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(Investigative Ophthalmology and Visual Science. 2008;49:295-303.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0612
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Murine Cytomegalovirus Infection and Apoptosis in Organotypic Retinal Cultures

Ming Zhang, Brendan Marshall, and Sally S. Atherton

From the Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. An organotypic retinal culture model was used to determine the pattern of murine cytomegalovirus (MCMV) infection and whether apoptosis is induced in MCMV-infected cultured retinas.

METHODS. Retinas harvested from C57BL/6 mice were individually cultured at 37°C on 3-µm filter inserts placed in 24-well plates. Some retinas were infected with MCMV (5 x 105 PFU/well). At days 4, 7, and 11 after infection (pi), the culture medium and cultured retinas were collected for examination.

RESULTS. Replicating virus was recovered and viral early antigen (EA)- and late antigen (LA)-positive cells were observed in the MCMV-infected retinal cultures. Most MCMV-infected cells were glia and horizontal cells. Infection resulted in atrophy of the photoreceptor cells and cytomegaly. Apoptosis of uninfected bystander cells, including photoreceptor cells and horizontal cells, was observed. TNF-{alpha} was produced by activated microglia during MCMV infection of the retina. Mouse apoptosis microarray studies, caspase activity studies, and RT-PCR studies showed that the genes involved in both the death receptor–mediated apoptotic pathway and the mitochondrial pathway were upregulated.

CONCLUSIONS. Many aspects of MCMV infection of retinal cultures parallel those observed during MCMV retinitis in mice. Thus, this in vitro system may be used to explore the role of apoptosis of uninfected retinal cells and the contribution of cytokines and other modulators to the pathogenesis of CMV retinitis.

Cytomegalovirus (CMV) infection in immunocompetent individuals usually causes a self-limiting, subclinical disease. However, in adult and pediatric patients who are immunosuppressed (IS) as a result of chemotherapy, malignancy, or the acquired immunodeficiency syndrome, CMV infections are a significant cause of morbidity and mortality.1 2 3 4 5 6 7 8 Before the introduction of highly active antiretroviral therapy (HAART), CMV retinitis was the most common sight-threatening opportunistic infection observed in patients with AIDS.4 5 6 7 8 CMV retinitis is also observed in newborn infants with congenital CMV infection.9 10 11 Although the ophthalmic features of CMV retinitis have been well characterized, the pathogenesis of ocular CMV infections has not been fully determined.

Although necrosis is a prominent feature of CMV retinitis, apoptotic cells have been observed during microscopic examination of biopsy specimens of eyes of patients with HCMV retinitis.12 13 In the mouse model of CMV retinitis used in our laboratory, both apoptotic cells and necrotic cells have been observed in the retina during the evolution of MCMV retinitis.14 15 16 Immunohistochemistry studies14 15 and electronic microscopic examinations16 have shown that most apoptotic cells are not virus infected. However, the apoptosis-inducing factor(s) and possible apoptosis pathway(s) involved remain to be identified. In this study, an in vitro MCMV-infected organotypic retinal culture system which has many features paralleling those observed during MCMV retinitis in mice was used to determine the pattern of MCMV infection; to see whether apoptosis occurs in MCMV-infected cultured retinas; and if apoptosis occurs, to identify the likely mechanism for its induction.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Virus and Virus Titration
The original stock of MCMV (K181 strain) was a generous gift of Edward S. Mocarski (Stanford University School of Medicine, Stanford, CA). Stocks of K181 were prepared in mouse embryo fibroblast (MEF) cells (BioWhittaker, Walkersville, MD) grown in tissue culture medium containing 5% fetal calf serum (HyClone, Logan, UT). The titer of MCMV in virus stocks was determined by duplicate plaque assay on monolayers of MEF cells and virus stocks were stored at –70°C. A fresh aliquot of stock virus was thawed and diluted to the appropriate concentration immediately before each experiment.

Mice
Female 3- to 4-week-old C57BL/6 mice (Taconic Inc., Germantown, NY) were used in all experiments. The mice were housed in accordance with National Institutes of Health guidelines. They were maintained on a cycle of 12 hours of light followed by 12 hours of dark and were given unrestricted access to food and water. The treatment of animals in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Institutional Animal Care and Use Committee of the Medical College of Georgia.

