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

Abstract

Purpose: Complement-mediated damage to the retinal pigment epithelium (RPE), Bruch membrane, and choroid has been associated with pathogenesis in age-related macular degeneration (AMD). The terminal step of complement activation involves lysis of cells by the insertion of the membrane attack complex (MAC) in the plasma membrane. The hypothesis that local overexpression of human CD59 (hCD59) delivered by an adenovirus (Ad) vector to primary murine RPE cells in vitro, RPE in vivo, or cornea ex vivo protects those cells from human MAC deposition and lysis was tested.

Methods: A humanized model of MAC deposition on murine cells and murine ocular tissues including RPE and cornea was developed to permit testing of human complement regulators in mice. A recombinant adenovirus-expressing hCD59 was generated, and this virus was injected into the subretinal space of adult mice. Subsequently, eyecups from these mice were exposed to human serum, and the levels of MAC deposition on the RPE were quantified. hCD59 was also expressed on murine cornea ex vivo and in murine hepatocytes, and primary RPE cells in vitro and levels of human MAC deposition and cell lysis were measured.

Results: Adenovirus-mediated delivery of hCD59 to the RPE, cornea, or cells in culture protects those cells from human MAC deposition and MAC-mediated damage and vesiculation.

Conclusions: The humanized model of MAC deposition on murine ocular tissues allows testing of human complement regulators that may have potential in the treatment of AMD or other diseases associated with complement activation.

Figure 1

Characterization of recombinant adenovirus constructs. (A) Adenovirus constructs used in this study. Expression cassettes were cloned in the deleted E1 region of an E1/E3-deleted adenovirus. (B) Western blot analysis of cell lysates and media from infected and unin-fected cells using an anti–hCD59 antibody. (C, D) Immunocytochemistry using the hCD59 antibody of mouse hepa-1c1c7 cells infected with 1 × 10³ virus particles/cell of the indicated adenovirus. Western blot and immunocytochemistry images are representative of three independent experiments. pA, polyadenylation signal; CAG, cytomegalovirus chicken β-actin β-globin promoter; Ψ, adenovirus-packaging signal; ITR, adenovirus-inverted terminal repeat; Δ, deleted; E, early region.

Figure 2

Adenovirus-delivered hCD59 protects mouse hepa-1c1c7 cells from human complement-mediated cell lysis as measured by FACS analysis of PI uptake. (A) Cell lysis after incubation of uninfected murine hepa-1c1c7 cells with various dilutions of human serum. Data were obtained from two independent experiments (n = 4 samples for each serum dilution). (B–D) Representative FACS histograms of uninfected cells (B) or cells preinfected with 1 × 10³ vp/cell of the AdCAGGFP (C) or the AdCAGCD59 (D) adenovirus and treated with 1% NHS or HI-NHS as indicated. (E) Quantification of cell lysis from FACS histograms as the ones shown in (B–D) expressed as percentages of complement-mediated cell lysis. Data were obtained from four independent experiments (n = 10 samples in each group). (F) Cell lysis analysis of cells preinfected with different amounts of the indicated adenovirus and subjected to serum treatment as in (B–E). Data were obtained from two independent experiments (n = 4 samples for each different amount and type of adenovirus). All data are expressed as mean ± SD. ***P < 0.0001; **P < 0.001. N.S., not significant; NHS, normal human serum; HI-NHS, heat-inactivated NHS; FL-3, channel for detection of PI fluorescence.

Figure 3

Human MAC deposition and trypan blue lysis assays on mouse hepa-1c1c7 cells. (A, B) Cells were treated with 10% NHS (A) or HI-NHS (B) for 5 minutes and subjected to immunocytochemistry for MAC (red) using an antibody against a neoepitope on the C5b-9 complex followed by an appropriate Cy3-conjugated secondary antibody. Cell nuclei were labeled with DAPI (blue). (C) Cells treated with 10% NHS or HI-NHS, followed by incubation in 0.1% trypan blue solution for 5 minutes. Images are representative of three independent experiments each for the MAC and trypan blue assay. NHS, normal human serum; HI-NHS, heat-inactivated NHS.

Figure 4

Protection of mouse hepa-1c1c7 cells from human MAC deposition and ensuing lysis by adenovirus-mediated delivery of hCD59. (A, B) Cells preinfected with 1 × 10³ vp/cell of AdCAGGFP (A) or AdCAGCD59 (B) were treated with 10% NHS for 3, 5, or 7 minutes, followed by immunocytochemistry for human MAC (red) as in Figure 3. Cell nuclei were labeled with DAPI (blue). (C) Cells preinfected as in (A) and (B) with the indicated adenovirus were treated with 10% NHS for 5 minutes and incubated in 0.1% trypan blue solution for 5 minutes. Images are representative of three independent experiments each for the MAC and trypan blue assay.

