Apical-To-Basolateral Transcytosis of Photoreceptor Outer Segments Induced by Lipid Peroxidation Products in Human Retinal Pigment Epithelial Cells

Retinal Cell Biology Apical-to-Basolateral Transcytosis of Photoreceptor Outer Segments Induced by Lipid Peroxidation Products in Human Retinal Pigment Epithelial Cells

Tim U. Krohne,1 Frank G. Holz,1 and Ju¨rgen Kopitz2

PURPOSE. Progressive accumulation of extracellular material at posit formation and drusen biogenesis in AMD. (Invest Oph- the basolateral side of the retinal pigment epithelium (RPE) is thalmol Vis Sci. 2010;51:553–560) DOI:10.1167/iovs.09-3755 a key event in the pathogenesis of age-related macular degeneration (AMD). The authors previously demonstrated that mod- rogressive deposition of extracellular material between the ifications with lipid peroxidation products, such as 4-hy- basolateral side of the retinal pigment epithelium (RPE) droxynonenal (HNE) and malondialdehyde (MDA), stabilize P and adjacent Bruch’s membrane is a hallmark of early-stage photoreceptor outer segment (POS) proteins against lysosomal age-related macular degeneration (AMD).1,2 Among the various degradation. Herein, they tested RPE cells for the basolateral histologically and clinically distinguishable manifestations of release of undegraded modified POS proteins. sub-RPE deposits, basal linear deposits (BLinD) and soft drusen METHODS. Polarized cultures of the human RPE cell line are recognized as specific for AMD.3,4 Both BLinD and soft ARPE-19 on permeable membranes were incubated with io- drusen represent accumulations of material described as mem- dine-125–labeled POS on the apical side. After 24 hours, radio- branous debris5 between the RPE basement membrane and the activity was quantified in apical medium, cell lysates, and inner collagenous layer of Bruch’s membrane and are, there- basolateral medium after separation of undegraded proteins by fore, considered diffuse and focal manifestations, respectively, precipitation. Protein composition of basolaterally released of the same lesion.1,3,6 The prognostic relevance of drusen size POS material was analyzed by two-dimensional gel electro- and number for the progression of AMD has been well estab- phoresis. C3a- and SC5b-9-specific enzyme-linked immunosor- lished.7–9 bent assays were used to assess complement activation by When Heinrich Mu¨ller initially coined the term drusen in modified POS. 1856, he already suggested that material deposition by the retinal pigment epithelium might contribute to their develop- RESULTS. The amount of phagocytic uptake was similar for 10 native and modified POS. Unmodified POS proteins were al- ment. Today, more than one and a half centuries later, the most completely (98.1%) degraded, whereas degradation of mechanisms of sub-RPE deposit formation are still not com- HNE- and MDA-modified POS proteins was significantly re- pletely resolved. However, proteomic studies revealed that duced (47.2%; 56.5%). Undegraded POS proteins accumulated drusen and aged Bruch’s membranes contain proteins that are intracellularly (14.2%; 12.1%) and were trafficked through the covalently modified by products of lipid peroxidation, and these modifications were more abundant in AMD eyes than in cells to be released into the basolateral medium (38.5%; 11 31.5%). Protein composition of basolaterally released material age-matched control eyes. Lipid peroxidation is a mechanism of oxidative damage that predominantly affects tissues, such as matched the original POS preparations. Protein modifications the outer retina, that are exposed to high levels of oxidative or did not confer increased complement-activating capacity to photooxidative stress and are highly enriched in polyunsatu- POS material. rated fatty acids (PUFAs). In lipid peroxidation, oxygen-derived CONCLUSIONS. Inhibition of lysosomal degradation by lipid per- free radicals interact with PUFA double bounds, resulting in oxidation-related protein modifications induces apical-to-baso- cleavage into a variety of highly reactive aldehydes, such as lateral transcytosis of undegraded POS proteins by human RPE malondialdehyde (MDA) and 4-hydroxynonenal (HNE).12 Once cells in vitro. This mechanism may contribute to sub-RPE de- formed, these lipid peroxidation products rapidly attach covalently to nearby proteins by forming adducts predominantly with cysteine, lysine, and histidine residues, a process that may interfere with protein functionality.12 Furthermore, modified From the 1Department of Ophthalmology, University of Bonn, Bonn, Germany; and the 2Department of Molecular Pathology, Univer- proteins, also referred to as advanced lipid peroxidation end- products (ALE), are prone to aggregation by cross-linking of the sity of Heidelberg, Heidelberg, Germany. 12 Supported by the German Research Foundation (DFG) Grant KR adducts. By these mechanisms, lipid peroxidation products 2863/6-1 (TUK); DFG Priority Program “Age-related macular degener- cause severe damage to cellular proteins and other macromol- ation” (SPP 1088) Grant HO 1926/2-1 (JK, FGH); the University of Bonn ecules and thus impair cellular functions and survival. BONFOR Program Gerok Fellowship (TUK); the German Ophthalmo- We previously detected modifications with MDA and HNE logical Society Alcon Retina Scholarship (TUK); and the Dr. Eberhard on proteins isolated from RPE-derived lipofuscin, thus demon- und Hilde Ru¨diger Foundation (TUK). strating the occurrence of these substances in the RPE lysoso- Submitted for publication March 23, 2009; revised June 13, 2009; mal compartment and suggesting their contribution to lysoso- accepted August 6, 2009. 13 Disclosure: T.U. Krohne, None; F.G. Holz, None; J. Kopitz, mal dysfunction and lipofuscinogenesis. Indeed, we None observed that modifications of photoreceptor outer segment Corresponding author: Tim U. Krohne, Department of Ophthal- (POS) proteins with MDA and HNE significantly reduced their 14 mology, University of Bonn, Ernst-Abbe-Strasse 2, D-53127 Bonn, Ger- degradation by RPE cells in vitro. After normal phagocytic many; [email protected]. uptake, modified POS proteins exhibited increased stability

