Cytokine Profiles and Inflammatory Cells during HSV-1–Induced Acute Retinal Necrosis

Abstract

purpose. To investigate infiltrating cells, cytokines, and kinetics of cytokine expression during acute retinal necrosis (ARN) in the uninoculated eye after inoculation of herpes simplex virus (HSV)-1 into the anterior chamber of one eye of BALB/c mice.

methods. At different time points after inoculation of 2 × 10⁴ plaque-forming units (PFU) HSV-1 (KOS strain) or an equivalent volume of Vero cell extract in cell culture medium, the uninoculated eyes were enucleated. RT-PCRs for TNFα, IFNγ, and IL-4 and immunohistochemical staining were performed to identify infiltrating cells and cytokines. Cytometric bead array was used to measure the levels of TNFα, IFNγ, and IL-4 protein.

results. CD4⁺ T cells, F4/80⁺ macrophages, Gr-1⁺ polymorphonuclear cells (PMNs), and CD19⁺ B cells were detected in the uninoculated eye of virus-infected mice. Furthermore, RPE65⁺ retinal pigment epithelial (RPE) cells and activated Müller cells were also detected in the ARN lesion. TNFα, IFNγ, and IL-4 mRNA and protein were upregulated during the evolution of ARN in HSV-1–infected contralateral eyes compared with levels in control subjects. Immunohistochemistry revealed that cytokines were produced by infiltrating cells as well as by resident retinal cells.

conclusions. The results of these studies support the idea that T cells and cytokines are actively involved in HSV-1 retinitis. They also suggest that PMNs, B cells, and/or macrophages, as well as resident retinal cells, such as RPE and activated Müller cells, also play a role in the pathogenesis of HSV-1 retinitis.

Acute retinal necrosis (ARN) syndrome is a well-known clinical entity that was first described by Urayama et al. in 1971. ¹ ARN is a potentially devastating necrotizing vaso-oclusive retinitis affecting both healthy and immunocompromised patients. ^{2 3 4} It is caused by several members of the herpes virus family including herpes simplex virus (HSV)-1, HSV-2, varicella zoster virus (VZV) and, rarely, cytomegalovirus (CMV). ^{2 5 6 7 8 9} Histopathologically, there is retinal necrosis and chronic inflammatory cell infiltration. Typical viral intranuclear inclusions have been observed in the retina. Although this syndrome was first described experimentally in rabbits by Von Szily, ¹⁰ Whittum et al. ¹¹ rekindled experimental interest in viral retinitis by showing that inoculation of the KOS strain of HSV-1 into the anterior chamber of one eye of BALB/c mice induces characteristic retinal changes, including a devastating inflammatory reaction that develops within the posterior segment of the uninoculated eye, resulting in pan-necrosis of the retina 10 to 14 days postinoculation (PI). 

Although the histopathological features of retinitis in the contralateral eye and the route by which the virus travels to cause disease in this eye have been well described, ^{12 13 14} the type of cytokines and the kinetics of their expression during the process of ARN have not been fully defined. In this study, we investigated the cells in the inflammatory milieu, including infiltrating cells; resident retinal cells, such as retinal pigment epithelial (RPE) cells; and activated Müller cells and cytokine profiles, by immunohistochemistry, to compare the retina of the uninoculated eye of HSV-1–infected mice with the retina of the uninoculated eye of mock-infected mice. The expression of TNFα, IFNγ, and IL-4 during the evolution of ARN in HSV-1–infected mice was examined by RT-PCR and cytometric bead array. 

Methods

Animals

All investigations with mice conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Six-week-old BALB/c mice were obtained from Taconic, Inc. (Germantown, NY). Experimental mice were anesthetized with an intramuscular injection of a mixture of 1.07 mg ketamine, 0.21 mg xylazine, and 0.04 mg acepromazine per 25 g body weight and 2 × 10⁴ plaque-forming units (PFU) HSV-1 contained in 2 μL was injected into the anterior chamber of the right eye. The uninoculated contralateral eye was examined by a stereomicroscope to verify the onset of the disease characterized by dilatation of the perilimbal vessels and moderate cell and flare in the anterior chamber, as previously described. ¹⁵ Control mice were anesthetized as just described, and the anterior chamber of the right eye was injected with an equal volume of uninfected Vero cell extract in cell culture medium. 