Organotypic Retinal Culture
Female C57BL/6 mice were killed by an overdose (5.0 mL/kg) of a mixture of 42.9 mg/mL ketamine, 8.57 mg/mL xylazine, and 1.43 mg/mL acepromazine. The eyes were removed and placed in ice-cold minimum essential medium (MEM; Invitrogen-Gibco, Carlsbad, CA). The anterior portion of the eye was removed by an incision along the ora serrata. After removal of the lens and vitreous, the retina was removed from the eye cup by inversion. Retinal explants were mounted with the photoreceptor side down on a 3.0-µm FCF filter (1 retina/filter) in a 12-mm culture plate insert (Millipore, Billerica, MA), covered with 1 drop of synthetic matrix (Matrigel; BD Biosciences, Bedford, MA), and kept at room temperature for 10 minutes, to allow coagulation of the matrix. A 26-gauge needle was used to make a hole in the periphery (to allow MCMV access to peripheral retinal cells). Each culture plate insert and retina was placed into one well of a 24-well plate containing 550 µL/well of Dulbecco’s MEM-F12 (supplemented with 10% horse serum and 5 mM glutamine and buffered with 20 mM HEPES [pH 7.4]), and incubated at 37°C. Culture medium was changed after 1 day and twice weekly thereafter. Some cultured retinas were inoculated with MCMV (5 x 105 PFU/well) for 3 hours. At days 4, 7, and 11 after infection (pi), culture medium was harvested for virus recovery, and some retinal cultures were collected and fixed in 4% paraformaldehyde for 5 minutes, immersed in 25% sucrose overnight, snap frozen, sectioned on a cryostat, and retained for further immunohistochemistry study. Some retinal cultures were prepared for caspase activity, apoptosis microarray, and RT-PCR studies.

Immunohistochemistry
A monoclonal antibody to an MCMV early antigen (EA)17 and the antibody rat anti-F4/80, which was used to identify activated microglia, were labeled with FITC (Sigma-Aldrich, St. Louis, MO) or biotinylated with sulfo-NHS-LC-biotin (Pierce, Rockford, IL), according to the manufacturer’s instructions. Mouse monoclonal antibody to an MCMV late antigen (LA) was kindly provided by John Shanley (University of Connecticut Health Center, Farmington, CT). Retinal glial cells including activated Müller cells and astrocytes were stained with mouse anti-glial fibrillary acidic protein (GFAP; BD-PharMingen, San Diego, CA) or with rabbit anti-GFAP (Chemicon, Temecula, CA). Mouse anti-Go-{alpha} (Chemicon) was used to identify both rod and cone bipolar cells. Rabbit anti-calbindin (Chemicon) was used to stain horizontal cells. A rabbit anti-{gamma}-aminobutyric acid (GABA; Sigma-Aldrich) was used to stain GABAergic amacrine cells. Mouse anti-neurofilament (NF; Sigma-Aldrich) was used to stain ganglion cells and some horizontal cells. FITC-labeled anti-TNF-{alpha} and rabbit anti-active caspase 3 was purchased from BD-PharMingen.

Double staining of MCMV EA and retinal cell antigens or TUNEL and triple staining of MCMV EA, GABA, and GFAP was performed as described previously.16 For double staining of TUNEL and retinal cell antigens, the sections were first detected with TUNEL staining15 and were then stained with a retinal cell–specific antibody followed by reaction with Texas red-avidin or Texas red–labeled anti-rabbit IgG, as described previously.16 For triple staining of MCMV EA, MCMV LA, and retinal antigens for which the antibodies had been made in rabbits (GABA, calbindin, and GFAP), the sections were stained first with one of the rabbit anti-retinal cell antibodies, and the reaction was developed by using AMCA-anti-rabbit IgG. The sections were then stained with anti-MCMV LA and reacted with biotinylated anti-mouse (ARK kit; Dako Corp., Carpinteria, CA).16 The immunolabeling was detected with Texas red-avidin. Finally, the slides were reacted with FITC-anti MCMV EA and mounted with mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and examined microscopically.

