In Vitro Susceptibility of Newborn Murine Retinal Cells to Herpes Simplex Virus Type 1 Infection

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1 Investigative Ophthalmology & Visual Science, Vol. 31, No. 7, July 199 Copyright Association for Research in Vision and Ophthalmology In Vitro Susceptibility of Newborn Murine Retinal Cells to Herpes Simplex Virus Type 1 Infection Michael J. Merges and Judith A. Whirrum-Hudson We have investigated the in vitro susceptibility of murine neural retinal cells to infection by herpes simplex virus Type 1 (HSV-1). Retinal cells obtained from newborn C57B1/6 mice were cultured for 6 days and infected with varying doses of HSV-1. Infection was determined by ABC immunoperoxidase staining of fixed cultures for HSV-1 antigens. Retinal neurons, including amacrine cells, were highly susceptible to infection, with 1% of the multipolar neurons expressing viral antigens after 12 hr of infection. Glial cells and retinal pigment epithelial (RPE) cells also were 1% infected within hr. Photoreceptor infection was not as fast, but all surviving photoreceptor cells and their precursors became infected by hr postinoculation. Since embryonic chick photoreceptors are highly resistant to HSV-1, these results demonstrate that mammalian (murine) photoreceptor cells differ from avian photoreceptor cells in their susceptibility to in vitro HSV-1 infection. In addition, our current results suggest that the in vivo resistance of adult C57B1/6 mice to herpetic retinitis may not reside at the level of the individual retinal cell populations, although apparent differences in susceptibility exist among the various retinal cell subpopulations. Invest Ophthalmol Vis Sci 31: ,199 Susceptibility of the eye to infection by herpes simplex virus type 1 (HSV-1) has been demonstrated both in humans and in experimental models. While the majority of ocular herpes infections occur in the cornea, 1 ' 2 an association of HSV-1 with retinitis and retinal necrosis has also been observed, particularly in immunosuppressed patients. 3 " 7 Other herpesviruses, including cytomegalovirus and varicella zoster virus, have been demonstrated in the retinas of some acquired autoimmune deficiency (AIDS) patients, 5-6 and may be the causative agent in some cases of acute retinal necrosis. 7 ' 8 In order to better understand the pathogenesis of herpetic retinitis and to prevent this potentially blinding course of infection, it is necessary to determine which cells of the neural retina are actually susceptible to HSV-1 infection. We previously demonstrated in the avian system the differential susceptibility of neural retinal cells to in vitro infection by HSV-1. 9 Embryonic chick photoreceptor cells (PhR) were completely resistant to HSV-1 infection, whereas neurons, glial cells, and retinal pigment epi- From the Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Supported in part by National Institutes of Health grant EY Presented in part at the 1988 meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida. Submitted for publication: July 11, 1989; accepted December 5, Reprint requests: Judith A. Whittum-Hudson, PhD, The Wilmer Institute, The Johns Hopkins Hospital, 6 N. Wolfe Street, Baltimore, MD thelial cells (RPE) were easily infected. We now have used an in vitro murine retinal cell culture system 1 " to determine whether mammalian (murine) cells exhibit similar differential susceptibility to in vitro infection by HSV-1. Our results indicate that retinal cell cultures established from eyes of newborn C57B1/6 mice do not exhibit the same pattern of resistance to HSV-1 as observed in the chick system. Partially differentiated, identifiable PhR as well as another population, which consisted of PhR precursors, neurons, and amacrine cells, were susceptible to HSV-1 infection. PhR are not fully differentiated since outer segments are not fully developed and opsin distribution does not become polarized before PhR cells degenerate over the 2-week culture period. 1 Neurons, glial-like flat cells, and RPE cells were 1% susceptible to infection. The details of these studies and their possible implications for in vivo retinal infections are presented in this report. Materials and Methods Mice Pregnant female C57B1/6 mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). Twenty-four to 48 hr after birth, mice were killed by decapitation and their eyes dissected under a binocular microscope to obtain either single cell preparations of neural retinal cells or RPE. Experiments with animals complied with the ARVO Resolution on the Use of Animals in Research. 1224