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against the proteolytic attack by lysosomal enzymes, resulting Paracellular Permeability Assays in their intracellular accumulation and long-term storage. In the present study, we further analyzed the processing of modified To assess RPE cell monolayer integrity, we measured transepithelial POS by human RPE cells in vitro and tested whether undegrad- electrical resistance (TEER) across cell monolayers using a volt-ohm- ⍀ 2 able POS components are disposed into the sub-RPE space, a meter (Millicell-ERS; Millipore, Billerica, MA). TEER ( cm ) was calcu- ⍀ 2 process that may contribute to local immune processes and lated as electrical resistance ( ) multiplied by cell culture area (cm ). sub-RPE deposit formation in vivo. Values derived from laminin-coated membranes without cells were subtracted as background. RPE monolayers in our culture system reached TEER values of 63 ⍀cm2 (Ϯ7) after 7 days of culture. MATERIALS AND METHODS Paracellular permeability was analyzed by means of marker dye leakage. We used FITC-dextran (molecular weight, 40 kDa; Sigma- RPE Cell Culture Aldrich) as the marker substance because it was previously shown not The human, spontaneously stable RPE cell line ARPE-19 (ATCC CRL- to be transported transcellularly by RPE cells.17 FITC-dextran was 2302) was obtained from American Type Culture Collection (Rockville, added to the medium on the apical cell side of membrane-cultured RPE MD) and was maintained in a 1:1 mixture of Dulbecco’s modified cell monolayers at a concentration of 100 ␮M. After 2 hours on a Eagle’s medium (DMEM) and Ham’s F-12 medium (PAA Laboratories, horizontal shaker (40 rpm) in a cell culture incubator, FITC-specific Co¨lbe, Germany) containing 2.5 mM L-glutamine, 0.5 mM sodium fluorescence in the basolateral medium was quantified using a micro- pyruvate, and 17.5 mM D-glucose and supplemented with 100 U/mL plate reader (excitation wavelength, 485 nm; detection wavelength, penicillin, 100 ␮g/mL streptomycin, and 10% heat-inactivated fetal 528 nm). Apparent permeability coefficient (Papp; cm/s) was calculated bovine serum (PAA Laboratories) at 37°C in a humidified atmosphere as marker dye flux across the cell monolayer (␮g/s) divided by cell 2 of 5% CO2 in air. Permeable polyester membrane cell culture inserts culture area (cm ) and initial marker dye concentration in the apical (Transwell-Clear, diameter 12 mm, pore size 0.4 ␮m; Corning, Lowell, medium (␮g/cm3). Experiments were performed in triplicate, and MA) were coated with 2 ␮g/cm2 laminin (Sigma-Aldrich, Munich, results are presented as mean (ϮSD). Germany) for 2 hours at 37°C according to the manufacturer’s recommendation. Postconfluent stationary cells were trypsinized, seeded on Electron Microscopy Studies the membranes at a density of 1.66 ϫ 105/cm2 as previously de- For transmission electron microscopy (TEM) analysis, cells were fixed scribed,15,16 and cultured for 7 additional days before use in experi- with Karnovsky’s solution in PBS (pH 7.3), postfixed in 1% OsO , ments. The upper and lower compartments of the cell culture inserts 4 dehydrated in ethanol, and embedded (Durcupan ACM Fluka; Sigma- were filled with 500 ␮L and 1500 ␮L culture medium, respectively, to Aldrich). Ultrathin sections were stained with uranyl acetate and lead achieve hydrostatic equilibrium. Under these culture conditions, RPE citrate and were examined with a transmission electron microscope cells formed monolayers of polygonal cells and exhibited features of (Zeiss EM 109; Carl Zeiss, Oberkochen, Germany). For scanning elec- polarized differentiation such as apical microvilli formation (Fig. 1). tron microscopy (SEM) analysis, the specimens were fixed and dehydrated as described and then were treated with hexamethyldisilazane, air dried, sputtered with gold, and examined with a scanning electron microscope (Zeiss DSM 950; Carl Zeiss).