Apoptosis Microarray and RT-PCR
RNA was extracted from MCMV-infected retinal cultures and control retinal cultures (day 7 pi) using extraction reagent (TRIzol; Invitrogen-Gibco) according to the manufacturer’s instructions. Apoptosis genes were detected with a gene microarray (Oligo GEArray Mouse Apoptosis Microarray; SuperArray Bioscience Corp., Frederick, MD) which profiles the expression of 112 genes involved in apoptosis according to the manufacturer’s instructions. Three retinas were pooled for each group. Gene expression was analyzed on computer (GEA Suite software; SuperArray Bioscience Corp.). The mean of selected control genes was adjusted to 100. The minimal positive value of gene expression was set as more than 5% of the mean of control genes. All the negative genes for which the value was equal to or less than 5% of the mean of control genes were counted as 5 for the calculation.

RNA-PCR was performed (Access RT-PCR System; Promega Corp., Madison, WI) according to the manufacturer’s instructions. The primers for TNF-{alpha}, caspase 3, caspase 8, BCL2-associated X protein (Bax), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and β-actin were as follows:

TNF-{alpha}: 5'-TTCTGTCTACTGAACTTCGG-3' (298-317), 5'-GTATGAGATAGCAAATCGG-3' (633-651); caspase 3: 5'-GGGCCTGTTGAACTGAAAAA-3' (457-476), 5'-CCGTCCTTTGAATTTCTCCA-3' (669-698); caspase 8: 5'-GGCCTCCATCTATGACCTGA-3' (1269-1288), 5'-GCAGAAAGTCTGCCTCATCC-3' (1461-1480); bax: 5'-ATCGAGCAGGGAGGATGGCT-3' (221-240), 5'-CTTCCAGATGGTGAGCGAGG-3' (671-691); ASC: 5'-GATGGACGCCATAGATCTCAC-3' (232-252), 5'-GTCTCTGCACGAACTGCCTG-3' (527-546); and β-actin: 5'-TCCTTCGTTGCCGGTCCACA-3' (44-63), 5'-CGTCTCCGGAGTCCATCACA-3' (534-552).

Caspase 3 Activity
The enzymatic activity of caspase 3 was measured by modified enzymatic assay using the fluorogenic peptide substrate carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD.AFC; Enzyme Systems Products, Dublin, CA).18 19 MCMV-infected and noninfected control cultured retinas were treated with 1% Triton X-100. Eighty micrograms of protein from each lysate was added to the enzymatic reactions containing 25 µm DEVD.AFC. After incubation at 37°C for 60 minutes, fluorescence at excitation 360 nm/emission 530 nm was monitored with a plate reader (GENios; Tecan US, Research Triangle Park, NC). For each measurement, a standard curve was constructed by using free AFC. Based on the standard curve, the fluorescence reading from each enzymatic reaction was translated into the nanomolar amount of liberated AFC to quantify caspase activity.

Western Blot Analysis for Caspase 8
Western blot analysis for caspase 8 was performed as previously described.20 Briefly, proteins were extracted from freshly isolated normal retinas and from MCMV infected and noninfected control cultured retinas by using modified radioimmunoprecipitation (RIPA) buffer with an inhibition cocktail (complete Mini Protease Inhibitor Cocktail; Roche, Basel, Switzerland). Eighty micrograms of protein from each sample was loaded onto a 12% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA). The separated proteins were transferred onto a nitrocellulose membrane (GE Healthcare, Piscataway, NJ) which was blocked with 5% nonfat dry milk for 1 hour at room temperature. The membrane was then incubated overnight at 4°C with rabbit anti-caspase 8 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Binding of HRP-conjugated secondary antibody (goat anti-rabbit IgG-HRP, 1:2000; BD-PharMingen) was performed for 1 hour at room temperature. The immune complex was detected by a chemiluminescence detection system (ECL; GE Healthcare, Piscataway, NJ) and exposure to x-ray film. To verify equal loading among lanes, the membranes were stained with the intrinsic protein actin (mouse monoclonal anti-β-actin antibody; Sigma-Aldrich) after staining for caspase 8.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Organotypic Retina Cultures
Cultured retinas were observed with an inverted microscope. Even after several exchanges of culture medium, the retinas remained attached to the filters. Hematoxylin and eosin staining of cultured retinas demonstrated that the overall architecture of the cultured retina was maintained throughout the culture period (Fig. 1A) . The staining patterns of calbindin (Fig. 2A) , Go-{alpha} (Fig. 2B) , GABA (Fig. 2C) , GFAP (Fig. 2D) , and NF (not shown), which are located in horizontal cells, bipolar cells, amacrine cells, glial, and ganglion cells, respectively, and the morphology of individual cells appeared to be identical with that of freshly isolated normal mouse retina. In addition, no F4/80-positive activated microglia were observed in uninfected cultured retinas (not shown).


