Oxidative Stress Renders Retinal Pigment Epithelial Cells Susceptible to Complement-mediated Injury
2009; Elsevier BV; Volume: 284; Issue: 25 Linguagem: Inglês
10.1074/jbc.m808166200
ISSN1083-351X
AutoresJoshua M. Thurman, Brandon Renner, Kannan Kunchithapautham, Viviana P. Ferreira, Michael K. Pangburn, Zsolt Ablonczy, Stephen Tomlinson, V. Michael Holers, Bärbel Rohrer,
Tópico(s)Retinal Imaging and Analysis
ResumoUncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H2O2 (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H2O2 reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H2O2 and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD. Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H2O2 (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H2O2 reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H2O2 and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD. Age-related macular degeneration (AMD) 6The abbreviations used are: AMDage-related macular degenerationRPEretinal pigment epitheliumTERtransepithelial resistanceDAFdecay accelerating factorVEGFvascular epidermal growth factorMCPmembrane cofactor proteinMACmembrane attack complexTUNELTdT-mediated dUTP nick-end labelingFACSfluorescence-activated cell sorterNHSnormal human serumELISAenzyme-linked immunosorbent assayBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolrhrecombinant factor H. is the leading cause of blindness in the elderly (1Gehrs K.M. Anderson D.H. Johnson L.V. Hageman G.S. Ann. Med. 2006; 38: 450-471Crossref PubMed Scopus (499) Google Scholar). Clinically, AMD is categorized as “dry” or “wet.” In the dry form of the disease, deposits (drusen) develop between the retinal pigment epithelium (RPE) and the underlying basement membrane (Bruch's membrane). The loss of photoreceptor function and vision observed in patients is attributed to atrophic changes in the RPE (1Gehrs K.M. Anderson D.H. Johnson L.V. Hageman G.S. Ann. Med. 2006; 38: 450-471Crossref PubMed Scopus (499) Google Scholar, 2Hogan M.J. Trans. Am. Acad. Ophthalmol. Otolaryngol. 1972; 76: 64-80PubMed Google Scholar). Wet AMD is characterized by choroidal neovascularization extending through Bruch's membrane and the RPE into the subretinal space. Subsequent leakage of exudative fluid and blood is thought to contribute to the eventual development of fibrosis characteristic of wet AMD. AMD is hypothesized to be a progressive disease, with the dry and wet forms likely representing different points on a spectrum of disease severity. Approximately 10–15% of patients with the less severe dry AMD go on to develop wet AMD (1Gehrs K.M. Anderson D.H. Johnson L.V. Hageman G.S. Ann. Med. 2006; 38: 450-471Crossref PubMed Scopus (499) Google Scholar). age-related macular degeneration retinal pigment epithelium transepithelial resistance decay accelerating factor vascular epidermal growth factor membrane cofactor protein membrane attack complex TdT-mediated dUTP nick-end labeling fluorescence-activated cell sorter normal human serum enzyme-linked immunosorbent assay 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol recombinant factor H. Several observations suggest that uncontrolled activation of the complement cascade contributes to the development and progression of AMD. Polymorphisms in complement factor H, a circulating inhibitor of the alternative pathway of complement, are strongly associated with the development of AMD (3Edwards A.O. Ritter 3rd, R. Abel K.J. Manning A. Panhuysen C. Farrer L.A. Science. 2005; 308: 421-424Crossref PubMed Scopus (2100) Google Scholar, 4Hageman G.S. Anderson D.H. Johnson L.V. Hancox L.S. Taiber A.J. Hardisty L.I. Hageman J.L. Stockman H.A. Borchardt J.D. Gehrs K.M. Smith R.J. Silvestri G. Russell S.R. Klaver C.C. Barbazetto I. Chang S. Yannuzzi L.A. Barile G.R. Merriam J.C. Smith R.T. Olsh A.K. Bergeron J. Zernant J. Merriam J.E. Gold B. Dean M. Allikmets R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7227-7232Crossref PubMed Scopus (1714) Google Scholar, 5Haines J.L. Hauser M.A. Schmidt S. Scott W.K. Olson L.M. Gallins P. Spencer K.L. Kwan S.Y. Noureddine M. Gilbert J.R. Schnetz-Boutaud N. Agarwal A. Postel E.A. Pericak-Vance M.A. Science. 2005; 308: 419-421Crossref PubMed Scopus (2091) Google Scholar, 6Klein R.J. Zeiss C. Chew E.Y. Tsai J.Y. Sackler R.S. Haynes C. Henning A.K. SanGiovanni J.P. Mane S.M. Mayne S.T. Bracken M.B. Ferris F.L. Ott J. Barnstable C. Hoh J. Science. 