POS Isolation Porcine eyes were obtained from a local slaughterhouse, and POS was isolated as previously described.18,19 Brieﬂy, the eyes were dissected at the equator, and the retinas were removed. Isolated retinas were

agitated in KCl buffer (0.3 M KCl, 10 mM HEPES, 0.5 mM CaCl2,1mM MgCl2) with 48% wt/vol sucrose at pH 7.0 and were centrifuged at 5000g for 5 minutes. Supernatant containing the POS was ﬁltered through sterile gauze, diluted 1:1 with KCl buffer without sucrose, and centrifuged at 4000g for 7 minutes The pelleted POS were repeatedly washed in PBS and stored at Ϫ80°C until use.

HNE and MDA Modification HNE and MDA were prepared by acid hydrolysis from (E)-4-hydroxynonenal-dimethylacetal (Sigma-Aldrich) and 1,1,3,3-tetrame- thoxypropane (Sigma-Aldrich), respectively, and were used for POS protein modification as described.14 Briefly, POS were reacted with freshly prepared 5 mM HNE or 20 mM MDA at room temperature overnight on a shaker. Unbound HNE and MDA was removed by repeated washes in PBS. Total protein content was measured by Bradford assay (Bio-Rad Laboratories, Munich, Germany). Modified POS were stored at Ϫ80°C until use.

Radioactive Labeling of POS Proteins POS (200 ␮g total protein) were incubated at room temperature for 10 minutes in PBS with 8.4 MBq carrier-free sodium iodide-125 (Na125I; FIGURE 1. In (A) transmission and (B) scanning electron microscopy studies, ARPE-19 cells cultured on laminin-coated permeable polyester Amersham-Pharmacia Biotech, Freiburg, Germany) in the presence of membranes exhibited monolayer formation and morphologic features one iodination reagent bead (Iodo-Bead; Pierce, Rockford, IL). Radio- of polarized differentiation such as apical microvilli development. Scale labeling was stopped by removal of the bead. Labeled POS were bar, 10 ␮m. separated from free iodine by repeated washes in PBS and were