Virus

The KOS strain of HSV-1 was used in all experiments. Stock virus was propagated by a low multiplicity-of-infection passage on Vero cells grown in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum and antibiotics. The titer of the virus stock was determined by standard plaque assay on Vero cells. Aliquots of stock virus were stored at −80°C, and a fresh aliquot was thawed and diluted for each experiment. Vero cell extract used for the injection of control mice was generated in parallel with virus propagation. In brief, instead of infecting the Vero cells with virus, only cell culture medium was added to the Vero cell culture. Vero cells were then harvested to generate Vero cell extract according to the same procedures used for harvesting HSV-1. 

Experimental Plan

Mice were anesthetized and injected with virus or Vero cell extract, as described. On days 6, 8, and 11 PI, the contralateral eyes of HSV-1– and mock-injected mice were enucleated and pooled, and total RNA was isolated (TRIzol Reagent; Invitrogen-Gibco, Grand Island, NY). For immunohistochemical staining, the contralateral eyes of virus- and mock-injected mice were enucleated at days 9 and 14 PI, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek USA, Inc., Torrance, CA) and sectioned at a thickness of 6 μm. For quantitation of cytokines using the cytometric bead array, the contralateral eyes of HSV-1–infected and mock-injected mice were enucleated. The enucleated eyes were dissected into anterior and posterior sections with the aid of a stereomicroscope. The anterior section containing the cornea and the iris was discarded, and the posterior sections of five eyes were pooled. The eye samples were digested in 600 U/mL collagenase IV (Sigma-Aldrich, St. Louis, MO) at 37°C in a 5% CO₂ incubator for 45 minutes. After collagenase treatment, the digested tissues were passed through a cell strainer (pore size, 70 μm; BD Biosciences-Falcon, Bedford, MA). The cells were then counted and placed into round-bottomed, 96-well plates at a concentration of 5 × 10⁵ cells per well in RPMI 1640 medium supplemented with 10% FCS. Coincident with enucleation of the uninoculated eye, submandibular lymph nodes were also removed, and lymphocytes were isolated and plated in round-bottomed, 96-well plates at a concentration of 5 × 10⁵ cells per well in RPMI 1640 medium with 10% FCS. The cells derived from the eye and submandibular lymph nodes were cultured at 37°C in a 5% CO₂ incubator. After 24 or 48 hours of culture, the supernatants were collected and stored at −80°C. 

Immunohistochemical Staining

Frozen sections were fixed with acetone and incubated with 0.3% hydrogen peroxide to eliminate endogenous peroxidase. Then the sections were blocked with 3% bovine serum albumin (Fisher Biotech, Fair Lawn, NJ). Biotinylated anti-mouse CD4, CD19, Gr-1 (BD Biosciences-PharMingen, San Diego, CA), F4/80 (Caltag Laboratories, Burlingame, CA); or nonbiotinylated rabbit anti-mouse retinal pigment epithelial antigen (RPE65; kindly provided by Michael Redmond, National Eye Institute, National Institutes of Health, Bethesda, MD); or nonbiotinylated rabbit anti-glial fibrillary acidic protein (GFAP; Sigma-Aldrich, Inc.) was added and the sections incubated at room temperature for 90 minutes. For RPE65 and GFAP staining, the sections were incubated with a biotinylated goat anti-rabbit secondary antibody. All sections were washed, and streptavidin HRP (DakoCytomation; Dako Corp., Carpinteria, CA) was applied. Liquid DAB chromogen (DakoCytomation; Dako Corp.) was added to develop the color reaction. The sections were counterstained with 1% methyl green in methanol, dehydrated, and examined microscopically. 