2005; 308: 385-389Crossref PubMed Scopus (3570) Google Scholar). Drusen-like lesions also develop in patients with dense deposit disease, a form of glomerulonephritis caused by dysregulation of the alternative pathway (7Duvall-Young J. MacDonald M.K. McKechnie N.M. Br. J. Ophthalmol. 1989; 73: 297-302Crossref PubMed Scopus (113) Google Scholar, 8Leys A. Vanrenterghem Y. Van Damme B. Snyers B. Pirson Y. Leys M. Graefes Arch. Clin. Exp. Ophthalmol. 1991; 229: 406-410Crossref PubMed Scopus (58) Google Scholar). Analysis of the composition of drusen demonstrates that they contain important complement proteins, including C3, C5, membrane attack complex (MAC), and endogenous complement regulatory proteins (7Duvall-Young J. MacDonald M.K. McKechnie N.M. Br. J. Ophthalmol. 1989; 73: 297-302Crossref PubMed Scopus (113) Google Scholar, 8Leys A. Vanrenterghem Y. Van Damme B. Snyers B. Pirson Y. Leys M. Graefes Arch. Clin. Exp. Ophthalmol. 1991; 229: 406-410Crossref PubMed Scopus (58) Google Scholar). Mice with a genetic deletion of factor H (cfh−/− mice) accumulate C3 throughout the RPE and the outer segment layer of the neuroretina, and lose visual function faster during aging than their wild type littermates (9Coffey P.J. Gias C. McDermott C.J. Lundh P. Pickering M.C. Sethi C. Bird A. Fitzke F.W. Maass A. Chen L.L. Holder G.E. Luthert P.J. Salt T.E. Moss S.E. Greenwood J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 16651-16656Crossref PubMed Scopus (176) Google Scholar). Furthermore, in a murine model of laser-induced choroidal neovascularization, blockade of signaling by C3a and C5a reduced the production of VEGF in the eye and reduced neovascularization (10Nozaki M. Raisler B.J. Sakurai E. Sarma J.V. Barnum S.R. Lambris J.D. Chen Y. Zhang K. Ambati B.K. Baffi J.Z. Ambati J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2328-2333Crossref PubMed Scopus (520) Google Scholar). Taken together, these studies suggest that in AMD, inadequate control of the alternative pathway 1) contributes to the structural changes observed in RPE and Bruch's membrane, including drusen formation; and 2) is upstream of VEGF-mediated mechanisms. The alternative pathway of complement is continually activated in the fluid phase, and inadequate inhibition of this pathway on tissue surfaces may permit spontaneous complement activation with rapid amplification and generation of pro-inflammatory activation fragments (11Thurman J.M. Holers V.M. J. Immunol. 2006; 176: 1305-1310Crossref PubMed Scopus (363) Google Scholar). In late-onset diseases such as AMD, local regulation of the alternative pathway may gradually be overwhelmed by cellular injury or the accumulation of debris (12Atkinson J.P. Goodship T.H. J. Exp. Med. 2007; 204: 1245-1248Crossref PubMed Scopus (66) Google Scholar, 13Elward K. Griffiths M. Mizuno M. Harris C.L. Neal J.W. Morgan B.P. Gasque P. J. Biol. Chem. 2005; 280: 36342-36354Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Several environmental factors contribute to a high level of oxidative stress at the RPE layer, and oxidative injury of the RPE cells may be an important cause of AMD (14Cai J. Nelson K.C. Wu M. Sternberg Jr., P. Jones D.P. Prog. Retin. Eye Res. 2000; 19: 205-221Crossref PubMed Scopus (550) Google Scholar). Therefore, we hypothesized that oxidative stress may impair the ability of the RPE to regulate complement on its surface. In the intact adult human eye, only one cell surface complement inhibitor, membrane cofactor protein (MCP; CD46), has been identified on RPE cells (15Vogt S.D. Barnum S.R. Curcio C.A. Read R.W. Exp. Eye Res. 2006; 83: 834-840Crossref PubMed Scopus (64) Google Scholar). In the current study, we investigated whether ARPE-19 cells express the three cell surface complement inhibitors, CD46, decay accelerating factor (DAF; CD55), and CD59; and whether oxidative stress of RPE cells in culture alters surface expression of the complement inhibitory proteins or reduces inhibition of the alternative pathway on the surface of the cells by factor H. Second, we tested the hypothesis that rather than causing cell lysis, sublytic activation of complement on RPE cells induces VEGF release by these cells, which is known to compromise barrier function. The goal of these studies was to construct a model whereby oxidative stress in the eye could be linked to the inflammatory events that cause AMD, including uncontrolled activation of complement. The reagents used in these studies included pooled normal human serum (NHS (Quidel)) as a source of complement proteins. To prevent formation of the terminal complement pathway, C7-depleted serum was used (Quidel), and purified C7 (250 μg/ml; Quidel) was added to the serum in some experiments. Primary antibodies to DAF (Chemicon International), CD59 (Chemicon International), MCP (Monosan), and C3 (ICN Pharmaceuticals) were used. Species-specific secondary antibodies were from Jackson ImmunoResearch and MP Biomedicals, Inc. To block the alternative pathway of complement activation, a targeted inhibitory protein (CR2-fH) was produced as previously described (16Huang Y. Qiao F. Atkinson C. Holers V.M. Tomlinson S. J. Immunol. 