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FIGURE 2. Polarized RPE cells on permeable membranes were incubated on their apical side with unmodified POS (Control), MDA-modified POS (MDA), HNE-modified POS (HNE), or unmodified POS and am-

monium chloride (NH4Cl). (A) After 0 and 24 hours of incubation, the radioactive POS label was quantiﬁed in extracts of total degraded (TCA- soluble) and undegraded (TCA-insoluble) proteins from apical medium, cell lysates, and basolateral medium. At 0 hour, labeled proteins were almost exclusively detected as undegraded proteins in the apical medium, whereas 24 hours later, both degraded and undegraded POS proteins were found in all compartments of the cell culture system. The intracellular, degraded protein fraction was also detectable but too small to be visible in the diagram. (B)Toil- lustrate the distribution of only the phagocytosed POS proteins, the fraction of apical, undegraded proteins was excluded in this alternative pre- sentation of results.

immediately used for experiments. Specific radioactivity was 10.4 For subsequent SDS-PAGE, the IPG strip was applied to a precast SDS MBq/mg protein. gradient gel (8–18 ExcelGel; Amersham Pharmacia Biotech), and separation was performed on a horizontal electrophoresis unit (Multiphor Transcytosis Assay II; Amersham Pharmacia Biotech). Phosphor screens were exposed to Polarized RPE cell cultures on permeable membranes were incubated the dried gels and scanned on a phosphor imaging system (Cyclon Plus with radiolabeled POS (10 ␮g/cm2 total POS protein; 11.7 kBq) in Phosphor Imager; PerkinElmer, Rodgau, Germany) using appropriate complete culture medium. In analogy to the in vivo situation, POS software (OptiQuant; AcroMetrix, San Francisco, CA). were added only to the upper (apical) compartment of the cell culture insert. Addition of 20 mM ammonium chloride (Sigma-Aldrich), acting C3a and SC5b-9 Enzyme-Linked as an inhibitor of lysosomal catabolic functions by raising lysosomal Immunosorbent Assays 20 pH , to both apical and basolateral medium was used in controls. After Human blood was drawn in anticoagulant-free tubes and was sedi- 24 hours, medium in the upper and lower (basolateral) compartment mented at room temperature for 30 minutes. Serum was separated by was collected separately. Cells were subjected to extensive washing to centrifugation (2000g, 5 minutes), and 100 ␮L serum was incubated remove noninternalized POS and subsequently were lysed in 0.5 M with 20 ␮L POS (10 ␮g total protein) in PBS. In parallel, serum was NaOH. In medium samples and cell lysates, proteins were precipitated incubated with 20 ␮L PBS without POS to serve as vehicle control. by addition of an equal volume of 10% trichloroacetic acid (TCA), and After 2 hours at 37°C, complement activation was assessed by detec- the resultant TCA-soluble (degraded) and TCA-insoluble (undegraded) tion of C3a and SC5b-9 in 1:104 and 1:10 diluted samples, respectively, protein fractions were separated by centrifugation (16,000g, 10 min- using specific enzyme-linked immunosorbent assays (Quidel, San Di- utes). Degraded and undegraded protein fractions were transferred to ego, CA) according to the manufacturer’s recommendation. Experi- liquid scintillation cocktail (Ultima Gold; PerkinElmer, Rodgau, Ger- ments were performed in triplicate, and results are presented as mean many), and radioactivity was measured in a liquid scintillation counter (ϮSD). (Tri-Carb 2500TR; PerkinElmer). Experiments were performed in triplicate, and results are presented as mean (ϮSD). RESULTS Two-Dimensional Gel Electrophoresis POS Phagocytosis and Degradation Transcytosis assays were performed exactly as described except that serum content of the culture medium was reduced to 1% during the Polarized RPE cell cultures on permeable membranes were 24-hour POS incubation period to reduce interference of high-abun- incubated with radiolabeled POS in the apical medium. At the dance serum proteins with the detection of POS proteins. For isoelec- beginning of the experiment, we confirmed that labeled POS tric focusing (IEF), basolateral medium samples resulting from these proteins in the apical medium consisted almost exclusively of transcytosis experiments and samples of the original radiolabeled POS undegraded proteins (99.6% of total POS protein; Fig. 2A). preparations were subjected to TCA protein precipitation. Proteins After 24 hours of incubation, approximately half the POS pro- were extracted from the TCA-insoluble fraction in 7 M urea, 2 M teins were phagocytosed by the cells, as determined by reduc- thiourea, 4% Triton X-100, 65 mM dithiothreitol, and 0.8% carrier tion of the apical undegraded POS protein fraction (Fig. 2A). ampholyte (Pharmalyte; Sigma-Aldrich). Equal amounts of protein were MDA and HNE modifications did not affect the amount of POS separated in an immobilized pH gradient (IPG) strip (Immobiline Dry- phagocytosis (51.8%; 50.9% of total POS protein) compared Strip, pH 3–10, 11 cm; Amersham Pharmacia Biotech, Freiburg, Ger- with unmodified POS (48.5%). When confining the analysis to many) using an IEF system (IPGphor; Amersham Pharmacia Biotech). phagocytosed POS proteins (by excluding the apical unde-

