Complement regulatory protein CD46 protects against choroidal neovascularization in mice.

Dysregulation of the complement system is increasingly recognized as a contributing factor in age-related macular degeneration. Although the complement regulator CD46 is expressed ubiquitously in humans, in mouse it was previously thought to be expressed only on spermatozoa. We detected CD46 mRNA and protein in the posterior ocular segment (neuronal retina, retinal pigment epithelium, and choroid) of wild-type (WT) C57BL/6J mice. Cd46(-/-) knockout mice exhibited increased levels of the membrane attack complex and of vascular endothelial growth factor (VEGF) in the retina and choroid. The Cd46(-/-) mice were also more susceptible to laser-induced choroidal neovascularization (CNV). In Cd46(-/-) mice, 19% of laser spots were positive for CNV at day 2 after treatment, but no positive spots were detected in WT mice. At day 3, 42% of laser spots were positive in Cd46(-/-) mice, but only 11% in WT mice. A fully developed CNV complex was noted in both Cd46(-/-) and WT mice at day 7; however, lesion size was significantly (P < 0.05) increased in Cd46(-/-) mice. Our findings provide evidence for expression of CD46 in the mouse eye and a role for CD46 in protection against laser-induced CNV. We propose that the Cd46(-/-) mouse has a greater susceptibility to experimental CNV because of insufficient complement inhibition, which leads to increased membrane attack complex deposition and VEGF expression.

Inhibition of complement alternative pathway suppresses experimental autoimmune anterior uveitis by modulating T cell responses.

The objective of the current study was to delineate the pathway of complement activation that is crucial for the induction of experimental autoimmune anterior uveitis (EAAU). We studied the development of EAAU in melanin-associated antigen (MAA)-sensitized Lewis rats treated with antibody against C4 or factor B. Control animals received isotype IgG control. Antibody against C4 had no effect on EAAU, and all of the animals developed EAAU similar to those injected with control IgG. In contrast, EAAU was completely inhibited in all MAA-sensitized Lewis rats injected with factor B antibody. Treatment with anti-factor B antibody resulted in suppression of ocular complement activation. Adoptive transfer of T lymphocytes harvested from draining lymph nodes of donor animals treated with anti-factor B did not transfer EAAU to naïve syngenic rats. Anti-factor B antibody inhibited the ability of MAA-specific CD4(+) T cells to proliferate (in vitro) in response to MAA in a dose-dependent manner. Level of TNF-α and IFN-γ decreased in the presence of anti-factor B. Collectively, our results provide the novel finding that complement activation via the alternative pathway contributes to intraocular inflammation in EAAU, and anti-factor B-mediated inhibition of EAAU is due to diminished antigen-specific CD4(+) T cell responses to MAA. Our findings explain the interactions between the complement system and T cells that are critical for the induction of EAAU and may lead to the development of therapy for idiopathic anterior uveitis based on selective blockade of the alternative pathway.

Relationship between complement membrane attack complex, chemokine (C-C motif) ligand 2 (CCL2) and vascular endothelial growth factor in mouse model of laser-induced choroidal neovascularization.

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

Recombinant membrane-targeted form of CD59 inhibits the growth of choroidal neovascular complex in mice.

This study was designed to explore the effect of recombinant, membrane-targeted CD59 (rCD59-APT542) on the growth and size of fully developed neovascular complex using the murine model of laser-induced choroidal neovascularization (CNV). CNV was induced by laser photocoagulation in C57BL/6 mice using an argon laser, and the animals received rCD59-APT542 via intravitreal (ivt) route. Western blot analysis, immunohistochemistry, and total complement hemolytic assay demonstrated that exogenously administered rCD59-APT542 was incorporated as well as retained in RPE and choroid and was functionally active in vivo. Single ivt injection during the growth of the CNV (i.e. at day 3 post-laser) resulted in ∼79% inhibition of the further growth of neovascular complex. The size of the CNV complex was significantly (p < 0.05) reduced by the administration of rCD59-APT542 after the CNV complex has fully developed (i.e. at day 7 post-laser). Treatment with rCD59-APT542 blocked the formation of membrane attack complex (MAC), increased apoptosis and decreased cell proliferation in the neovascular complex. On the basis of results presented here we conclude that recombinant membrane targeted CD59 inhibited the growth of the CNV complex and reduced the size of fully developed CNV in the laser-induced mouse model. We propose that a combination of two mechanisms: increased apoptosis and decreased cell proliferation, both resulting from local inhibition of MAC, may be responsible for inhibition of CNV by rCD59-APT542.