For immunofluorescence double staining, the sections were fixed in 4% paraformaldehyde and incubated with biotinylated anti-mouse TNFα, IFNγ, or IL-4 antibody (BD Biosciences-PharMingen). Sections were then washed and incubated with avidin D-Texas red (Vector Laboratories, Inc., Burlingame, CA) and washed again. Sections were then incubated with one of the following antibodies: FITC-conjugated rat anti-mouse CD4, FITC-conjugated rat anti-mouse Ly-6G (Gr-1), Ly-6C (RB6-8C5) monoclonal, FITC-conjugated anti-mouse F4/80 (Caltag Laboratories), FITC-conjugated rat anti-mouse CD19 monoclonal (BD Biosciences-PharMingen), or rabbit anti-mouse RPE65. For sections reacted with RPE65 or GFAP, FITC-conjugated goat anti-rabbit (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was added as the second antibody. Sections were then washed, mounted with antifade medium (Vectashield and DAPI; Vector Laboratories, Inc.), and examined by fluorescence microscopy. For quantitative analysis of retinal infiltrating and activated resident cells, contralateral eyes were removed from three randomly selected HSV-1–infected mice at day 9 PI. Four or five noncontiguous sections of each eye were examined by fluorescence microscopy. The number of marker-positive cells and the number of cytokine-expressing cells was determined by counting the number of cells in the central (approximate) one-third of the retina. 

Cytokine Bead Array Analysis

The mouse Th1/Th2 cytokine cytometric bead array (CBA) kit (BD Biosciences-PharMingen) was used to measure the protein levels of TNFα, IFNγ, and IL-4 in supernatants from eye-derived cultures after 24 and 48 hours of incubation. Cells were collected from the uninoculated eyes at days 6, 9, and 14 PI, respectively. The kit contains three bead populations of distinct fluorescence intensities. The beads were coated with capture antibodies specific for IL-4, IFNγ, and TNFα proteins and were mixed with PE-conjugated detection antibodies and incubated with recombinant standards or with the supernatant from the cultured cells to form sandwich complexes. The Th1/Th2 CBA was resolved in the FL3 channel of the flow cytometer (FACS; BD Biosciences), and the results were generated in graphic and tabular format by using the CBA analysis software (BD Biosciences-PharMingen). The assay sensitivities for TNFα, IL-4, and IFNγ are 6.3, ≤5.0, and 2.5 pg/mL, respectively (BD Biosciences-PharMingen). The experiment was repeated three times, and the results were analyzed for significant differences between experimental and control groups by paired t-test. P ≤ 0.05 was considered to be statistically significant. 

Cytokine RT-PCR

At days 6, 8, and 11 after HSV-1 anterior chamber inoculation, the contralateral eyes were collected. The posterior portion of the contralateral eye was isolated, and the posterior portions of five eyes were pooled at each time point. The samples were immediately put in extraction reagent (TRIzol; Invitrogen-Gibco), and total RNA was extracted according to the manufacturer’s instructions. RNA was then reverse transcribed (Superscript II RNAase H⁻ reverse transcriptase; Invitrogen, Carlsbad, CA) for RT-PCR. Oligo(DT)12 -18 (500 ng) and 1 μL of 10 mM dNTP mix were added to 1 ng to 5 μg total RNA (10 μL), and H₂O was added to a final volume of 20 μL/reaction. The mixture was incubated at 65°C for 5 minutes and cooled quickly on ice. After brief centrifugation at 1600g for 20 seconds, 4 μL of 5× first strand buffer, 2 μL 0.1 M dithiothreitol [DTT], 1 μL of ribonuclease inhibitor (RNase OUT; Invitrogen), and 1 μL of recombinant ribonuclease inhibitor were mixed gently and incubated at 42°C for 2 minutes. Reverse transcriptase (Superscript II, Invitrogen) was added and the mixture was incubated at 42°C for 50 minutes. The reaction was inactivated at 70°C for 15 minutes. The concentration of the resultant cDNAs was determined with a spectrometer (MBA 2000; PerkinElmer Life and Analytical Sciences, Inc., Boston, MA), and the concentration of the samples was adjusted and normalized. 

The PCR reaction was performed according to the manufacturer’s PCR protocol (Invitrogen) with 35 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 90 seconds. After 35 cycles, the reaction was maintained at 72°C for an additional 10 minutes. 