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nium chloride-treated cells, the amount of undegraded proteins was significantly higher, but the distribution between intracellular (42.3%) and basolateral (55.1%) compartments followed the same trend. To analyze whether the difference in basolateral occurrence of undegraded POS proteins between the unmodified and modified POS groups resulted from paracellular leakage in the latter, we compared paracellular permeability of cell monolayers for the marker substance FITC-dextran (Fig. 3). We detected no significant differences in apparent permeability co-

efficients (Papp) after 24 hours of incubation with MDA- and HNE-modified POS (4.4 ϫ 10Ϫ6 cm/s; 3.8 ϫ 10Ϫ6 cm/s) compared with unmodified POS (4.3 ϫ 10Ϫ6 cm/s). Similarly, TEER values after POS incubation were similar in all groups (data not shown). In summary, these results suggests that the observed FIGURE 3. Paracellular permeability of membrane-cultured RPE cell apical-to-basolateral translocation of undegraded modified POS monolayers for FITC-dextran was assessed after 24 hours of incubation was not a result of paracellular leakage but rather of transcel- with unmodified POS (Unmodified), MDA-modified POS (MDA), HNE- lular transport processes such as transcytosis. modified POS (HNE), or unmodified POS and ammonium chloride (NH Cl). Laminin-coated but cell-free membranes served as positive 4 Characterization of Modified POS Material control (Control). Papp, apparent permeability coefficient. To assess whether transcytosis and basolateral release of modified POS material was selective for specific POS proteins, we graded protein fraction; Fig. 2B), unmodified POS proteins compared protein compositions of the original, purified POS were almost completely degraded (98.1% of total phagocy- preparations with those of basolaterally released, undegraded tosed POS protein), whereas protein degradation of MDA- and POS material using two-dimensional gel electrophoresis (Fig. HNE-modified POS was significantly reduced (56.5%; 47.2%). 4). Given that the previous experiment had already established Unspecific inhibition of lysosomal enzymes by ammonium that virtually no undegraded POS proteins are released after chloride also reduced degradation (2.6%). In each group, total incubation with unmodified POS, the two-dimensional gel anal- ϫ 5 radioactivity of all protein fractions combined (6.9 10 cpm) ysis was confined to the groups of HNE- and MDA-modified was in good agreement with radioactivity of initially added POS POS. The use of radiodetection for imaging of the gels ensured ϫ 5 (7.0 10 cpm), confirming that protein loss during the that the visualized proteins were derived exclusively from the experimental procedure was minimal. radiolabeled POS. The analysis demonstrated a virtually identical protein com- Basolateral Release of Undegraded POS position of POS material in the basolateral medium and the In cells incubated with MDA- and HNE-modified POS, some original POS preparations, indicating that POS proteins are undegraded POS proteins accumulated intracellularly (12.1%; basolaterally released in a nonselective fashion. Because pro- 14.2% of total phagocytosed POS protein; Fig. 2B). In both tein extraction for this analysis was performed by TCA precip- groups, however, most undegraded POS proteins were de- itation, low-molecular weight protein degradation fragments tected in the basolateral medium (31.5%; 38.6%). In ammo- were eliminated in the process and, thus, do not appear on the

