- Email: [email protected]
Pre-transplant ficolin-3 levels are associated with kidney allograft survival
Abstracts / Immunobiology 217 (2012) 1129–1222
1193
gangrene healed with loss of the end phalanges. The patient was continued on three-weekly eculizumab maintenance infusions. aHUS activity has subsided completely, and some late recovery of kidney function has occurred within the past 10 months with an increase in measured GFR from 1 to 6 ml/min/1.73 m2 . In rare cases aHUS can present by thrombotic macroangiopathy of small peripheral arteries. Eculizumab effectively prevents or interrupts the course of this complication.
182
http://dx.doi.org/10.1016/j.imbio.2012.08.182
A major complement regulator on most murine cells is Crry, a homolog of human CD46. Genetic knockout of the Crry gene is embryonic lethal with demise at day 9.5 (d). The cause is maternal complement attack on the developing placenta leading to failure of the allantois to fuse with the chorion. Crry is expressed on trophoblast cells by at least d7.5, whereas DAF is not expressed until d10.5. Maternal C3, factor B, factor D, and Properdin are required for lethality, but not C4 or antibodies (Abs). Interesting, unlike other mouse models of fetal loss, C5 deficiency does not rescue, suggesting that C3b deposition and/or C3a release influences embryo development. To further analyze the events leading to embryo loss, we used cobra venom factor (CVF) to deplete the alternative pathway (AP) between d4.5 and d8.5. CVF treatment rescues Crry−/− embryos. Chorio-allantoic fusion occurs at d8.5, and histological analysis reveals that this defect is present at d8.5 and Crry−/− embryos are smaller at that point. This is consistent with our CVF treatments, which must be given prior to d8.5. Additionally, blocking the AP with a function blocking Ab against mouse properdin rescues, if given at d6.5 and d7.5. Extensive trials were also conducted depleting neutrophils with an anti-Gr1 mAb but it did not mitigate embryo loss. This model of fetal loss differs from other C5 dependent models in that it shows no dependence on neutrophils. The failure of “allantoic fusion” phenocopies the developmental defect seen in the Integrin Alpha 4 (ITGA4)−/− and VCAM1−/− embryos. These two proteins are expressed on chorion and allantois, respectively. qPCR utilizing cDNA from whole embryos suggests that Crry−/− embryos fail to upregulate ITGA4 expression at d8.5. We are investigating if AP activation is linked to this defect. Additionally, we are well evaluating the potential role of the C3a receptor, utilizing the C3aR−/− mouse.
181 Phagocytosis of Escherichia coli in human whole blood: Early complement dependency is succeeded by late CD14 dependency over time Dorte Christansen 1 , Jørgen Stenvik 2 , Ole-Lars Brekke 1,3 , John D. Lambris 4 , Terje Espevik 2 , Tom Eirik Mollnes 1,3,5 1 Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway 2 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway 3 Faculty of Health Sciences, University of Tromsø, Norway 4 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 5 Institute of Immunology, Oslo University Hospital and University of Oslo, Norway
To study the dependency of complement and CD14 in Escherichia coli phagocytosis over time in a human whole blood model. Alexalabeled bacteria were used to study phagocytosis after 15 and 120 min in leukocytes using flow cytometry. Cytokines including Interleukin (IL)-1b, IL-6, IL-8 and Tumor Necrosis Factor (TNF)-a were measured after 120 min using multiplex technique and Interferon (IFN)-b by ELISA. The C3 inhibitor compstatin, human genetic C5 deficient blood and a monoclonal antibody were used to outline the time-dependent role of complement (C3, C5) and CD14 in the phagocytosis process. Lepirudin blood was obtained from human healthy donors and a human genetic C5 deficient individual. The actin filament inhibitor Cytochalacin D inhibited phagocytosis of E. coli at both time-points. Phagocytosis measured after 15 min showed a strong dependency of complement, since phagocytosis in both compstatin-inhibited normal blood and in C5 deficient human blood was virtually abolished, whereas anti-CD14 alone had only a modest effect. In contrast, after 2 h the phagocytosis was virtually abolished by anti-CD14 alone, whereas complement played a minor role. Notably, the combination of compstatin and anti-CD14 was the most effective regimen to inhibit phagocytosis, approaching complete blocking at both time points. TNF-a, IL-1b, IL-6 and IL-8 were all substantially reduced when compstatin and anti-CD14 were used in combination, whereas Cytochalacin D had only a minor inhibitory effect. In contrast, IFN-b was not inhibited by compstatin or C5 deficiency, but was largely reduced by Cytochalacin D, and virtually abolished by anti-CD14 alone. Phagocytosis of E. coli in human whole blood was initially dependent on complement, succeeded by a dependency of CD14. The combination of compstatin and anti-CD14 was effective at all time points. CD14-dependent phagocytosis of E. coli seemed to be required for induction of IFN-b. http://dx.doi.org/10.1016/j.imbio.2012.08.183