2 No. 7 VITRO SUSCEPTIBILITY OF RETINAL CELLS TO HSV-1 / Merges and Whirrum-Hudson 1225 Culture Preparation The technique of Politi et al 1 was used to establish the neural retinal cultures. Briefly, the retinas of four to six eyes were dissected and subjected to trypsin (.25%) digestion, followed by washing in Basal Medium Eagle containing 1% bovine serum albumin and.25% soybean trypsin inhibitor. Six hundred thousand cells were seeded onto 35-mm Petri dishes that had been pretreated with polyornithine (5 Mg/ml) and 25% Schwannoma-conditioned medium to facilitate neurite formation. 1 Neural retinal cells were cultured in a serum-free defined medium containing Dulbecco's modified essential medium (DMEM, Gibco, Long Island, NY) supplemented with penicillin (1, U/liter), glutamine (2 mm), cytidine 5'-diphosphocholine (2.56 mg/liter), cytidine 5'-diphosphoethanolamine (1.28 mg/liter), hydrocortisone (1 nm), and the Nl supplement (insulin, transferrin, progesterone, putrescine, and sodium selenite) at twice the concentrations recommended by Bottenstein and Sato. 12 After cellular attachment and differentiation, the cultures consisted of a low-density, nonconfluent mixture of three cell types: 1-2% multipolar neurons, 2-3% PhR, and 6-7% round cells, all of which are postmitotic. 1 Fewer than 1% of the cells in these cultures were glial-like and no RPE cells were present. To obtain cultures containing exclusively glial-like flat cells, neural retinal cells were seeded at the higher density of 9, cells onto untreated 35-mm tissue culture dishes. 13 The glial culture medium consisted of DMEM, 2 mm glutamine, 1% fetal calf serum (FCS), and penicillin (1, U/liter). Under these culture conditions, neurons and PhR cells did not survive longer than 1-3 days, and glial cells were confluent after 5-7 days. RPE was obtained by a modification of the method of Mayerson et al. 14 Briefly, whole eyes from 1-5- day-old mice were subjected to two 6-min digestions, first with a mixture of collagenase (.5%) and hyaluronidase (5 U/ml), and then with.1% trypsin. Sheets of RPE cells were peeled from the anterior portion of the eyecup, subjected to additional trypsin digestion for 2-3 min, treated with trypsin inhibitor (.25%), and washed in Hanks' balanced salt solution (HBSS, Gibco). Cells were cultured in DMEM containing 2 mm glutamine, 1, U/liter penicillin, and 1% FCS until cells became confluent (1-2 weeks), at which time cells were used in virus infection assays as described below. In Vitro HSV-1 Infection HSV-1 of the KOS strain was propagated and titrated in VERO cells by standard methods as described previously. 15 Five- to six-day retinal cell cultures were infected with varying multiplicities of infection (MOI) of live HSV-1 by adding 5 p\ virus suspension to eight-well chamber slides or 33 ix\ per 35-mm dish. Time course experiments were performed by infection at a constant MOI of 1, followed by culture for varying lengths of time from -72 hr. These cultures were low-density and nonconfluent to favor survival of PhR cells, round cells, and neurons rather than overgrowth by glial cells. Since PhR and round cells would not tolerate a typical adsorption step because of light adherence and the toxic effects of serum in the virus suspension, virus was added to the existing cultures. As shown by the stability of total cells per culture (Table 1), the numbers of cells remained stable over at least 24 hr. Cultures then were gently washed with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde for 45 min, followed by additional PBS washes. Cells were identified and enumerated based on welldefined morphologic criteria. Neurons, PhR, and amacrine cells present in these cultures were identified by a variety of different morphologic and biochemical criteria. 1 Three hundred cells (approximately 1 high-power fields [HPF]) were counted in each culture. Table 1. Time course study of HSV-1 antigens on neural retinal cells Time post inoculation (hr) Neurons Cell type (mean total number ± SD) Round PhR Neurons Cells HS V-1 -antigen-positive (%) Round PhR ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1 ± 1 ± 9 ± 2.* 32 ± 4.6* 63 ± 7.8* 1 ± 2± 1.7* 32 ±5.1* 66 ± 6.9* 1 ± Neural retinal cell cultures were prepared on day from 1-day-old neonatal C57B1/6 eyes; on day 5, replicate cultures were infected with HSV-1 (MOI = 1) for 8-48 hr, at which time cultures were fixed and stained for viral antigens by the ABC immunoperoxidase technique. See Materials and Methods for details of staining. Each time point represents data from two to five cultures. * Significantly different from neurons, P <.4.