FIGURE 4. In RPE cells incubated with modified POS, protein compositions of POS protein extracts were analyzed by two-dimensional gel electrophoresis. The original HNE- and MDA-modified POS preparations (A, B) added to the apical medium at the start of incubation period were compared with POS material extracted from the basolateral medium after 24 hours of incubation with HNE- and MDA-modified POS (C, D). Gels were imaged by radiodetection to visualize radiolabeled POS proteins.

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FIGURE 5. Complement activation was assessed by quantification of complement factor 3 activation frag- ment C3a and terminal complement complex SC5b-9 in human serum after incubation with vehicle alone (Control), unmodified POS (Unmodi- fied), HNE-modified POS (HNE), and MDA-modified POS (MDA).

gels. Larger protein degradation fragments and cross-linked that POS proteins modified with HNE and MDA become stabi- protein aggregates were not detected because of their random lized against the proteolytic attack in the RPE lysosomal com- molecular weights and isoelectric points. partment and reduce activities of lysosomal proteases by a Finally, we evaluated the capability of modified POS mate- competitive mechanism.14 Our finding that phagocytosis of rial to activate the complement system. We measured genera- modified POS was unaffected while internalized POS were no tion of two key complement components, C3 activation frag- longer degraded raised the question of how the RPE cells deal ment C3a and terminal complement complex SC5b-9, after with the increasing intracellular load of modified POS material. incubation of POS with human serum (Fig. 5). Unmodified, In the present study, we addressed this question in an in vitro HNE-modified, and MDA-modified POS significantly amplified model using radiolabeled POS. the generation of C3a (4.5-, 4.7-, 4.2-fold) and SC5b-9 (2.2-, 1.8-, Polarized differentiation and sufficient barrier properties of 2.0-fold) compared with vehicle control. However, comple- the cellular monolayer are prerequisites for meaningful analysis ment activation was not increased for modified POS compared of transcytosis processes in vitro. Our experiments were per- with native POS. formed using the cell line ARPE-19, a nontransformed human RPE cell line that forms polarized monolayers and intercellular tight junctions under the culture conditions used in this DISCUSSION study15,16 and that is capable of phagocytosis and degradation 33 Transcytosis is the vesicular transport of molecules and parti- of POS in vitro. Although prolonged culture periods and the 21 use of specialized culture media were reported to further cles across cellular barriers. After uptake by endocytosis on 15 one side of the cell, the cargo is moved through the cell and increase barrier properties of this cell line, we found our released on the opposite side by fusion of the transport vesicle culture conditions to be sufficient to induce morphologic fea- with the plasma membrane. Transcytosis is a common feature tures of a polarized RPE monolayer and barrier properties that of various epithelial cell types, such as intestinal epithelium, resulted in almost complete blockage of paracellular passage of microvascular endothelium, and respiratory epithelium.21 Sev- undegraded POS proteins from the apical to the basolateral eral examples of transcytosis of phagocytosed particles across compartment (99.5%; Fig. 2, control group). The degree of POS epithelial barriers exist,22–25 and regulated exocytosis of phago- protein modifications yielded by the methods used in this study 14 somal contents by macrophages has also been reported.26 In has been reported previously and was chosen to correspond RPE cells, transcytosis of undegraded residual material derived to the range of carbonyl modifications detected in human cells 34 from phagocytosed POS has previously been described mor- in vivo, such as aged human erythrocytes, because quantita- phologically in different animal models.27,28 In the present tive data for human POS modifications in vivo is not yet avail- study, we provide evidence that polarized human RPE cells in able. vitro make use of transcytosis of undigested POS material when In accordance with our previous results, we found phago- lysosomal capacity has been exceeded and that these processes cytosis to be unaffected by POS modifications, as evident from can be induced by lipid peroxidation-related modifications of similar amounts of POS uptake after incubation with unmodi- phagocytosed POS proteins. fied, MDA-, and HNE-modified POS. After phagocytosis, unmod- Several lines of evidence indicate an involvement of lipid ified POS proteins were almost completely degraded. In con- peroxidation processes in the development of the outer retinal trast, degradation of modified POS proteins was markedly changes observed in AMD. Protein modifications with the lipid reduced; consequently, a significant amount of undegraded peroxidation product carboxyethylpyrrole (CEP) were de- POS proteins accumulated within the RPE cells. The predom- tected in drusen isolated from AMD eyes,11 HNE-derived pro- inant fraction of undegraded POS proteins, however, was de- tein modifications in retinal tissue have been demonstrated as tected in the medium on the basolateral side of the cells, a result of light damage and aging,29,30 and peroxidized lipids indicating translocation of undegraded POS material across the have been shown to accumulate in Bruch’s membrane with RPE cell monolayer. Similar results were obtained when cells age.31 HNE and MDA represent two of the most abundant were incubated with unmodified POS in the presence of am- products of lipid peroxidation and are predominantly gener- monium chloride, an inhibitor of intralysosomal proteolysis.20 ated from docosahexaenoic acid (MDA only), arachidonic acid, Paracellular permeability assays did not yield evidence for in- and linoleic acid,12 the three most prevalent PUFAs in POS creased paracellular leakage and thus imply transcellular trans- membranes.32 Our previous observation that RPE-derived lipo- port as the underlying mechanism. Protein composition of fuscin contains proteins modified with HNE and MDA docu- basolaterally released material from RPE cells incubated with ments that these substances occur within the RPE lysosomal modified POS matched that of the original HNE- and MDA- compartment and suggests their contribution to lysosomal dys- modified POS preparations, suggesting that the apical-to-baso- function and lipofuscinogenesis.13 Indeed, we demonstrated lateral POS transport was not specific for selected proteins but