Figure 1
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  		FIGURE 1. Photomicrographs of hematoxylin and eosin–stained sections of a control (noninfected) (A) and an MCMV-infected (B) cultured retina at day 11 pi. The overall architecture of the retina in the control cultures was well preserved (A). Photoreceptor atrophy and loss and CMV-infected cells were observed in the MCMV-infected retina (arrows) (B). The retinas are oriented so that the filter/photoreceptor layer is at the top of each micrograph. Original magnification, x200.

 

Figure 2
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  		FIGURE 2. Photomicrographs of double staining for DAPI (A2, B2, C2, D2) and calbindin (A1), Go-{alpha} (B1), GABA (C1), and GFAP (D1), which are located in horizontal cells, bipolar cells, amacrine cells, and glia, respectively, in a control (noninfected) cultured retina. (A3, B3, C3, D3) Merged images. Original magnification, x200.

 
MCMV Infection of the Cultured Retina
After inoculation of MCMV, replicating virus was recovered from cultures (Fig. 3) and MCMV-EA and -LA–positive cells were observed in the retina. As early as day 4 pi, a few MCMV-infected cells were observed in the peripheral retina and by day 11, infected cells were observed not only in peripheral retina but also in the central retina. As shown in Figure 4 , most of the MCMV-infected cells in the cultured retina were GFAP-positive glia. Some virus-infected cells were calbindin-positive horizontal cells (Fig. 5) . Many GABA-positive cells were also observed; however, because these cells were also GFAP positive, they were glia and not amacrine cells (Fig. 6) . A similar pattern of staining of infected cells has been observed in the retinas of MCMV-infected immunosuppressed mice.16 F4/80-positive activated microglia, which were usually EA negative, were also observed in the retina (not shown). Photoreceptor atrophy and virus-infected cytomegalic cells were observed only in MCMV-infected retinas (compare Fig. 1A with Fig. 1B ).


Figure 3
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  		FIGURE 3. Titer of MCMV (log10 ± SEM; PFU/mL) in the MCMV infected cultured retinas at day 4, 7, and 11 days after inoculation of MCMV.

 

Figure 4
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  		FIGURE 4. Photomicrographs of staining for MCMV EA, LA, and GFAP in an MCMV-infected cultured retina at day 11 pi. Many MCMV-infected cells in the cultured retina were GFAP-positive glia (arrows) (EA, A; LA, B; GFAP, C; merge, D). Original magnification, x200.

 

Figure 5
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  		FIGURE 5. Photomicrographs of staining for MCMV EA and calbindin in an MCMV-infected cultured retina at day 11 pi. Some MCMV-infected cells in the cultured retina were calbindin-positive horizontal cells (arrows) (EA, A; calbindin, B; DAPI, C; merged image, D). Original magnification, x200.

 

Figure 6
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  		FIGURE 6. Photomicrographs of (A) MCMV EA–, (B) GFAP-, and (C) GABA-positive cells (arrows) in an MCMV-infected cultured retina at day 11 pi. As shown in the merged image (D), most of the MCMV-infected, GABA-positive cells were also GFAP positive. Original magnification, x200.