Pinpointing the time and cause of fetal demise in the Crry−/− embryo Michael P. Triebwasser, Xiaobo Wu, Dennis Hourcade, John P. Atkinson Department of Rheumatology, Washington Univ. School of Medicine, Saint Louis, MO, USA
http://dx.doi.org/10.1016/j.imbio.2012.08.184 183 Pre-transplant ficolin-3 levels are associated with kidney allograft survival Jakob T. Bay 1 , Estrid Hein 1 , Søren S. Sørensen 2 , Jesper Melchior Hansen 3 , Peter Garred 1 1
Laboratory of Molecular Medicine, Department of Clinical Immunology, Sect 7631, Rigshospitalet, Copenhagen, Denmark 2 Department of Nephrology P, Rigshospitalet, Copenhagen, Denmark 3 Department of Nephrology, Herlev Hospital, Copenhagen, Denmark Ficolin-3 is an initiator of the lectin complement pathway. Activation of the complement system is a mediator of the pathophysiology of graft rejection in kidney transplantation, but the role of ficolin-3 in this process is unknown. Using a prospective study design, 527 kidney transplanted patients with a followup period of 5 years were included. 97 blood donors served as controls. Ficolin-3, C4 and C3 were measured in pre-transplant as well as in control serum samples. In the controls, deposition of ficolin-3, C4, C3 and the terminal complement complex (TCC) was measured in an assay based on acetylated albumin as matrix. The concentration of ficolin-3, C4 and C3 was increased in the patients compared with the controls (p ≤ 0.001). The ficolin-3 levels
1194
Abstracts / Immunobiology 217 (2012) 1129–1222
correlated with the serum levels of C4 and C3 both in the patients (rho: 0.44, p ≤ 0.0001, rho: 0.53, p ≤ 0.0001, respectively) and in the controls (rho: 0.30, p ≤ 0.0038, rho: 0.50, p ≤ 0.0001, respectively). The serum levels of ficolin-3 correlated with ficolin-3 deposition (rho: 0.66, p ≤ 0.0001), C4 deposition (rho: 0.45, p ≤ 0.0001), C3 deposition (rho: 0.46, p ≤ 0.0001) and formation of TCC (rho: 0.24, p = 0.0197). In a multivariable regression analysis adjusted for confounders a high ficolin-3 level was a predictor of death censored graft survival (HR = 3.18, 95% CI: 1.48–6.81) (p = 0.003), while this was not seen for C4 and C3. Ficolin-3 is involved in the pathophysiology of kidney graft rejection. The ficolin-3 levels correlated closely with the concentration and deposition of down-stream serum complement components, illustrating the importance of ficolin-3 as an initiator molecule of complement activation. http://dx.doi.org/10.1016/j.imbio.2012.08.185 184 Premolis semirufa envenomation, disease affecting rubber tappers of the Amazon: Searching for caterpillar-bristles toxic components acting on the complement system Isadora Maria Villas Boas 1 , Rute Maria Gonc¸alves de Andrade 1 , Giselle Pidde-Querioz 1 , Fernanda C.V. Portaro 1 , Carmen W. van den Berg 2 , Denise V. Tambourgi 1 1 2
Immunochemistry Laboratory, Butantan Institute, S.P., Brazil School of Medicine, Cardiff University, Cardiff, UK
Pararama, the popular name of the larval form of the moth Premolis semirufa, inhabits rubber plantations in the Amazon region and the accidental contact of the skin with the caterpillar’s bristles or cocoons results in immediate and intense heat, pain, edema and itching, which last for three to seven days. However, after multiple contacts, it may induce joint-space narrowing and bone alteration, as well as degeneration of the articular cartilage and immobilization of the affected joints. Specific treatment for this disease does not exist, but corticosteroids are frequently administered. Despite the public health hazard of P. semirufa caterpillar poisoning, little is known about the nature of the toxic components involved in the induction of the pathology. In the present study we have investigated the action of the Pararama caterpillar bristles extract on the complement system in in vitro studies. The bristle extract is able to activate the human complement system with the generation of anaphylatoxins. Aiming to isolate the component responsible for C-activation, P. semirufa’s