2008; 181: 8068-8076Crossref PubMed Scopus (108) Google Scholar). This agent targets the inhibitory domain of factor H to sites of C3d deposition and effectively blocks alternative pathway activation. To block cell surface complement inhibition by factor H, we used a protein referred to as recombinant factor H domains 19–20 (rh-19–20) (17Ferreira V.P. Herbert A.P. Hocking H.G. Barlow P.N. Pangburn M.K. J. Immunol. 2006; 177: 6308-6316Crossref PubMed Scopus (127) Google Scholar). This protein is a recombinant form of the 19th and 20th short consensus repeats of factor H and contains the polyanion and C3b-binding region, but not the N-terminal complement regulatory region of the full-length factor H protein. It competitively blocks interaction of native factor H with cell surfaces, thereby preventing regulation of complement activation and amplification by factor H on those surfaces. The VEGFR-1/2 inhibitor, SU5416 (Chemicon), was used to block the effects of VEGF. SU5416 (Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone) is a lipophilic synthetic receptor tyrosine kinase inhibitor, which inhibits VEGFR-1/2 by binding to the ATP binding pocket within the kinase domain of the receptor. SU5416 has been shown to inhibit VEGF-dependent endothelial cell proliferation in vitro and in animal models. These experiments were performed using ARPE-19 cells, a human retinal pigment epithelial cell line that displays the differentiated phenotype of RPE cells, and form a polarized monolayer on Transwell filters (Costar) (18Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. Exp. Eye Res. 1996; 62: 155-169Crossref PubMed Scopus (1067) Google Scholar, 19Dunn K.C. Marmorstein A.D. Bonilha V.L. Rodriguez-Boulan E. Giordano F. Hjelmeland L.M. Invest. Ophthalmol. Vis. Sci. 1998; 39: 2744-2749PubMed Google Scholar). These cells were grown in Dulbecco's modified Eagle's medium, F12 (Invitrogen) with 10% fetal bovine serum, and 1× penicillin/streptomycin. In some of the experiments the cells were grown as monolayers on Transwell filters. For those experiments, fetal bovine serum was removed completely for the final 5–7 days (2–3 media changes) prior to measurements, which we have previously shown does not alter survival or monolayer formation in these cells (20Ablonczy Z. Crosson C.E. Exp. Eye Res. 2007; 85: 762-771Crossref PubMed Scopus (113) Google Scholar). Transepithelial resistance (TER) of the cell monolayer on the Transwell filters was determined by measuring the resistance across the monolayer with an EVOM volt-ohmmeter (World Precision Instruments). The value for cell monolayers was determined by subtracting the TER for filters without cells and then multiplying by the surface area of the filters. Cell monolayers were considered stable when TER was repeatedly measured as ∼40–45 Ω/cm2 (20Ablonczy Z. Crosson C.E. Exp. Eye Res. 2007; 85: 762-771Crossref PubMed Scopus (113) Google Scholar). TER measurements, which are proportional to membrane permeability, are an accepted readout for the barrier function of an RPE monolayer (18Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. Exp. Eye Res. 1996; 62: 155-169Crossref PubMed Scopus (1067) Google Scholar, 20Ablonczy Z. Crosson C.E. Exp. Eye Res. 2007; 85: 762-771Crossref PubMed Scopus (113) Google Scholar). In parallel experiments, cells were grown on plates or glass slides for ∼2 weeks after the cells reached confluence to mimic the conditions in the Transwell plates. Cells from these long-term cultures were used for flow cytometry (plates) or immunofluorescence microscopy (glass slides). As a model of oxidative stress, stable ARPE-19 cell monolayers were treated with 0.5 mm of H2O2. It has previously been reported that doses of up to 1 mm are not cytotoxic, and do not lead to disruption of barrier function in these cells (21Bailey T.A. Kanuga N. Romero I.A. Greenwood J. Luthert P.J. Cheetham