Role of ocular complement factor H in a murine model of choroidal neovascularization.

The objective of this study was to explore the relationship between local (ie, ocular) complement factor H (CFH) and choroidal neovascularization (CNV) associated with wet age-related macular degeneration (AMD), a leading cause of irreversible blindness, in laser-treated C57BL/6 mice. Immunohistochemical and RT-PCR analysis of retinal pigmented epithelium (RPE)-choroid sclera revealed that the expression of CFH was down-regulated on day 1 with a dramatic increase on days 5 and 7 postlaser injury. Flat mount and Western blot analysis further revealed that membrane attack complex (MAC) expression was up-regulated on days 1 and 3 postlaser injury; however, MAC was down-regulated on days 5 and 7 postinjury but was still higher than in non-injured mice. Similar patterns for CFH and MAC were observed for RPE cells when serial paraffin sections of the laser spots were analyzed. Subretinal injection of siRNA directed against CFH resulted in a threefold suppression of CFH in the RPE and choroid without affecting either CFH levels in the liver or the functional activity of the alternative pathway in the peripheral blood. Ocular knock-down of CFH resulted in increased MAC deposition, which leads to the early onset as well as exacerbation of laser-induced CNV. In conclusion, our findings provide evidence that CFH present on RPE and choroid regulates local MAC formation that is critical for the development of laser-induced CNV.

Proteolytic cleavage of type I collagen generates an autoantigen in autoimmune uveitis.

This study was initiated to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats by melanin-associated antigen (MAA; 22-kDa fragment of type I collagen alpha2 chain) derived from rat iris and ciliary body (CB), to localize MAA within the eye, and to investigate the possible mechanism of MAA generation in vivo. The EAAU model replicates idiopathic human anterior uveitis. Lewis rats sensitized to rat MAA developed anterior uveitis, and EAAU induced by rat MAA can be adoptively transferred to naive syngenic rats by MAA-primed T cells. Animals immunized with rat MAA developed cellular immunity to the antigen. MAA was detected only in the iris and CB of the eye. Iris and CB were the major source of matrix metalloproteinase-1 (MMP-1) in the naive eye, and ocular expression of MMP-1 was up-regulated, whereas expression of tissue inhibitor of metalloproteinase 1 decreased before the onset of EAAU. These results demonstrated that EAAU can be induced by autologous MAA. Uveitogenic antigen is present only in the iris and CB of the eye, and the imbalance between MMP-1 and tissue inhibitor of metalloproteinase 1 may play a role in the generation of MAA in vivo. Collectively, the evidence presented here suggests that MAA is an autoantigen in EAAU. These observations may extend to idiopathic human anterior uveitis and facilitate the development of antigen-specific therapy.

Antigen-specific tolerance inhibits autoimmune uveitis in pre-sensitized animals by deletion and CD4+CD25+ T-regulatory cells.

The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

A strategy towards the synthesis of superhydrophobic/superoleophilic non-fluorinated polypyrrole nanotubes for oil-water separation.

Superhydrophobic/superoleophilic materials have shown great potential for applications in oil/water separation. However, practical applications of these materials are restricted due to their toxicity and complicated, expensive, and non-eco-friendly fabrication procedures. Here, we have successfully developed an easy, simple, cost-effective, and environmentally friendly strategy towards the synthesis of superhydrophobic and superoleophilic porous polypyrrole nanotubes. Such wettability has been introduced into polypyrrole by co-doping with sodium dodecylbenzenesulfonate, a surfactant for lowering surface energy and controlling the morphology of the nanotubes. These non toxic and environment friendly polymer nanotubes exhibit oil absorption capability from oil/water mixtures with a reasonable efficiency with good reusability.

Tolerance to melanin-associated antigen in autoimmune uveitis is mediated by CD4+CD25+ T-regulatory cells.