3 1226 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 199, Vol. 31 Immunohistochemical Staining for HSV-1 Antigens and Cell-Associated Markers The ABC immunoperoxidase technique was used to detect viral and cellular antigens using methodology which has been described previously. 9 Briefly, fixed monolayers of retinal cells or control VERO cells were incubated for 2 min with a blocking antibody (normal goat IgG; 1:5). After blotting excess fluid, the primary rabbit anti-hsv-1 antibody (1:1; Accurate, Westbury, NY) was applied for 3 min. For detection of interphotoreceptor retinolbinding protein (IRBP) on PhR and round cells, an affinity-purified polyclonal antibody (kindly provided by G. Chader, National Eye Institute, National Institutes of Health, Bethesda, MD) was used. Neurons were IRBP-negative. Additional cultures were stained for opsin expression with a sheep antiopsin antibody (provided by L. Politi, Johns Hopkins University, Baltimore, MD). An anti-glial fibrillary acidic protein (GFAP) antibody (rabbit anti-bovine GFAP, 1:1) was used on fixed, permeabilized cultures to identify glial cells. Normal IgG from the relevant species was used as a negative primary antibody control in each case, and known positive sections or cultures were included for each antibody. After washes in PBS, the appropriate biotinylated secondary antibodies (1:2; Vector) were applied for 3 min, followed by the avidin-biotin-peroxidase complex (Vector) for an additional 45 min. The reaction product was visualized by development in aminoethylcarbazole (AEC) in dimethyl sulfoxide containing.1 % hydrogen peroxide, which gives a red reaction product. Cells were counterstained with Harris' hematoxylin (Harleco, American Scientific Products, McGraw Park, IL) and mounted with Gelvatol (Monsanto, Springfield, MA). In some experiments, double staining was performed using an alkaline phosphatase-conjugated secondary antibody and the substrate Kit III provided by Vector. This resulted in a blue reaction product, which contrasted with the red reaction product from AEC, and no counterstain was used. Cells of each population in the mixed cultures were enumerated as described above, and the number of positively staining cells within the total counted population was determined. Duplicate or triplicate cultures were counted for each data point. Results represent the mean of two or three experiments, and indicate the percentage of cells expressing the antigen of interest. Results Susceptibility of Murine Neural Retinal Cells to HSV-1 Infection Retinal cell cultures were infected with varying concentrations of HSV-1 and cultured for hr. Uninfected cultures of each cell type examined served as cell controls. Fixed cultures were stained by the ABC immunoperoxidase method for the presence of viral antigens; the antigen-positive cells were identified as PhR, round cells, or multipolar neurons. Double-staining for opsin or IRBP and HSV-1 antigens confirmed that PhR and round cells were infected (data not shown). A representative neural retinal cell culture with immunohistochemical evidence of viral antigens is shown in Figure 1A. A view of uninfected PhR under phase optics is shown for comparison in Figure IB. Dose-response experiments were performed to determine whether there was differential susceptibility 4 A B 4 Fig. 1. Presence of viral antigens 18 hr after in vitro infection of neural retinal cells with HSV-1. Neonatal retinal cell cultures were infected with HSV-1 (MOI = 5) for 18 hr. (A) Positive immunoperoxidase staining for HSV-1 antigens is associated with neurons (open arrows), photoreceptors (solid arrows), and round cells (hematoxylin counterstain, X1). (B) Higher-power phase view of uninfected photoreceptor cells (X25O).

4 No. 7 IN VITRO SUSCEPTIBILITY OF RETINAL CELLS TO HSV-1 / Merges ond Whirrum-Hudson 1227 among the murine neural retinal populations to in vitro HSV-1 infection. One hundred percent of neurons were infected within hr over a broad range of MOI. Whereas all subpopulations became infected in a dose-dependent fashion, 1% of PhR and round cells were not viral-antigen positive even at the highest doses of virus (MOI of 5-1) at 18 hr postinfection (PI). The percentage of neurons infected was significantly higher than for round and PhR cells at all virus concentrations (P <.5). These results suggest that differences in susceptibility to HSV-1 do exist for these populations of retinal cells. The results of a typical 18-hr dose-response experiment are shown in Figure 2. Time Course of HSV-1 Infection of Retinal Cells Replicate 35-mm cultures were infected with 6 X 1 6 PFU of HSV-1 (MOI = 1). At varying times after infection, cultures were fixed and stained for viral antigens. By 8-1 hr PI, viral antigens were detectable in neurons (5-8% infected), with a lag in antigen expression observed for round cells and PhR since only 1-3% of these cells expressed HSV-1 antigens at this time. In the experiment shown in Figure 3, round cells appeared to express viral antigens more slowly than PhR until hr after initiation of infection. At 18 hr PI, 1% of neurons were expressing viral antigen, whereas approximately 3% of round cells and 45% of PhR were positive. By 24 hr PI, only 4% of the round cells expressed HSV-1 an Time Post-Inoculation (hrs) Fig. 3. Time course of HSV-1 infection of murine neural retinal cells. Replicate cultures