M.E. Invest. Ophthalmol. Vis. Sci. 2004; 45: 675-684Crossref PubMed Scopus (211) Google Scholar). After treatment with H2O2, monolayers were exposed to 25% NHS as a source of complement proteins. In some experiments NHS was replaced with C7-deficient HS, or the complement system in NHS was inhibited by heat inactivation of NHS (30 min at 52 °C) or by treatment with CR2-fH. VEGF effects through VEGFR-1/2 activation were inhibited by treating the cells with SU5416. Surface expression of the various complement regulatory proteins was examined by flow cytometry. ARPE-19 cells were grown in long-term cultures. The cells were released from the plates by treatment with Accutase (Innovative Cell Technologies, Inc.), washed in phosphate-buffered saline, and treated with H2O2 or rH-19–20 as described in the specific experiments. For complement activation experiments, the cells were then incubated in 10% NHS at 37 °C for 1 h. Staining of surface proteins was performed by incubating the cells with primary antibody at 4 °C for 1 h, followed by washing the cells in phosphate-buffered saline, and incubating them with appropriate secondary antibodies when necessary. Cells were then washed and resuspended in 500 μl of phosphate-buffered saline, run on a FACSCalibur machine (BD Biosciences), and analyzed with CellQuest Pro software (BD Biosciences). Fluorescence was expressed in relative fluorescence units. Surface expression of DAF and CD59 on ARPE-19 cells was examined by immunofluorescence microscopy. Cells were plated on glass coverslips and grown for 2 weeks after reaching confluence to generate polarized monolayers. After washing with phosphate-buffered saline, the cells were fixed and permeabilized by immersion in cold acetone/methanol (1:1) for 5 min. Nonspecific binding was blocked by treatment of the cells with Superblock (ScyTek Laboratories), followed by incubation with anti-DAF, anti-MCP, anti-CD59, or anti-Na+K+-ATPase antibodies (1:400 for 1 h at 4 °C). The cells were washed, and then incubated with appropriate secondary antibody. As a negative control, primary antibodies were omitted. Staining of the cells was examined using a Nikon T-2000, inverted microscope equipped for confocal microscopy and analyzed with Slidebook 4.2 software (Intelligent Imaging Innovations). TUNEL (TdT-mediated dUTP nick-end labeling) staining of cells on Transwell filters was performed according to the protocol provided by the manufacturer (Roche Diagnostics) as published previously (22Kunchithapautham K. Rohrer B. Autophagy. 2007; 3: 433-441Crossref PubMed Scopus (152) Google Scholar). In short, monolayers were fixed in 2% paraformaldehyde for 2 h at 4 °C followed by TUNEL labeling and DNA strand-break visualization with fluorescein. To measure production of VEGF by the cells, cell culture supernatants were concentrated (Amicon Ultra-4, 3000 Da cutoff; Millipore), solubilized in CellLytic MT (mammalian tissue lysis/extraction reagent; Sigma), and centrifuged at 20,000 × g for 5 min. Microplates were coated with the VEGF capture antibody (Antigenix America, Inc.) and 100 μl of the concentrated supernatant was added. The captured proteins were detected with the same VEGF-specific antibody conjugated to horseradish peroxidase, followed by development with the chromogenic substrate OPD (Sigma). Product development was assayed by measuring absorbance at 492 nm. Aliquots were assayed in duplicate, and values compared with a VEGF dose-response curve. To measure C5b-9 formed during complement activation, cell supernatants were diluted 1:10 and an ELISA for C5b-9 (Quidel) was performed according the manufacturer's instruction. Cell culture supernatants were separated by electrophoresis on a 10% BisTris polyacrylamide gel (Invitrogen), and proteins were transferred to a nitrocellulose membrane. The membrane was probed with polyclonal antibody to C3 (ICN Pharmaceuticals) and antibody binding was visualized using a chemiluminescence detection kit (Amersham Biosciences). Data are expressed as mean ± S.D. Statview software was used for statistical analysis. The Fisher protected least significant difference (PLSD) was used to compare trends over time. Comparison of two conditions was performed using the Student's t test. ARPE-19 cells were grown as monolayers on Transwell filters and TER was monitored until a stable value was achieved. TER of 40–45 Ω/cm2 is the result of the establishment of an epithelial barrier function based on the formation of tight junctions. Changes in