The primers for TNFα, IL-4, IFNγ, and β-actin were as follows: TNFα: 5′-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC 3′ and 5′-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3′; IL-4: 5′-GACGGCACAGAGCTATTGATGGGTC-3′ and 5′-TAGGCTTTCCAGGAAGTCTTTCAGTGATGTG-3′; IFNγ: 5′-GGCTGTTTCTGGCTGTTACTG-3′ and 5′-GACTCCTTTTCCGCTTCCTGA-3′; and β-actin: 5′-TCCTTCGTTGCCGGTCCACA-3′ and 5′CGTCTCCGGAGTCCATCACA-3′. 

Results

Inflammatory Cells

At day 9 PI during the acute phase of retinitis, ¹⁵ CD4⁺ or CD19⁺ cells were observed in the retina, most of them located in the photoreceptor and outer nuclear layers (Figs. 1A 1J) . The distribution of Gr-1⁺ cells was similar to that of F4/80⁺ cells, and both cell types were observed in the inner nuclear and ganglion cell layers of the retina (Figs. 1D 1G) . At day 14 PI, although the retinal structure was disrupted, CD4⁺, F4/80⁺, Gr-1⁺, and CD19⁺ cells were still observed within the remnants of the retina (Figs. 1B 1E 1H 1K) . CD4⁺, Gr-1⁺, F4/80⁺, and CD19⁺ cells were not found in the contralateral eyes of mock-infected mice at any of the time points (Fig. 1C 1F 1I 1L) . 

RPE Cells and Activated Müller Cells

In the contralateral eyes of mock-infected mice, RPE65⁺ RPE cells were observed in their normal anatomic location subjacent to the photoreceptors (Fig. 1O) . In contrast, in the uninoculated contralateral eyes of HSV-1–infected mice, there appeared to have been migration of RPE cells, and RPE65⁺ cells were observed in the photoreceptor layer and in the outer and inner nuclear layers of the inflamed retina (Figs. 1M 1N) . The extent of migration of the RPE65⁺ cells corresponded with the severity of the inflammation in the retina, and the more inflamed the eye, the more migration of RPE65⁺ cells was observed. 

In the contralateral eyes of mock-infected mice, only radial processes and some horizontal fibers of Müller cells located in the nerve fiber layer and ganglion cell layers of the retina were GFAP⁺ (Fig. 1R) . In contrast, in the retina of the contralateral eyes of HSV-1–infected mice, GFAP⁺ staining was observed in the nerve fiber, ganglion cell, inner nuclear, and outer nuclear layers of the infected retina (Figs. 1P 1Q) . 

Patterns of Cytokine mRNA and Protein

As shown in Figure 2 , the level of mRNA for TNFα, IFNγ, and IL-4 in the contralateral eye of mock-infected mice was low at day 6 PI. Although only day 6 results are shown, the mRNA for TNFα, IL-4, and IFNγ in mock-inoculated mice did not vary significantly throughout the course of the experiment. In contrast, the pattern of mRNA expression was different for TNFα, IL-4, and IFNγ in the uninoculated eye of HSV-1–injected mice. Compared with mock-infected eyes, the level of TNFα, which was elevated on day 6 PI, decreased thereafter. IFNγ mRNA was moderately elevated on day 6 PI, increased slightly between days 6 and 8 PI, and then increased slightly again between days 8 and 11 PI. On days 6 and 8 PI, the level of mRNA for IL-4 was only slightly above that observed in the uninjected eyes of mock-infected mice. Between days 8 and 11 PI, the amount of IL-4 mRNA increased approximately threefold. 

