated the presence of CD59 on human NT2-N neurons. In this study, we investigated the functional role of CD59 for NT2-N cell survival after IgM-mediated complement activation. Complement activation was induced on NT2-N neurons with human serum following incubation with the IgM monoclonal antibody A2B5 reacting with a neuronal cell membrane epitope. Deposition of C1q and C5b-9 was detected on the cell membrane and sC5b-9 in the culture supernatant. Specific inhibition of complement was obtained by the C3 inhibitor compstatin, and by anti-C5/C5a MoAb. CD59 was blocked by the MoAb BRIC 229. Membrane damage of propidium iodide-stained NT2-N cells was confirmed by immunofluorescence microscopy and degeneration of neuronal processes was shown with crystal violet staining. A2B5, but not the irrelevant control IgM antibody, induced complement activation on NT2-N neurons after incubation with a human serum, as detected by the deposition of C1q. A marked membrane deposition of C5b-9 on NT2-N neurons with accompanying cell death and axonal degeneration was found after the blocking of CD59 with MoAb BRIC 229 but not with an isotype-matched control antibody. Compstatin and anti-C5 monoclonal antibodies which blocked C5 activation efficiently inhibited complement activation. In conclusion, CD59 is essential for protecting human NT2-N neurons against complement-mediated damage, which is known to occur in a number of clinical conditions including stroke.

[Update on pathophysiologic and immunotherapeutic approaches for the treatment of multiple sclerosis]. / Multiple-Sklerose-Update zur Pathophysiologie und neuen immuntherapeutischen Ansätzen.

Multiple sclerosis (MS) is a chronic disabling disease with significant implications for patients and society. The individual disease course is difficult to predict due to the heterogeneity of clinical presentation and of radiologic and pathologic findings. Although its etiology still remains unknown, the last decade has brought considerable understanding of the underlying pathophysiology of MS. In addition to its acceptance as a prototypic inflammatory autoimmune disorder, recent data reveal the importance of primary and secondary neurodegenerative mechanisms such as oligodendrocyte death, axonal loss, and ion channel dysfunction. The deepened understanding of its immunopathogenesis and the limited effectiveness of currently approved disease-modifying therapies have led to a tremendous number of trials investigating potential new drugs. Emerging treatments take into account the different immunopathological mechanisms and strategies, to protect against axonal damage and promote remyelination. This review provides a compilation of novel immunotherapeutic strategies and recently uncovered aspects of known immunotherapeutic agents. The pathogenetic rationale of these novel drugs for the treatment of MS and accompanying preclinical and clinical data are highlighted.

Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization.

Sublytic C5b-9 has been described as a pro-inflammatory mediator that triggers cell activation rather than inducing cell death. Dendritic cells (DC) play a critical role in controlling antigen-specific immune responses. Although DC maturation induced by various stimuli has been well characterized, the role of C5b-9 in DC function has not been described. In this report, we use in vitro assembled functional C5b-9 based on purified distal complement protein to show that DC maturation is promoted by sublytic C5b-9. This was demonstrated by up-regulation of CD83, HLA-antigens and costimulatory molecules, including CD80, D86, B7-H1, B7-H3, B7-H4 and BTLA. In addition, secretion of cytokines such as interleukin (IL)-12 and tumor necrosis factor-alpha was increased while the capacity for antigen uptake (FITC-Dextran and Lucifer Yellow) was reduced in C5b-9-treated DC. Mixed lymphocyte reactions indicated that C5b-9-activated DC acted as stimulators that significantly promoted CD4+ T cell activation and elicited production of cytokines, including interferon-gamma and IL-2. Interestingly, C5b-9-treated DC also orient CD4+ CD45RA+ naïve T cells toward Th1 polarization. Our results are the first to report that DC are potential immunoregulatory targets of C5b-9, suggesting that C5b-9 bridges innate and acquired immunity by inducing DC maturation.

The inflammatory reaction pattern distinguishes primary dysferlinopathies from idiopathic inflammatory myopathies: an important role for the membrane attack complex.

Degenerative muscle changes in dysferlinopathies are often accompanied by inflammatory infiltrates and may even mimic primary idiopathic inflammatory myopathies. In the present study, the inflammatory reaction pattern with respect to the cellular composition of the infiltrates and the expression of potent cytokines was characterized in dysferlinopathies and in idiopathic inflammatory myopathies. Cellular infiltrates in dysferlinopathies mainly consisted of CD4+CD25- T cells and macrophages. We noted a prominent expression of interferon-gamma which may contribute to the marked upregulation of MHC class I antigen observed on the vast majority of muscle fibres. Furthermore, membrane attack complex positive deposits were found on intact as well as necrotic muscle fibres. Collectively, our study indicates that the inflammatory reaction pattern in dysferlinopathies is distinct from the one in idiopathic inflammatory myopathies. In particular, membrane attack complex deposits and a pro-inflammatory milieu in the absence of interleukin-10 expression may contribute to progressive muscle damage in dysferlinopathies.