TER measurements can then be used to probe for barrier function integrity as an early marker for RPE injury. Exposure of the cells to 25% NHS (as a source of complement proteins) did not alter TER (Fig. 1A). Likewise, exposure of the cells to 0.5 mm H2O2 to induce oxidative stress did not significantly alter TER, confirming previous results by Bailey and co-workers (21Bailey T.A. Kanuga N. Romero I.A. Greenwood J. Luthert P.J. Cheetham M.E. Invest. Ophthalmol. Vis. Sci. 2004; 45: 675-684Crossref PubMed Scopus (211) Google Scholar). However, treatment of the cells with 0.5 mm H2O2 and 25% NHS together did cause a significant reduction in TER in a time-dependent manner (p < 0.01). Heat inactivation of the serum to deplete complement activity significantly attenuated the drop in TER induced by exposure of oxidatively stressed cells to serum (p < 0.01). To confirm that the effect of serum was due to complement activation and not some other heat-labile component, C7-depleted serum was used in place of complement-sufficient NHS (Fig. 1B). As with heat inactivation, C7-depleted serum was found to be ineffective in reducing TER, indicating an important role for the membrane attack complex in reducing TER. Addition of purified C7 to the C7-depleted serum restored the ability of the serum to decrease TER, confirming that the lack of effect with the C7-depleted serum was due to the absence of this factor. Application of H2O2 and serum to the apical surface caused a greater change in TER than application to the basal surface (Fig. 1C; p = 0.02), indicating that the apical surface is more susceptible to complement-mediated injury after oxidative stress. Based on TUNEL staining, apoptotic cells were not observed with any of the treatment protocols, including the combined treatment of the cells with H2O2 and serum (Fig. 1D). To induce apoptosis in stable monolayers it was necessary to increase the H2O2 concentration 10-fold. Thus, exposure of oxidatively stressed cells to serum impairs the barrier function of the cellular monolayer, but in this system the drop in TER is not due to direct cytotoxicity. We next sought to determine whether oxidative stress impairs the ability of ARPE-19 cells to regulate complement activation on their surface by performing flow cytometry to measure fixation of C3 activation fragments (such as C3b and C3d) to the cell surface as a marker of complement activation. We found that treatment of ARPE-19 cells with H2O2 caused increased surface C3b/d staining even in the absence of exogenous complement proteins (Fig. 2A). The production and release of complement components by stressed or injured epithelial cells is well described (23Pratt J.R. Basheer S.A. Sacks S.H. Nat. Med. 2002; 8: 582-587Crossref PubMed Scopus (432) Google Scholar). Next, we examined the effects of oxidative stress on complement regulation by cells that are exposed to NHS as a source of complement. Exposure of the cells to 10% NHS for 1 h caused fixation of C3b/d on the cells, but levels of surface C3b/d were higher in cells treated with H2O2 prior to exposure to serum (Fig. 2B). This increase in surface C3b/d was concomitant with a drop in intact C3 levels in the supernatant (Fig. 2C). Levels of intact (∼185 kDa) C3 were reduced by contact with cells previously treated with H2O2 in a dose-dependent manner. In other words, oxidative stress of the cells caused increased consumption of intact C3 later added to the supernatant. An ELISA for C5b-9 in cell supernatants confirmed that detectable levels of MAC were formed when serum was exposed to oxidatively stressed cells, but not when the serum was exposed to unmanipulated cells (Fig. 2D). It has previously been shown that the expression of complement regulatory proteins may be reduced on apoptotic and necrotic cells (13Elward K. Griffiths M. Mizuno M. Harris C.L. Neal J.W. Morgan B.P. Gasque P. J. Biol. Chem. 2005; 280: 36342-36354Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), which could account for the increase in C3b/d binding seen here. C3b/d deposition was compared in viable, apoptotic and necrotic populations identified by forward-scatter and side-scatter, as well as by staining with Annexin V and propidium iodide (data not shown). Increased C3b/d deposition after combined treatment with H2O2 and serum was seen on a population of apoptotic and necrotic cells always present after release of the cells using enzymatic treatment. However, more importantly, surface C3b/d was increased on viable