bristles extract was submitted to FPLC gel-filtration chromatography and a fraction containing a component of 82 kDa, endowed with serine protease activity, was able to induce a strong activation of the alternative pathway and partially to activate classical and lectin pathways. This protease was able to induce the generation of C3a, C4a and C5a in human serum, not only through C-activation but also through the direct cleavage of the C3, C4 and C5 components. These data suggest that a serine protease present in P. semirufa’s bristles extract is responsible for complement activation, which can play an important role in the inflammatory process in humans after envenomation. Financial support: Fapesp, CNPq, INCTTox. http://dx.doi.org/10.1016/j.imbio.2012.08.186
185 Production of functional full-length recombinant human C1q Isabelle Bally 1 , Sarah Ancelet 1 , Christine Moriscot 1 , Florence Bonnet 2 , Regis Daniel 2 , Guy Schoehn 1 , Gerard J. Arlaud 1 , Nicole M. Thielens 1 1
Institut de Biologie Structurale, Grenoble, France Laboratoire Analyse et Modelisation pour l’Environnelment, Evry, France 2
la
Biologie
et
Human C1q is a versatile recognition protein with the ability to sense a wide variety of targets, such as antibodies, pentraxins, and numerous non-immune targets, including pathogens and damaged structures from self. Most of the C1q ligands are recognized through its globular heads, triggering sequential activation of the associated proteases C1r and C1s. Precise mapping of the specific binding sites of C1q for its various ligands and for the C1s-C1r-C1r-C1s tetramer has been precluded so far due to the unavailability of recombinant C1q, a complex protein assembled from three polypeptide chains encoded by three different genes. For the first time, we have successfully generated a stable mammalian cell line producing full-length recombinant human C1q. Purification of the recombinant protein was facilitated by the presence of an affinity tag on one of the three chains. SDS-PAGE analysis of the purified material revealed a band pattern similar to that of serumderived C1q with typical A-B and C-C dimers under non-reducing conditions and the three A, B and C chains under reducing conditions. Mass spectrometry analysis of recombinant C1q showed that the three chains had undergone the expected post-translational modifications. Importantly, electron microscopy analysis revealed the typical bouquet-like shape of the molecule. The interaction properties of recombinant C1q with immunoglobulins, C-reactive protein and the C1s-C1r-C1r-C1s tetramer were investigated by surface plasmon resonance spectroscopy. Kinetic analyses of the binding data yielded affinities comparable to those of plasmaderived C1q for all ligands. The C1 complex reconstituted from recombinant C1q and the serum-derived proenzyme tetramer was able to self-activate in a typical manner. These results demonstrate that a method allowing production of a fully functional recombinant human C1q molecule is now available, opening the way to in-depth analysis of its interaction properties by site-directed mutagenesis. http://dx.doi.org/10.1016/j.imbio.2012.08.187 186 Prolonged voltage-gated calcium channel activation by sublytic complement activation in retinal pigment epithelium, a mechanism for controlling VEGF secretion Bärbel Rohrer 1 , Kannan Genewski 2 , Olaf Strauss 2 1 2
Kunchithapautham 1 , Andreas
Medical University of South Carolina, Charleston, SC, USA Klinikum der Universitaet Regensburg, Germany
Age-related macular degeneration (AMD) is a slowly progressing multifactorial disease involving genetic abnormalities and environmental insults. Genetic studies have demonstrated that polymorphisms in different complement proteins each increase the risk for developing AMD. As the alternative pathway is continuously activated in the fluid phase, tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. To explore the hypothesis that AMD pathology results from uncontrolled activation of the alternative pathway of complement, we have utilized stable RPE monolayers challenged with H2 O2 and
1193
gangrene healed with loss of the end phalanges. The patient was continued on three-weekly eculizumab maintenance infusions. aHUS activity has subsided completely, and some late recovery of kidney function has occurred within the past 10 months with an increase in measured GFR from 1 to 6 ml/min/1.73 m2 . In rare cases aHUS can present by thrombotic macroangiopathy of small peripheral arteries. Eculizumab effectively prevents or interrupts the course of this complication.