 
Apoptosis in MCMV-Infected, Cultured Retinas
To determine whether apoptosis was induced and to identify which retinal cells underwent apoptosis after MCMV infection, cultured retinas were double stained with TUNEL and with antibodies specific for retinal cell antigens or for MCMV antigens. In the absence of MCMV infection, only a few apoptotic cells were noted in the cultured retina (Fig. 7) . Many TUNEL-positive cells were observed in all layers of the MCMV infected retina (Fig. 8) . Consistent with previous observations in vivo,14 15 16 most of the apoptotic cells were noninfected bystander retinal cells (not shown). Many apoptotic cells observed in the outer nuclear layer were photoreceptor cells (Fig. 8) whereas some of them were horizontal (Fig. 8) or bipolar cells (not shown). Because both caspase dependent and independent apoptosis have been reported in retinal apoptosis,21 we examined slides of MCMV-infected retina and control retina with active caspase 3. As shown in Figure 9 , active caspase 3-positive cells were observed in the MCMV-infected retina (Fig. 9B) , but not in the control retina (Fig. 9A) . Although TUNEL-positive cells were observed in all layers of the MCMV-infected retina (Fig. 8) , most of the active caspase 3-positive cells were noted only in the inner nuclear and ganglion cell layers (Fig. 9B) .


Figure 7
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  		FIGURE 7. Photomicrographs of TUNEL staining in a control retina after 11 days of culture. Isolated areas containing a few apoptotic cells were observed in the cultured retina (circle) (TUNEL, A; DAPI, B). Original magnification, x200.

 

Figure 8
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  		FIGURE 8. Photomicrographs of staining for TUNEL and calbindin in an MCMV-infected cultured retina at day 11 pi. Many apoptotic cells observed in the outer nuclear layer were photoreceptor cells; rare calbindin-positive horizontal cells were also present (arrow) (TUNEL, A; calbindin, B; DAPI, C; merge, D). Original magnification, x200.

 

Figure 9
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  		FIGURE 9. Photomicrographs of noninfected cultured retina (A1–A3) after 11 days of culture and MCMV-infected cultured retina (B1–B3) at day 11 pi. Active caspase 3 (A1, B1) and DAPI (A2, B2) staining. The merged images of the noninfected and infected retinas are shown in (A3) and (B3), respectively. Original magnification, x200.

 
A mouse apoptosis microarray was used to confirm that apoptosis was induced and to explore the possible inducer(s) of apoptosis in cultured MCMV-infected retinas. Of the 112 genes analyzed, 29 were upregulated more than twofold and 5 were downregulated more than twofold by MCMV infection. Among the 29 upregulated genes, 19 are directly involved in apoptosis (Table 1) . Some genes involved in the death receptor–mediated apoptotic pathway, such as caspase 3, caspase 8, TNF-{alpha}, and Apaf1,22 were upregulated. Although Fas and Tnfrsf22, a TRAIL receptor, were upregulated, Fas ligand and TRAIL were not detected in MCMV-infected retinas. Some genes involved in the mitochondrial pathway of apoptosis—for example, Bid, Bax, and Trp5323 —were also upregulated. ASC, an adaptor molecule with a role in caspase-1 activation23 was strongly upregulated (17.59-fold), whereas caspase 1 was increased 7.6-fold in the MCMV-infected retinas. Other upregulated apoptosis genes included caspase 12 which is usually activated by oxidative stress,24 three genes (BCL10, Card6, and Card14) which mediate activation of NF-{kappa}B,25 26 and two BCL2-like genes (Bcl2L11, Bcl2L14) which are initiators of apoptosis.27


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  		TABLE 1. Increased Expression of mRNA of Apoptosis-Related Genes during MCMV Infection of the Cultured Retina

 
To confirm the microarray results that indicated that genes involved in the death receptor–mediated apoptotic pathway and mitochondrial pathway as well as the ASC gene were upregulated, expression of apoptotic genes including caspase 3, caspase 8, Bax and ASC was analyzed by RT-PCR. The results were similar to microarray results. As shown in Figure 10 , more apoptotic gene mRNA was detected in the MCMV-infected cultured retinas, compared with the mRNA level in the normal retinas and in the noninfected control retinal cultures. In addition, protein was extracted from the MCMV-infected retinas and control retinas and detected with caspase 3 activity and caspase 8 cleavage. The results showed that caspase 3 activity in MCMV-infected retinas was 0.06 nanomoles/mg protein/hour, whereas the level of caspase activity in control mice was undetectable. Caspase 8 cleavage was measured with Western blot analysis. Elevated cleaved caspase 8 was found in the MCMV-infected retinas (Fig. 11) .