C5b-9-induced endothelial cell proliferation and migration are dependent on Akt inactivation of forkhead transcription factor FOXO1.

Migration and proliferation of aortic endothelial cells (AEC) are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1.

C5b-9 terminal complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and up-regulating FLIP.

Activation of the terminal complement cascade involving C5 to C9 proteins has a beneficial role for oligodendrocytes (OLG) in experimental allergic encephalomyelitis, an animal model of multiple sclerosis, by protecting them from apoptotic cell death. We have previously shown that sublytic C5b-9 complexes, through posttranslational regulation of Bad, inhibit the mitochondrial pathway of apoptosis induced by serum deprivation. In the present study, we examined the possible involvement of the caspase-8 and Fas pathway in OLG apoptosis and the role of C5b-9 in this process. In a serum-free defined medium, OLG undergo apoptosis and differentiation concomitantly. Under this condition, we found that caspase-8 processing was increased in association with Bid cleavage and markedly reduced expression of cellular FLIP long isoform protein. The caspase-8 inhibitor Z-IETD-FMK inhibited cell death associated with differentiation in a dose-dependent manner. Exposure to C5b-9 induced an inhibition of caspase-8 activation, Bid cleavage, and a significant increase in expression of cellular FLIP long isoform. These C5b-9 effects were reversed by PI3K inhibitor LY294002. C5b-9 also down-regulated the expression of FasL and the Fas-induced apoptosis. These data suggest that C5b-9 through PI3K signaling can rescue OLG from Fas-mediated apoptosis by regulating caspase-8 processing.

Complement activation in angiotensin II-induced organ damage.

We tested whether or not complement activation participates in angiotensin (Ang) II-induced vasculopathy. We used double transgenic rats harboring human renin and angiotensinogen genes (dTGR) with or without losartan or the human renin inhibitor aliskiren. Sprague-Dawley (SD) rats were controls. DTGR had increased blood pressure at week 5 that increased further by week 7. Albuminuria was absent at week 5 but increased markedly in weeks 6 and 7. C-reactive protein (CRP) elevation, macrophages, T cells, tumor necrosis factor (TNF)-alpha, C1q, C3, C3c, and C5b-9 expression preceded albuminuria. C1q, C3, C3c, and C5b-9 were observed in the dTGR vessel media. C5b-9 colocalized with interleukin (IL)-6. Losartan and aliskiren reduced albuminuria and complement expression. We also studied vascular smooth muscle cells (VSMC) from dTGR compared VSMC from SD. C3 and IL-6 mRNA were analyzed after Ang II, TNF-alpha, and CRP stimulation. VSMC from dTGR showed increased proliferation and C3 expression compared with SD. Ang II did not induce C3 mRNA in either VSMC type. However, TNF-alpha and CRP induced C3 mRNA slightly in SD VSMC but markedly in dTGR VSMC, whereas IL-6 induction was similar in both. Thus, complement activation and cell infiltration occurred before the onset of albuminuria in Ang II-mediated renal damage. TNF-alpha and CRP played a major role in C3 activation. VSMC from dTGR are more sensitive for C3 activation. Our data show that, in this Ang II-induced model, complement activation is a major participant and suggest that TNF-alpha and CRP may play a role in its induction.

The role of c5b-9 terminal complement complex in activation of the cell cycle and transcription.

Activation of the complement system plays an important role in innate and acquired immunity. Activation of complement and subsequent formation of C5b-9 channels on the surface of cellular membranes leads to cell lysis. When the number of channels assembled on the surface of nucleated cells is limited, C5b-9 does not cause lysis, but instead can induce cell-cycle progression by activating signal transduction pathways, transcription factors, and key components of the cell-cycle machinery. Cell-cycle induction by C5b-9 is dependent on the activation of phosphatidylinositol 3-kinase and the ERK1 pathway in a Gi protein-dependent manner. Cell-cycle activation is regulated, in part, by activation of proto-oncogene c-jun and AP1 DNA binding activity. C5b-9 induces sequential activation of CDK4 and CDK2, leading to G1/S-phase transition and cellular proliferation. RGC-32 is a novel gene whose expression is induced by C5b-9. RGC-32 may play a key role in cell-cycle activation by increasing cyclin B1-CDC2 activity. C5b-9-mediated cell-cycle activation plays an important role in cellular proliferation and protection from apoptosis.