Figure 5

Human MAC deposition on the RPE of murine eyecups and on primary murine RPE cells. Flatmounts of eyecups (A–C) and primary RPE cells (D, E) incubated with (+) or without (−) a complement-activating anti–mouse emmprin antibody, followed by treatment with the indicated NHS or HI-NHS concentration at 37°C for the specified time and followed by immunohistochemistry for human MAC (red). Primary RPE cell nuclei were labeled with DAPI (blue). Images are representative of at least three independent experiments each for eyecups (n = 4 eyecups for each condition) and primary RPE cells. NHS, normal human serum; HI-NHS, heat-inactivated NHS.

Figure 6

Inhibition of MAC-dependent vesiculation on primary mouse RPE cells by adenovirus-mediated delivery of hCD59. (A, B) Three days after infection with the indicated adenovirus mixture (4:1 ratio, total of 1 × 10³ vp/cell), primary mouse RPE cells were treated with the anti–mouse emmprin antibody and NHS, as in Figure 5D, and were photographed immediately after washing and fixation. Cells were coinfected with AdCAGGFP to allow easy visualization of vesicles (arrows). Images are representative of three separate experiments.

Figure 7

hCD59 expression on the RPE of in vivo adenovirus-injected mouse eyes. (A, B) Subretinal injections of the indicated adeno-virus (total, 3 × 10⁸ vp/eye) were performed in C57Bl/6J mice. Six days after injection, eyes were harvested, and eyecups were subjected to immunohistochemistry for hCD59 (red). (B, bottom) GFP expression on the RPE of the AdCAGGFP-injected eyecup. Images are representative of at least three eyes injected with each adenovirus.

Figure 8

Protection from MAC deposition on the RPE of murine eyecups and on primary murine RPE cells by in vivo or in vitro adenovirus-mediated delivery of hCD59. (A, B) Flatmounts of eyecups from mouse eyes injected subretinally with the indicated adenovirus mixtures and subjected to the MAC deposition assay, as in Figure 5A, 6 days after injection. Immunohistochemistry for human MAC (red) shows reduced staining at the area of GFP expression of the AdCACD59+ AdCAGGFP-injected eyecup (B) compared with the control injected eyecup (A). Images are representative of at least three independent experiments (n = 10 eyecups for each group of injections). (C) Quantification of MAC immunofluorescence intensity (arbitrary units) at the area of GFP expression of eyecups from eyes injected as indicated or from random areas of uninjected eyecups. Graph includes data from experiments shown in (A) and (B), Figures 5A and 5B, Supplementary Figures S3A and S3B, and data not shown. Indicated times specify length of serum treatment. Values from the 7.5-minute serum treatment are plotted on the left y-axis, whereas values from the 15-minute serum treatment are plotted on the right y-axis. (D, E) MAC immunostaining (red) on primary RPE cells subjected to the MAC deposition assay (as in Figs. 5D and 6) 3 days after infection with the indicated adenovirus mixtures. Primary RPE cell nuclei were labeled with DAPI (blue). Images are representative of three separate experiments. Data are expressed as mean ± SEM. *P < 0.01; **P < 0.001; ***P < 0.0001. N.S., not significant; NHS, normal human serum; HI-NHS, heat-inactivated NHS.

Figure 9

Immunohistochemistry for mouse emmprin on adenovirus-infected murine RPE cells. (A, B) Flatmounts of eyecups from eyes injected as in Figures 8A and 8B with the indicated adenovirus mixture, followed by emmprin immunostaining (red) 6 days after injection. Images are representative of three eyecups for each group of injection. (C, D) Double-labeling experiment showing no hCD59 (D, green)-dependent differences on anti–mouse emmprin (red) antibody-binding compared with GFP (C, green). Three days after infection with the indicated adenovirus, cells were subjected to immunocytochemistry with the first primary goat anti–mouse emmprin antibody, followed by the first secondary Cy3-conjugated donkey anti–goat IgG antibody. After washings and brief fixation of these antibodies, cells were incubated with the second primary mouse anti– hCD59 antibody, followed by the second secondary Cy2-conjugated goat anti–mouse IgG antibody. Cell nuclei were labeled with DAPI. Images are representative of three separate experiments.