Figure 10
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  		FIGURE 10. mRNA of apoptosis-related genes in MCMV-infected cultured retinas at day 7 pi. (A) RNA from normal retinas and from non–MCMV-infected retinal cultures was used as the control. More apoptotic gene mRNA was detected in the MCMV-infected cultured retinas. Amplification of β-actin was used to verify loading. (B) Ratio of target gene mRNA/β-actin mRNA.

 

Figure 11
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  		FIGURE 11. (A) Western blot of caspase-8 cleavage in MCMV infected cultured retinas at day 7 pi. Protein from normal retinas and from non–MCMV-infected retinal cultures was used as the control. Cleaved caspase 8 was detected in the MCMV-infected cultured retinas. β-Actin was used to verify loading. (B) Ratio of cleaved caspase 8 to β actin.

 
Several antiapoptosis genes were also upregulated (Table 2) . Some of these genes function as neuronal apoptosis inhibitor proteins, such as the Bag4, Cflar, and Birc serial proteins.28 29 30 31 32 The gene for IL-10, an anti-inflammatory cytokine, was also upregulated 4.86-fold. In contrast, as shown in Table 3 , downregulated genes were those that are usually highly expressed in the normal retina and perform functions such as regulating cell growth (Ark1),33 epithelial sodium transport (Tsc22d3),34 and modulating ATP metabolism (Mapk8ip1).35


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  		TABLE 2. Increased Expression of mRNA of Antiapoptosis Genes during MCMV Infection of the Cultured Retina

 

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  		TABLE 3. Genes for Which mRNA Expression Was Decreased during MCMV Infection of the Cultured Retina

 
TNF-{alpha} in MCMV-Infected Cultured Retinas
To determine that the apoptosis inducer TNF-{alpha} was produced during MCMV infection of the retina, expression of TNF-{alpha} mRNA was analyzed by RT-PCR. As shown in Figure 12 , TNF-{alpha} mRNA was observed in MCMV-infected retinas but not in noninfected control retinas. To determine whether TNF-{alpha} was produced by the activated microglia that were observed in the MCMV-infected retinas, the retinas were stained for TNF-{alpha} and F4/80. TNF-{alpha}-positive cells were indeed observed in MCMV-infected retinas, and most of these cells were activated microglia that were F4/80 positive (Fig. 13) .


Figure 12
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  		FIGURE 12. TNF-{alpha} mRNA in MCMV-infected cultured retinas at day 7 pi. TNF-{alpha} mRNA was detected in MCMV-infected retinas but not in control retinas. RNA from MCMV-infected eyes of immunosuppressed (IS) and RNA from non-IS mice were used as the TNF-{alpha}-positive control. No TNF-{alpha} mRNA was detected in MCMV-infected or noninfected RPE cells. A 1-kb ladder was used to identify TNF-{alpha} RNA, and amplification of β-actin was used to verify loading.

 

Figure 13
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  		FIGURE 13. Photomicrographs of staining of TNF-{alpha} and F4/80 in an MCMV-infected cultured retina at day 11 pi. Most of the TNF-{alpha}-positive cells were F4/80-positive activated microglia (arrow) (TNF-{alpha}, A; F4/80, B; DAPI, C; merge, D). Original magnification, x200.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Organotypic retinal cultures have been developed and used for investigation and experimental manipulation of the retina under controlled biochemical and treatment conditions.36 37 38 In contrast to cultures of dissociated retinal cells, organotypic cultures have the advantage of possessing the highest degree of architecture preservation, retaining cell–cell contacts and interactions among different cell populations. Results from the MCMV-infected organotypic retinal cultures described herein are similar to observations of the retina from studies using MCMV-infected mice.14 15 16 For example, both in culture and in animals,15 16 most of the MCMV-infected cells were glia and horizontal cells. MCMV infection in vivo and in organotypic retinal culture resulted in atrophy of the photoreceptor cells and the appearance of cytomegalic MCMV-infected cells.