of C57B1/6 retinal cells were infected with HSV-1 (MOI = 1) for 6-72 hr, after which cultures were fixed, stained for viral antigens, and the percentage of positive neurons (circles), round cells (triangles), and photoreceptor cells (squares) determined for two to four cultures at each time point. Results for neurons were significantly different from round and PhR cells at 8-24 hr (P <.1). The results are similar to those obtained in three independent experiments. tigens, although 8% of cells had become antigenpositive (Fig. 3). The percentage of neurons infected from 8-24 hr PI was significantly higher (P <.1) than values for round and PhR cells. In one time course experiment, some cultures were stained 16 hr after infection for the presence of IRBP as well as HSV-1 antigens. As expected, no neurons expressed IRBP, but 9-96% of HSV antigen-positive round and PhR cells exhibited positive staining for IRBP (data not shown). The distribution of IRBP-positive cells was identical in control uninfected retinal cell cultures. The results of another typical time course experiment are summarized in Table 1. As shown by the total cell counts, numbers of all subpopulations remained stable through 24 hr PI. Between 36 and 48 hr PI, substantial loss of round and PhR cells occurred. Politi et al 1 have reported the short-term survival of these cells in vitro, and it is of note that 1% of all three surviving cell populations were infected. None 36. Virus Concentration (x 1 pfu) Fig. 2. Concentration dependence of susceptibility to in vitro HSV-1 infection. Retinal cell cultures were infected for 18 hr with varying concentrations of HSV-1 (KOS), representing 2-fold dilutions from an MOI of 4 at the highest virus concentration. Cultures werefixedand stained for HSV-1 antigens, and positive cells enumerated. The percentages of positive neurons (circles), round cells (triangles), and photoreceptor cells (squares) at each virus concentration are shown. Each data point represents the mean of two to four individual cultures. These results are representative of three separate dose-response experiments. Susceptibility of Murine Retinal Glial-like Cells to HSV-1 Infection We determined the susceptibility of retinal gliallike cells to HSV-1 by infecting confluent cultures of glial (flat) cells (MOI = 1) for 18 hr, after which fixed cultures were stained for the presence of viral antigens and GFAP. Figure 4A shows a typical culture in which 1% of the cells are positive for HSV-1 antigens. Viral antigens were expressed diffusely throughout the glial-like cells, including the nucleus. One hundred percent of these cells also stained posi-

5 *# * * INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 199 Vol. 31 t r IFig. 4. Susceptibility of C67BI/6 mouse glial cells to in vitro HSV-1 infection, under bright-field optics. (A) Confluent gliaj-like cells were infected with HSV-1 (MOI = 1-5) for 18 hr, after which cultures werefixedand stained for viral antigens by the ABC immunoperoxidase double staining technique without counterstain. One hundred percent of confluent glial-like cells expressed viral antigens after 18 hr of infection. All gray and granularity is positive stain for HSV-1. (B) Uninfected culture with hematoxylin counterstain (X1). tively for GFAP (not shown). Identical results were obtained in three separate experiments. At higher MOI, or at the later times after infection, cells showed typical cytopathic effects (rounding up; lysis). Susceptibility of RPE to HSV-1 Infection In the intact mouse model of herpetic retinitis, RPE underlying necrotic retina express viral antigens. 16 In vitro susceptibility of RPE to HSV-1 infection was assessed with purified viable RPE cells obtained from 1-5-day-old C57B1/6 mice. After 18 hr of in vitro infection (MOI = 1), fixed cultures were stained for viral antigens. Regardless of whether the RPE contained pigment granules or not, 1% of cells expressed HSV-1 antigens (Figs. 5A,B). Viral antigens were observed throughout the cytoplasm. The RPE nuclei appeared to be free of viral antigens. Discussion We have determined the in vitro susceptibility of newborn C57B1/6 mouse neural retinal cells to infection with HSV-1. Murine photoreceptors are highly susceptible to HSV-1 at early stages of differentiation. At the time of infection (5-6 days in vitro), many photoreceptors have differentiated to express opsin and the IRBP (characteristic of PhRs 17 ), and have the typical PhR morphology of a basal neurite ending in a spherule. 1 ' 17 In contrast, round cells, lacking processes, comprise a population which expresses both opsin and IRBP, and presumably includes photoreceptor precursors. 1 The susceptibility of these immature photoreceptor populations to HSV-1 infection was nearly equal that of PhR cells. Larger, multipolar neurons of several different morphologies were easily distinguishable from PhR and sf-' m "W I**' Fig. 5. Susceptibility of C57B1/6 retinal pigment epithelial cells to HSV-1 infection. (A) Confluent RPE cultures were infected overnight with HSV-t, fixed, and stained for HSV-1 antigens by the immunoperoxidase method. One hundred percent of the cells showed the blue alkaline phosphatase-associated reaction product indicative of viral antigens (gray). Pigment granules appear black. (B) Uninfected confluent culture with no counterstain. Only pigmented cells are visible (X1).