182
http://dx.doi.org/10.1016/j.imbio.2012.08.182
A major complement regulator on most murine cells is Crry, a homolog of human CD46. Genetic knockout of the Crry gene is embryonic lethal with demise at day 9.5 (d). The cause is maternal complement attack on the developing placenta leading to failure of the allantois to fuse with the chorion. Crry is expressed on trophoblast cells by at least d7.5, whereas DAF is not expressed until d10.5. Maternal C3, factor B, factor D, and Properdin are required for lethality, but not C4 or antibodies (Abs). Interesting, unlike other mouse models of fetal loss, C5 deficiency does not rescue, suggesting that C3b deposition and/or C3a release influences embryo development. To further analyze the events leading to embryo loss, we used cobra venom factor (CVF) to deplete the alternative pathway (AP) between d4.5 and d8.5. CVF treatment rescues Crry−/− embryos. Chorio-allantoic fusion occurs at d8.5, and histological analysis reveals that this defect is present at d8.5 and Crry−/− embryos are smaller at that point. This is consistent with our CVF treatments, which must be given prior to d8.5. Additionally, blocking the AP with a function blocking Ab against mouse properdin rescues, if given at d6.5 and d7.5. Extensive trials were also conducted depleting neutrophils with an anti-Gr1 mAb but it did not mitigate embryo loss. This model of fetal loss differs from other C5 dependent models in that it shows no dependence on neutrophils. The failure of “allantoic fusion” phenocopies the developmental defect seen in the Integrin Alpha 4 (ITGA4)−/− and VCAM1−/− embryos. These two proteins are expressed on chorion and allantois, respectively. qPCR utilizing cDNA from whole embryos suggests that Crry−/− embryos fail to upregulate ITGA4 expression at d8.5. We are investigating if AP activation is linked to this defect. Additionally, we are well evaluating the potential role of the C3a receptor, utilizing the C3aR−/− mouse.
181 Phagocytosis of Escherichia coli in human whole blood: Early complement dependency is succeeded by late CD14 dependency over time Dorte Christansen 1 , Jørgen Stenvik 2 , Ole-Lars Brekke 1,3 , John D. Lambris 4 , Terje Espevik 2 , Tom Eirik Mollnes 1,3,5 1 Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway 2 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway 3 Faculty of Health Sciences, University of Tromsø, Norway 4 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 5 Institute of Immunology, Oslo University Hospital and University of Oslo, Norway
To study the dependency of complement and CD14 in Escherichia coli phagocytosis over time in a human whole blood model. Alexalabeled bacteria were used to study phagocytosis after 15 and 120 min in leukocytes using flow cytometry. Cytokines including Interleukin (IL)-1b, IL-6, IL-8 and Tumor Necrosis Factor (TNF)-a were measured after 120 min using multiplex technique and Interferon (IFN)-b by ELISA. The C3 inhibitor compstatin, human genetic C5 deficient blood and a monoclonal antibody were used to outline the time-dependent role of complement (C3, C5) and CD14 in the phagocytosis process. Lepirudin blood was obtained from human healthy donors and a human genetic C5 deficient individual. The actin filament inhibitor Cytochalacin D inhibited phagocytosis of E. coli at both time-points. Phagocytosis measured after 15 min showed a strong dependency of complement, since phagocytosis in both compstatin-inhibited normal blood and in C5 deficient human blood was virtually abolished, whereas anti-CD14 alone had only a modest effect. In contrast, after 2 h the phagocytosis was virtually abolished by anti-CD14 alone, whereas complement played a minor role. Notably, the combination of compstatin and anti-CD14 was the most effective regimen to inhibit phagocytosis, approaching complete blocking at both time points. TNF-a, IL-1b, IL-6 and IL-8 were all substantially reduced when compstatin and anti-CD14 were used in combination, whereas Cytochalacin D had only a minor inhibitory effect. In contrast, IFN-b was not inhibited by compstatin or C5 deficiency, but was largely reduced by Cytochalacin D, and virtually abolished by anti-CD14 alone. Phagocytosis of E. coli in human whole blood was initially dependent on complement, succeeded by a dependency of CD14. The combination of compstatin and anti-CD14 was effective at all time points. CD14-dependent phagocytosis of E. coli seemed to be required for induction of IFN-b. http://dx.doi.org/10.1016/j.imbio.2012.08.183