Apoptosis is a consistent finding in HCMV infection of the retina in human patients12 13 and in the studies of MCMV retinitis in the mouse model.14 15 16 20 The results from the current studies of MCMV-infected cultured retina confirmed that apoptosis is induced after MCMV retinal infection and most of the TUNEL-positive apoptotic cells, including photoreceptors and horizontal cells, were not infected. TNF-{alpha}, as well as some genes involved in apoptotic death receptor–mediated pathways, such as caspase 3, caspase 8, and Apaf1, were upregulated during MCMV infection of cultured retina. These results correlate well with our previous observation that TNF-{alpha}, which was produced mainly by activated microglia in MCMV-infected retinas, plays a role in apoptosis in MCMV-infected retinas.20 Because the retinal cultures lack inflammatory cells that migrate into the eye during MCMV infection, the results of these studies using cultured retinas provide additional support for our previous suggestion that retinal apoptosis during MCMV infection is not T-cell dependent.14 20 In addition, some genes involved in the mitochondrial pathway, such as Bid, Bax, and Trp53, were also upregulated. It has been shown that mitochondrial activation mediated by Bid, a BH3-only pro-death Bcl-2 family protein and the major molecule linking the two pathways, is responsible for the prompt progress of TNF-{alpha}-induced apoptosis.39 40 41 42 43 44 45 46 Bid, which is activated by caspase 8 after TNF-{alpha} stimulation,39 40 can activate mitochondria via direct interaction with the multidomain, prodeath molecule Bax or Bak40 41 42 43 44 or via the cathepsin B and caspase 2 pathways.45 TNF-{alpha} may activate mitochondria in our model, because high levels of mRNA for TNF-{alpha}, Bid, caspase 8, and Bax were detected. However, the relative contribution of caspase-dependent apoptosis versus caspase-independent apoptosis during MCMV retinal infection cannot be determined from these experiments.

Other factors in addition to TNF-{alpha} may contribute to retinal apoptosis. An interesting result is the highly increased expression of ASC in the MCMV-infected retina. ASC is a proapoptotic protein originally found as a component of a "speck" in apoptotic cells and contains an N-terminal pyrin domain (PD) and a C-terminal caspase-recruitment domain (CARD).47 Both CARD and PD are protein–protein interaction domains and are structurally related to the death domain (DD) and death effector domain (DED).48 49 Recent studies have shown that ASC is key to caspase-1 activation which, in turn, is essential for the processing and release of biologically active IL-1β,50 51 52 53 54 55 a proinflammatory cytokine with critical roles in innate and adaptive immunities.56 IL-1β is also known to be an inducer of cellular NO production through activation of iNOS which could induce retinal apoptosis in some retinal diseases.57 58 59 60 In addition to caspase 1 activation, one recent study showed that ASC mediates induction of multiple cytokines, including TNF-{alpha}, by Porphyromonas gingivalis via a caspase-1- independent pathway.61 The exact role of ASC in apoptosis of MCMV-infected retina remains to be determined.

Some antiapoptosis genes were also elevated. These genes probably have two functions. First, many of these genes—for example, Bag4, Cflar, and Birc serial proteins—function as inhibitors of neuronal apoptosis,27 28 29 30 31 which could counter proapoptotic stimuli and contribute to preventing apoptosis of noninfected retinal neurons. Second, elevated antiapoptosis gene expression in MCMV-infected cells induced by viral infection or by specific viral genes could prevent apoptosis in MCMV-infected cells. Recently, it has been shown that HCMV IE2 induces expression of cellular Cflar (c-FLIP), an antiapoptotic molecule that blocks the direct downstream executor caspase 8 of the FasL/Fas pathway, and prevents HCMV-infected cells from apoptosis.62

In summary, results of the MCMV-infected retinal culture system correlate with results from studies of the retina in MCMV-infected immunosuppressed mice. In the future, this in vitro system could be used to explore the role of apoptosis of noninfected retinal cells as well as the those of cytokines and other modulators in the pathogenesis of CMV retinitis. The retinal culture system may also be used to test new approaches to prevent or reduce viral replication, since it permits manipulation and examination in carefully controlled experimental conditions.


   Footnotes
 
Supported by National Eye Institute Grant EY009169.

Submitted for publication May 24, 2007; revised August 6 and September 5, 2007; accepted November 21, 2007.

Disclosure: M. Zhang, None; B. Marshall, None; S.S. Atherton, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Sally S. Atherton, Department of Cellular Biology and Anatomy, Medical College of Georgia, R and E Building, Room CB2915, Augusta, GA 30912; satherton{at}mail.mcg.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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