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may involve basolateral exocytosis of complete POS material. resultant generation of C3a and SC5b-9 have been reported.46 In summary, our findings demonstrate that RPE cells in vitro Other studies, however, detected no, or only minor, comple- use transcellular apical-to-basolateral transport (transcytosis) of ment activation by oxidized LDL.47–49 We analyzed the effects undegradable POS proteins with subsequent release into the of modified POS on key components of the complement sys- sub-RPE space. tem and found no evidence that modifications with HNE and Because of the remaining nonphagocytosed POS in the MDA confer increased complement-activating capacity to POS apical medium at the end of the experiments, apical release of material. undegraded POS proteins could not be tested for and, thus, Although these results suggest that lipid peroxidation-re- cannot be excluded. However, our data indirectly indicate that lated modifications may not directly act as complement activa- apical release did not contribute significantly, if it did at all, to tors, they could, however, generate new antigens that induce the overall release of undegraded POS material. In cells incu- the formation of autoantibodies and subsequent immune com- bated with unmodified POS, degradation of phagocytosed POS plex-mediated complement activation. Autoantibody formation proteins was almost complete; hence, all remaining unde- against lipid peroxidation-derived modifications on LDL has graded POS proteins in the apical medium (51.5%) most likely been demonstrated, and the resultant immune complexes have resulted from nonuptake because of exceeded phagocytic ca- been implicated in chronic local inflammation in atherosclero- pacity. The amount of undegraded apical POS proteins was sis.50 A role for immune complex-mediated complement acti- similar for MDA- and HNE-modified POS (48.2%; 49.1%), sug- vation in AMD has previously been suggested based on the gesting that this assumption applies as well to these groups colocalization of immunoglobins and C5b-9 in drusen.51 Fur- given their previously established similar rate of phagocytic thermore, serum from patients with AMD has been reported to POS uptake.14 These results imply that in our experimental contain increased levels of autoantibodies against the lipid setting, release of undegraded POS occurred predominantly on peroxidation product CEP,52 and mice immunized with CEP- the basolateral cell side. modified albumin develop sub-RPE complement depositions Previous studies have demonstrated that lipid peroxidation and RPE cell damage.53 Clearly, more research is needed to products interfere with the cellular vesicle transport machin- identify the mechanisms that trigger complement activation in ery by blocking vesicular fusion events or inducing trafficking patients with AMD. defects.35–38 Interestingly, in an experimental model similar to In the present study, we provide evidence that human RPE ours, simultaneous challenge of RPE cells with POS and lipid cells in vitro use apical-to-basolateral transcytosis as a means of peroxidation product/protein complexes (oxidized LDL) also disposing undegraded POS material in situations when lysoso- resulted in the inhibition of POS degradation and the release of mal capacity has been exceeded. Furthermore, we demon- phagosomal contents such as pro-cathepsin D into the extra- strate that lipid peroxidation-related modifications of POS pro- cellular space.35,37 Furthermore, intracellular accumulation of teins are capable