Pinpointing the time and cause of fetal demise in the Crry−/− embryo Michael P. Triebwasser, Xiaobo Wu, Dennis Hourcade, John P. Atkinson Department of Rheumatology, Washington Univ. School of Medicine, Saint Louis, MO, USA
http://dx.doi.org/10.1016/j.imbio.2012.08.184 183 Pre-transplant ficolin-3 levels are associated with kidney allograft survival Jakob T. Bay 1 , Estrid Hein 1 , Søren S. Sørensen 2 , Jesper Melchior Hansen 3 , Peter Garred 1 1
Laboratory of Molecular Medicine, Department of Clinical Immunology, Sect 7631, Rigshospitalet, Copenhagen, Denmark 2 Department of Nephrology P, Rigshospitalet, Copenhagen, Denmark 3 Department of Nephrology, Herlev Hospital, Copenhagen, Denmark Ficolin-3 is an initiator of the lectin complement pathway. Activation of the complement system is a mediator of the pathophysiology of graft rejection in kidney transplantation, but the role of ficolin-3 in this process is unknown. Using a prospective study design, 527 kidney transplanted patients with a followup period of 5 years were included. 97 blood donors served as controls. Ficolin-3, C4 and C3 were measured in pre-transplant as well as in control serum samples. In the controls, deposition of ficolin-3, C4, C3 and the terminal complement complex (TCC) was measured in an assay based on acetylated albumin as matrix. The concentration of ficolin-3, C4 and C3 was increased in the patients compared with the controls (p ≤ 0.001). The ficolin-3 levels
1194
Abstracts / Immunobiology 217 (2012) 1129–1222
correlated with the serum levels of C4 and C3 both in the patients (rho: 0.44, p ≤ 0.0001, rho: 0.53, p ≤ 0.0001, respectively) and in the controls (rho: 0.30, p ≤ 0.0038, rho: 0.50, p ≤ 0.0001, respectively). The serum levels of ficolin-3 correlated with ficolin-3 deposition (rho: 0.66, p ≤ 0.0001), C4 deposition (rho: 0.45, p ≤ 0.0001), C3 deposition (rho: 0.46, p ≤ 0.0001) and formation of TCC (rho: 0.24, p = 0.0197). In a multivariable regression analysis adjusted for confounders a high ficolin-3 level was a predictor of death censored graft survival (HR = 3.18, 95% CI: 1.48–6.81) (p = 0.003), while this was not seen for C4 and C3. Ficolin-3 is involved in the pathophysiology of kidney graft rejection. The ficolin-3 levels correlated closely with the concentration and deposition of down-stream serum complement components, illustrating the importance of ficolin-3 as an initiator molecule of complement activation. http://dx.doi.org/10.1016/j.imbio.2012.08.185 184 Premolis semirufa envenomation, disease affecting rubber tappers of the Amazon: Searching for caterpillar-bristles toxic components acting on the complement system Isadora Maria Villas Boas 1 , Rute Maria Gonc¸alves de Andrade 1 , Giselle Pidde-Querioz 1 , Fernanda C.V. Portaro 1 , Carmen W. van den Berg 2 , Denise V. Tambourgi 1 1 2
Immunochemistry Laboratory, Butantan Institute, S.P., Brazil School of Medicine, Cardiff University, Cardiff, UK
Pararama, the popular name of the larval form of the moth Premolis semirufa, inhabits rubber plantations in the Amazon region and the accidental contact of the skin with the caterpillar’s bristles or cocoons results in immediate and intense heat, pain, edema and itching, which last for three to seven days. However, after multiple contacts, it may induce joint-space narrowing and bone alteration, as well as degeneration of the articular cartilage and immobilization of the affected joints. Specific treatment for this disease does not exist, but corticosteroids are frequently administered. Despite the public health hazard of P. semirufa caterpillar poisoning, little is known about the nature of the toxic components involved in the induction of the pathology. In the present study we have investigated the action of the Pararama caterpillar bristles extract on the complement system in in vitro studies. The bristle extract is able to activate the human complement system with