Experimental autoimmune anterior uveitis (EAAU) serves as an animal model for human idiopathic AU, the most common form of intraocular inflammation of significant morbidity whose recurrence can lead to permanent vision loss. This study was undertaken to inhibit EAAU by inducing tolerance to melanin-associated antigen (MAA) and to investigate the underlying mechanisms responsible for tolerance induction. Intravenous administration of MAA both induced tolerance and inhibited EAAU in Lewis rats. Flow cytometric analysis revealed that the proliferation of lymph node cells in response to antigenic stimulation was drastically reduced in the state of tolerance both in vivo and in vitro. Our results from co-culture experiments demonstrated that intravenous administration of MAA led to the generation of T-regulatory cells that suppress T-cell proliferative responses and induce tolerance. Expression levels of both interleukin-10 and transforming growth factor-beta2 were elevated whereas reduced levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-2 were detected in tolerance-induced animals. Tolerance was reversed by replenishing these animals with recombinant interleukin-2. Tolerance could be adoptively transferred by removing lymph node cells from tolerance-induced donors and giving them to recipient rats. Interestingly, adoptive transfer of tolerance failed when lymph nodes cells were depleted of CD4(+)CD25(+) T cells. In conclusion, T-cell nonresponsiveness because of active suppression mediated by T-regulatory cells facilitates the development of tolerance to MAA in EAAU.

Alcohol and nicotine consumption exacerbates choroidal neovascularization by modulating the regulation of complement system.

The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups. Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.

The role of complement system in ocular diseases including uveitis and macular degeneration.

In the normal eye, the complement system is continuously activated at low levels and both membrane-bound and soluble intraocular complement regulatory proteins tightly regulate this spontaneous complement activation. This allows protection against pathogens without causing any damage to self-tissue and vision loss. The complement system and complement regulatory proteins control the intraocular inflammation in autoimmune uveitis and play an important role in the development of corneal inflammation, age-related macular degeneration and diabetic retinopathy. The evidence derived from both animal models and patient studies support the concept that complement inhibition is a relevant therapeutic target in the treatment of various ocular diseases. Currently, several clinical trials using complement inhibitors are going on. It is possible that, in the near future, complement inhibitors might be used as therapeutic agents in eye clinics.

Crucial role of apoptosis in the resolution of experimental autoimmune anterior uveitis.

PURPOSE: Experimental autoimmune anterior uveitis (EAAU) serves as an animal model of human idiopathic anterior uveitis. This study was undertaken to investigate the role of apoptosis in the resolution of EAAU. METHODS: EAAU was induced in Lewis rats by bovine melanin-associated antigen (MAA). Animals were killed at different time points during EAAU, and apoptosis of the inflammatory cells within the eye was monitored. RESULTS: Flow cytometry, TUNEL staining, and light microscopy demonstrated that CD11b/c(+) and CD4(+) T cells undergo apoptosis during EAAU. Electron microscopic analysis demonstrated that the macrophages remove these apoptotic infiltrating cells from the eye by phagocytosis. Caspase-3 levels peaked during the resolution of EAAU, and the upregulation of caspase-8 and -9 preceded that of caspase-3, suggesting that both extrinsic and intrinsic pathways of apoptosis are involved. There was an inverse relationship between the expression of proapoptotic protein Bax and antiapoptotic protein Bcl-2 during EAAU. Cytochrome c was present in the cytoplasm of the infiltrating cells undergoing apoptosis. CONCLUSIONS: These results demonstrate that extrinsic and intrinsic pathways of apoptosis are involved in the resolution of EAAU. They further suggest that apoptosis followed by phagocytosis plays a critical role in the clearance of infiltrating cells from eyes with uveitis and leads to the resolution of EAAU.

The complement system plays a critical role in the development of experimental autoimmune anterior uveitis.