6 No. 7 VITRO SUSCEPTIBILITY OF RETINAL CELLS TO HSV-1 / Merges and Whirrum-Hudson 1229 round cells in these cultures. The time course data suggested that neurons are significantly more susceptible to HSV-1 infection than either PhR cells or round cells; multiprocessed neurons exhibited viral antigens earlier in infection and at consistently lower virus concentrations than observed for the latter cell types. Whether this is a result of their greater surface area exposed to potential infectious virions, of more receptors for HSV-1, or of a greater extent of intracellular viral replication is not known. Some of the infected multipolar neurons had the morphology associated with amacrine cells, 1 and these neuronal cells in particular survived through hr of infection. Fewer neuronal cells of other morphologies persisted this long, suggesting differential susceptibility of retinal neuron subpopulations to HSV-1 infection. This question will be the subject of additional studies. The murine photoreceptor cells in these cultures have been shown by Politi et al 1 to be rods, in contrast to the avian photoreceptor cell culture system in which most of the PhR cells are cones. 18 This latter observation is only one difference between the avian and murine cell culture systems, since the chick retinal cell cultures contained fetal calf serum, whereas the murine retinal cell culture medium was serumfree and chemically defined. In addition, embryonic chick retinas were used in the avian system, whereas newborn mice served as a source for the murine culture system. 1 ' 18 Whether the presence of serum contributes to the differences in in vitro susceptibility of PhR cells to HSV-1 observed for avian and murine cells remains to be determined. Murine glial-like cells and RPE were 1% susceptible to HSV-1 infection, a result consistent with reports from other laboratories. 19 ' 2 Earlier, Politi et al obtained similar results using avian RPE and glial-like cells. 9 In vivo studies of herpes-induced retinitis in the mouse from our laboratory 1521 " 23 and those of others 24 ' 25 have shown that BALB/c mice are susceptible to the development of herpesvirus-associated retinitis, while C57B1/6 are highly resistant to development of retinal pathology after intracameral or intravitreal inoculation of HSV-1. Our current in vitro findings suggest that C57B1/6 resistance to retinitis is not determined at the level of neural retinal or RPE cells, but by other genetic and immunologic constraints. The rapidity with which the neurons became infected suggests that these retinal cells are exquisitely sensitive to HSV-1 infection. Such rapid neuronal infection in vitro is paralleled in vivo, in immunosuppressed susceptible mouse strains, where virus is detectable throughout both retinas in the absence of inflammation or detectable infection of the photoreceptor layers. 16 Other investigators have shown that non-ocular cells, such as fibroblasts, from mouse strains resistant to in vivo HSV-1 infection can readily be infected in vitro. 19 One obvious limitation of this culture system is that it is much less complex than the intact adult retina, and there may be other cell populations in the adult retina of C57B1/6 mice which are resistant to HSV-1 infection. However, if the neurons, PhR, RPE, and glial (Miiller) cells are all susceptible to HSV-1 infection, the integrity of the in vivo retina would probably be disrupted by the infection and lysis of any one of these retinal cell populations. Having determined that several retinal cell subpopulations obtained from the newborn C57B1/6 mouse retina are susceptible to HSV-1 infection, we are now in a position to examine potential interactions of the immune system with these retinal populations. Future studies may enable us to define methods to avoid virus- and immune-mediated damage within the posterior segment of the eye, or ultimately to protect the eye from HSV-1 infection by immunologic and anti-viral interventions. Key words: herpes simplex virus Type 1, retina, retinal pigment epithelium, photoreceptor, glial-like cells, resistance, mouse Acknowledgments The authors thank Drs. Luis Politi and Ruben Adler for supplying reagents, as well as for helpful discussions and readings of this manuscript. Liddian Lindenmuth's excellent help in manuscript preparation is also acknowledged. Figures were drawn with Sigmaplot (Jandel Software, Corte Madera, CA). References 1. 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