the generation of anaphylatoxins. Aiming to isolate the component responsible for C-activation, P. semirufa’s bristles extract was submitted to FPLC gel-filtration chromatography and a fraction containing a component of 82 kDa, endowed with serine protease activity, was able to induce a strong activation of the alternative pathway and partially to activate classical and lectin pathways. This protease was able to induce the generation of C3a, C4a and C5a in human serum, not only through C-activation but also through the direct cleavage of the C3, C4 and C5 components. These data suggest that a serine protease present in P. semirufa’s bristles extract is responsible for complement activation, which can play an important role in the inflammatory process in humans after envenomation. Financial support: Fapesp, CNPq, INCTTox. http://dx.doi.org/10.1016/j.imbio.2012.08.186
185 Production of functional full-length recombinant human C1q Isabelle Bally 1 , Sarah Ancelet 1 , Christine Moriscot 1 , Florence Bonnet 2 , Regis Daniel 2 , Guy Schoehn 1 , Gerard J. Arlaud 1 , Nicole M. Thielens 1 1
Institut de Biologie Structurale, Grenoble, France Laboratoire Analyse et Modelisation pour l’Environnelment, Evry, France 2
la
Biologie
et
Human C1q is a versatile recognition protein with the ability to sense a wide variety of targets, such as antibodies, pentraxins, and numerous non-immune targets, including pathogens and damaged structures from self. Most of the C1q ligands are recognized through its globular heads, triggering sequential activation of the associated proteases C1r and C1s. Precise mapping of the specific binding sites of C1q for its various ligands and for the C1s-C1r-C1r-C1s tetramer has been precluded so far due to the unavailability of recombinant C1q, a complex protein assembled from three polypeptide chains encoded by three different genes. For the first time, we have successfully generated a stable mammalian cell line producing full-length recombinant human C1q. Purification of the recombinant protein was facilitated by the presence of an affinity tag on one of the three chains. SDS-PAGE analysis of the purified material revealed a band pattern similar to that of serumderived C1q with typical A-B and C-C dimers under non-reducing conditions and the three A, B and C chains under reducing conditions. Mass spectrometry analysis of recombinant C1q showed that the three chains had undergone the expected post-translational modifications. Importantly, electron microscopy analysis revealed the typical bouquet-like shape of the molecule. The interaction properties of recombinant C1q with immunoglobulins, C-reactive protein and the C1s-C1r-C1r-C1s tetramer were investigated by surface plasmon resonance spectroscopy. Kinetic analyses of the binding data yielded affinities comparable to those of plasmaderived C1q for all ligands. The C1 complex reconstituted from recombinant C1q and the serum-derived proenzyme tetramer was able to self-activate in a typical manner. These results demonstrate that a method allowing production of a fully functional recombinant human C1q molecule is now available, opening the way to in-depth analysis of its interaction properties by site-directed mutagenesis. http://dx.doi.org/10.1016/j.imbio.2012.08.187 186 Prolonged voltage-gated calcium channel activation by sublytic complement activation in retinal pigment epithelium, a mechanism for controlling VEGF secretion Bärbel Rohrer 1 , Kannan Genewski 2 , Olaf Strauss 2 1 2
Kunchithapautham 1 , Andreas
Medical University of South Carolina, Charleston, SC, USA Klinikum der Universitaet Regensburg, Germany
Age-related macular degeneration (AMD) is a slowly progressing multifactorial disease involving genetic abnormalities and environmental insults. Genetic studies have demonstrated that polymorphisms in different complement proteins each increase the risk for developing AMD. As the alternative pathway is continuously activated in the fluid phase, tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. To explore the hypothesis that AMD pathology results from uncontrolled activation of the alternative pathway of complement, we have utilized stable RPE monolayers challenged with H2 O2 and