PURPOSE: The role of complement in ocular autoimmunity was explored in a experimental autoimmune anterior uveitis (EAAU) animal model. METHODS: EAAU was induced in Lewis rats by immunization with bovine melanin-associated antigen. Complement activation in the eye was monitored by Western blot for iC3b. The importance of complement to the development of EAAU was studied by comparing the course of intraocular inflammation in normal Lewis rats (complement-sufficient) with cobra venom factor-treated rats (complement-depleted). Eyes were harvested from both complement-sufficient and complement-depleted rats for mRNA and protein analysis for IFN-gamma, IL-10, and interferon-inducible protein (IP)-10. Intracellular adhesion molecule (ICAM)-1 and leukocyte-endothelial cell adhesion molecule (LECAM)-1 were detected by immunofluorescent staining. OX-42 was used to investigate the importance of iC3b and CR3 interaction in EAAU. RESULTS: There was a correlation between ocular complement activation and disease progression in EAAU. The incidence, duration, and severity of disease were dramatically reduced after active immunization in complement-depleted rats. Complement depletion also completely suppressed adoptive transfer EAAU. The presence of complement was critical for local production of cytokines (IFN-gamma and IL-10), chemokines (IP-10), and adhesion molecules (ICAM-1 and LECAM-1) during EAAU. Furthermore, intraocular complement activation, specifically iC3b production and engagement of complement receptor 3 (CR3), had a significant impact on disease activity in EAAU. CONCLUSIONS: The study provided the novel finding that complement activation plays a central role in the pathogenesis of ocular autoimmunity and may serve as a potential target for therapeutic intervention.

Vaccinia virus complement control protein ameliorates collagen-induced arthritic mice.

The main objective of this study was to investigate the therapeutic efficiency of recombinant vaccinia virus complement control protein (rVCP) on collagen-induced arthritis (CIA) in DBA-1/J mice. Arthritis was induced in DBA-1J mice by injecting bovine collagen emulsified in complete Freunds adjuvant. We used rVCP to block complement activation and investigated its effect on different aspects of CIA including osteoclast formation and bone destruction. The osteoclast-like cells were detected using immunohistochemistry. Joint destruction was studied using X-ray of the intact knee joints. Cartilage destruction was monitored by staining the paraffin sections with toluidine blue. ELISA was used to measure the cytokine levels in the serum. Blocking complement activation in DBA/1J arthritic mice with rVCP resulted in significant inhibition of the clinical progression of the disease and reduction in joint destruction as revealed by X-ray analysis and toluidine blue staining of the joint sections. Inhibition of complement reduced the production of proinflammatory cytokines and the number of osteoclast-like cells in arthritic joints. In conclusion, blocking of complement in CIA by rVCP inhibits the inflammation and the formation of osteoclast-like cells and reduces cartilage destruction.

Vaccinia complement control protein: multi-functional protein and a potential wonder drug.

Vaccinia virus complement control protein (VCP) was one of the first viral molecules demonstrated to have a role in blocking complement and hence in the evasion of host defense. Structurally it is very similar to the human C4b-BP and the other members of complement control protein. Functionally it is most similar to the CR1 protein. VCP blocks both major pathways of complement activation. The crystal structure of VCP was determined a little over a year ago and it is the only known structure of an intact and complete complement control protein. In addition to binding complement, VCP also binds to heparin. These two binding abilities can take place simultaneously and contribute to its many function and to its potential use in several inflammatory diseases, e.g. Alzheimer's disease (AD), CNS injury, xenotransplantation, etc. making it a truly fascinating molecule and potential drug.

Complement mediated apoptosis leads to the loss of retinal ganglion cells in animal model of glaucoma.

This study investigated the role of complement in the protection of retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma. Intraocular pressure (IOP) was elevated in the right eye of Lewis rats by laser photocoagulation (two treatments, 7days apart) of episcleral and limbal veins. Left eye did not receive laser treatment and served as control. Animals were injected with cobra venom factor every fifth day starting day 7 after first laser, to deplete the complement system. Animals were sacrificed at 6-week post-laser. Levels of C3 split products and membrane attack complex (MAC) were elevated in the retina of eyes with increased IOP and complement depletion reduced the loss of Brn3a(+) RGCs accompanied by decreased expression of GFAP and reduced MAC deposition. In complement depleted rats with increased IOP, reduced TUNEL(+) cells in ganglion cell layer, and decreased levels of active caspase-8 and active caspase-9 was observed compared to PBS treated complement sufficient rats with increased IOP. Interestingly, complement depletion also resulted in reduction of calcium influx and levels of BAD in the retinal cells of the eyes with increased IOP. Together, our results provide evidence that complement mediated apoptosis plays a pivotal role in the loss of RGCs in chronic ocular hypertension model of glaucoma.