cells, indicating that complement deposition on oxidatively stressed ARPE-19 cells is a result of non-cytotoxic changes to the surface of the cells. The data in Fig. 2 are gated to show C3b/d deposition on viable cells. It has previously been reported, based on immunohistochemistry, that human RPE cells express MCP, whereas DAF and CD59 could not be detected (15Vogt S.D. Barnum S.R. Curcio C.A. Read R.W. Exp. Eye Res. 2006; 83: 834-840Crossref PubMed Scopus (64) Google Scholar). Here, we identified the presence of all three membrane-bound inhibitors on human ARPE-19 cells, using FACS analysis (Fig. 3). Immunofluorescence microscopy was performed together with three-dimensional reconstruction to identify whether surface expression of DAF and CD59 are polarized (Fig. 4). The apical side was verified by labeling with the apical marker Na+K+-ATPase. In unmanipulated, polarized monolayers, DAF and CD59 expression was concentrated on both the apical and basolateral sides; the antibody against CD46 was found to be unsuitable for immunofluorescence.FIGURE 4DAF and CD59 are expressed on the apical and basolateral surface of ARPE-19 cells. Cells were grown to confluence on coverslips. They were then permeabilized and stained for DAF (A), CD59 (B), and Na+K+-ATPase (C). Best focus views of DAF and CD59 demonstrated apical and basolateral localization in the plasma membrane, and three-dimensional reconstructions confirmed apical and basolateral concentration of DAF and CD59 compared with Na+K+-ATPase, which is known to be only apically concentrated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Complement activation on oxidatively stressed ARPE-19 cells could be a result of increased local concentrations of complement proteins or activating proteases (11Thurman J.M. Holers V.M. J. Immunol. 2006; 176: 1305-1310Crossref PubMed Scopus (363) Google Scholar). Alternatively, we hypothesized that reduced surface levels of complement regulatory proteins could account for increased complement activation on the cells after treatment with H2O2. Surface levels of DAF decreased after treatment with H2O2 (Fig. 3, A and D) to ∼70% of those seen on unmanipulated cells (p < 0.001); whereas surface levels of MCP did not detectably change on the cells after treatment with H2O2 (Fig. 3B). Measurement of CD59 on unmanipulated cells revealed high and low expressing populations of cells (Fig. 3C). Treatment with H2O2 caused a downward shift in surface CD59 in the high-, but not the low-expressing population of cells. Mean CD59 expression for all cells fell to ∼60% of those on unmanipulated cells (Fig. 3D; p < 0.001). Mechanistically, DAF accelerates the decay of C3 convertases and reduces cleavage of C3 by the classical and alternative pathway of C3 convertases, so reduced levels of DAF are consistent with the observed increases in surface C3b/d deposition (Fig. 2). To further explore this relationship, cells were treated with H2O2 and exposed to serum, stained for both DAF and C3b/d, and examined by FACS analysis. The cells were gated on whether they expressed high or low levels of DAF. C3b/d deposition on the low DAF cells was ∼2.5-fold higher than that on the high DAF cells (Fig. 3E). CD59 inhibits formation of MAC on cell surfaces, but does not inhibit cleavage of C3. As shown in Fig. 1B, MAC formation is necessary for the drop in TER observed after treatment with H2O2 and serum. Hence, reduction in DAF or CD59 would make the RPE cells potentially more vulnerable to complement attack. In addition to the cell surface complement inhibitors, serum factor H may regulate the alternative pathway on ARPE-19 cells (24Pangburn M.K. Immunopharmacology. 2000; 49: 149-157Crossref PubMed Scopus (148) Google Scholar). To determine whether factor H contributes to regulation of complement activation on the surface of ARPE-19 cells, we incubated the cells with rH-19–20, a recombinant protein that inhibits factor H activity on cell surfaces by blocking its binding. As shown in Fig. 2, exposure of cells to 10% serum alone leads to C3b/d deposition on the cell surface. The addition of 50 μg of rH-19–20 per ml of serum caused an increase in the amount of C3b/d deposited on the surface of the cells (Fig. 5A). The mean fluorescence with rH-19–20 was on average 40% higher than that in cells without rH-19–20 (n = 3, p < 0.05). These results indicate that under normal conditions, serum-derived factor H limits complement activa
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