of inducing this process, most likely by their oxidized LDL inhibited phagosome maturation and phago- previously demonstrated capacity to induce lysosomal dysfunc- some/lysosome fusion,35,38 raising the possibility that similar tion in RPE cells. Future studies will have to examine whether effects of HNE- and MDA-modified POS proteins may contrib- these mechanisms can be generalized from the in vitro to the ute to POS transcytosis in our model. However, our observa- in vivo situation and to determine their role in sub-RPE deposit tion that lysosomal inhibition by ammonium chloride alone is formation in AMD. The further elucidation of the mechanisms sufficient to induce POS transcytosis, even in the absence of of drusen biogenesis and associated complement activation is lipid peroxidation-related protein modifications, argues for ly- crucial for the understanding of AMD pathogenesis and the sosomal dysfunction as the predominant underlying factor for identification of novel therapeutic targets for prophylaxis and the induction of transcytosis in our system. treatment of this blinding disease. Whether and to what extent RPE cell transcytosis contrib- utes to AMD pathogenesis in vivo remains to be elucidated. Acknowledgments The lack of detectable POS-specific proteins in drusen, at least in one study,11 and differences in the cholesterol composition The authors thank Claudine Strack and Sigrun Himmelsbach for expert between drusen and POS39 may argue against a substantial technical assistance. direct contribution of unprocessed POS material to sub-RPE deposits. However, damage by lipid peroxidation and related References mechanisms as well as intracellular processing during transcy- 1. Green WR, Enger C. Age-related macular degeneration histopatho- tosis could result in extensive alterations to POS components logic studies: the 1992 Lorenz E. Zimmerman Lecture. Ophthal- that might have precluded their detection in drusen in the past. mology. 1993;100(10):1519–1535. Furthermore, modified POS components may not necessarily 2. Abdelsalam A, Del Priore L, Zarbin MA. Drusen in age-related contribute quantitatively to drusen material but rather trigger macular degeneration: pathogenesis, natural course, and laser pho- local immune processes, such as complement activation, that tocoagulation-induced regression. Surv Ophthalmol. 1999;44(1): result in chronic low-grade inflammation with progressive dep- 1–29. osition of additional, inflammation-related material in the sub- 3. Sarks S, Cherepanoff S, Killingsworth M, Sarks J. Relationship of RPE space and proangiogenic stimuli that eventually induce basal laminar deposit and membranous debris to the clinical pre- choroidal neovascularization.40–42 sentation of early age-related macular degeneration. Invest Oph- Complement activation has been implicated in the patho- thalmol Vis Sci. 2007;48(3):968–977. genesis of AMD based on the detection of activated comple- 4. Curcio CA, Millican CL. Basal linear deposit and large drusen are ment components such as C3b and C5b-9 in drusen40–43 and specific for early age-related maculopathy. Arch Ophthalmol. 1999;117(3):329–339. the association of AMD with genetic variants in the comple- 44,45 5. Sarks JP, Sarks SH, Killingsworth MC. Evolution of geographic ment factors CFH, CFB, C2, and C3. Damaged cellular atrophy of the retinal pigment epithelium. Eye. 1988;2(5):552– components, such as modified lipids and proteins, sequestered 577. from compromised RPE cells into the sub-RPE space have been 6. 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