The use of topical honey in the treatment of corneal abrasions and endotoxin-induced keratitis in an animal model.

PURPOSE: To investigate the effect of topically applied honey on intact corneas, surgically induced corneal abrasions and endotoxin induced keratitis. MATERIALS AND METHODS: The effect of honey on the cornea was investigated by application of honey on intact corneas, wounded corneas and endotoxin-induced keratitis in Lewis rats. The corneas were wounded by creating an epithelial defect using a surgical blade, and the keratitis was induced by topically applying Pseudomonas aeruginosa endotoxin to scarified corneas. After treatment rats were sacrificed and cornea harvested in each case. Corneas were processed for paraffin embedding for histological and immuno-fluorescence staining. Corneas were also harvested and processed for total ribonucleic acid (RNA) isolation for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for various growth factors and inflammatory chemokines/cytokines). RESULTS: Histological analysis revealed that no inflammation or morphological changes occurred after honey treatment in naive intact corneas. Vascular endothelial growth factor (VEGF) levels were also not altered after honey treatment. Topical application of honey to injured corneas resulted in faster epithelial healing and decreased expression of VEGF, transforming growth factor beta (TGF-ß), interferon gamma (IFN-γ), interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-α) in injured corneas. Our results also established that honey treatment reduced the inflammation in endotoxin-induced keratitis by reducing the levels of angiogenic factors (VEGF and TGF-ß), inflammatory cytokines (IL-12) and chemokines (CC chemokine receptor 5(CCR-5)). CONCLUSION: Short term use of honey on intact corneas can be safe. Honey has anti-angiogenic and anti-inflammatory properties that can be explored in several corneal inflammatory and infectious conditions.

Suppression of complement activation by recombinant Crry inhibits experimental autoimmune anterior uveitis (EAAU).

This study was initiated to explore the effect of recombinant rat Crry linked to the Fc portion of rat IgG2a (Crry-Ig) on the induction of experimental autoimmune anterior uveitis (EAAU) and on established disease. EAAU was induced in Lewis rats by immunization with bovine melanin-associated antigen (MAA). MAA sensitized animals received Crry-Ig, rat IgG2a (isotype control) or PBS separately before the onset of EAAU or after the onset of clinical disease. Administration of Crry-Ig suppressed the induction of EAAU while all animals injected with IgG2a or PBS developed the normal course of EAAU. Treatment with Crry-Ig resulted in the suppression of ocular complement activation as well as the functional activity of complement in the peripheral blood. At the peak of EAAU, levels of IFN-γ, IP-10, ICAM-1 and LECAM-1 were significantly reduced within the eyes of Crry-Ig treated Lewis rats. Importantly, administration of Crry-Ig even after the onset of EAAU resulted in a sharp decline in the disease activity and early resolution of EAAU. Collectively, the evidence presented here demonstrate that inhibition of complement by Crry-Ig results in low levels of inflammatory molecules-C3 activation products, MAC, cytokines, chemokines and adhesion molecules in the eye. Down-regulation of these molecules affects the infiltration and recruitment of inflammatory cells to the eye resulting in the inhibition of EAAU.

The role of complement in ocular pathology.

Functionally active complement system and complement regulatory proteins are present in the normal human and rodent eye. Complement activation and its regulation by ocular complement regulatory proteins contribute to the pathology of various ocular diseases including keratitis, uveitis and age-related macular degeneration. Furthermore, a strong relationship between age-related macular degeneration and polymorphism in the genes of certain complement components/complement regulatory proteins is now well established. Recombinant forms of the naturally occurring complement regulatory proteins have been exploited in the animal models for treatment of these ocular diseases. It is hoped that in the future recombinant complement regulatory proteins will be used as novel therapeutic agents in the clinic for the treatment of keratitis, uveitis, and age-related macular degeneration.