To assess the levels of cytokine protein in HSV-1–infected contralateral eyes, the posterior portion of the eye (five eyes per time point) was collected on days 6, 9 and 14 PI, and the tissue was digested as described in the Methods section. Ocular cells were cultured for 24 or 48 hours. The supernatant was collected, and the amount of TNFα, IL-4, and IFNγ was determined with the cytometric bead array kit. Although supernatants from 24 and 48-hour cultures were assayed, only the higher levels of cytokines observed at 48 hours are shown in Figure 3 . At days 6, 9, and 14 PI, the level of TNFα in the supernatant of cells recovered from the contralateral eye of HSV-1–infected mice was significantly higher (P < 0.05) than from the uninoculated eye of mock-infected mice (day 6 PI: 38.4 ± 4.2 pg/mL vs. 13.3 ± 0.4 pg/mL; day 9 PI: 40.8 ± 6.8 pg/mL vs. 12.5 ± 3.9 pg/mL; day 14 PI: 54.2 ± 4.5 pg/mL vs. 19.8 ± 6.8 pg/mL). 

At all time points, IFNγ was significantly higher (P < 0.05) in supernatants of cells recovered from the uninoculated eye of HSV-1–infected mice than from the uninoculated eye of control mice (day 6: 52.3 ± 12.8 pg/mL vs. 24.4 ± 3.0 pg/mL; day 9: 40.1 ± 2.7 pg/mL vs. 22.0 ± 2.1 pg/mL; day 14: 53.2 ± 13.3 pg/mL vs. 14.3 ± 1.9 pg/mL). The level of IL-4 was not significantly different between control and HSV-1–infected eyes at day 6 PI However at days 9 and 14 PI, the level of IL-4, although lower than that of either TNFα or IFNγ, was significantly (P < 0.05) higher than that of control eyes (day 9 PI: 9.0 ± 2.0 pg/mL vs. 4.9 ± 0.2 pg/mL; day 14 PI: 18.0 ± 2.5 pg/mL vs. 6.5 ± 1.4 pg/mL). 

Inflammatory Cells and Cytokine Production

As shown in Figures 4A 4D 4G 4J 4M , TNFα⁺ cells were observed in all layers of the inflamed retina at day 9 PI. Between 65% and 100% of the CD4⁺, Gr-1⁺, CD19⁺, and F4/80⁺ cells were also TNFα⁺ (Figs. 4C 4F 4I 4L , Table 1 ). IL-4⁺ cells were observed throughout the retina at day 9 PI (Figs. 5A 5D 5G 5J) . Most of the CD4⁺, Gr-1⁺, CD19⁺ and F4/80⁺ cells expressed IL-4 (Figs. 5C 5F 5I 5L , Table 1 ). IFNγ⁺ cells were also observed throughout the retina (Figs. 6A 6D 6G 6J) . Most of the IFNγ⁺ staining colocalized with CD4⁺, Gr-1⁺, CD19⁺, or F4/80⁺ cells (Figs. 6C 6F 6I 6L ; Table 1 ). 

RPE Cells, Activated Müller Cells, and Cytokine Production

As shown in Table 1and Figure 4O , 33% to 41% of RPE65⁺ cells also expressed TNFα at day 9 PI. Between 28% and 41% of RPE65⁺ cells expressed IL-4 (Table 1 , Fig. 5O ). Between 26% and 44% of RPE65⁺ cells expressed IFNγ (Table 1 , Fig. 6O ) at day 9 PI. As shown in Table 1and Figure 4R , only 4% to 14% of GFAP⁺-activated Müller cells also expressed TNFα at day 9 PI. Between 2% and 9% of GFAP⁺-activated Müller cells expressed IL-4 (Table 1 , Fig. 5R ). Between 4% and 14% of GFAP⁺-activated Müller cells expressed IFNγ (Table 1 , Fig. 6R ) at day 9 PI. 

Discussion

The interaction between virus and specific immune effectors, such as virus-specific T cells, and nonspecific effector modalities, such as cytokines in patients with ARN, is a complex process. The murine model of HSV-1 ARN provides a well-defined system for studying these complex interactions and by judicious interpretation of results using the mouse model, for providing insight into the pathogenesis of ARN in humans. 

Previous studies of the BALB/c mouse model of ARN have shown that after uniocular anterior chamber inoculation of HSV-1, the virus infects the retina of the uninoculated contralateral eye at day 7 PI and the peak titer of virus is observed in this eye on day 9 or 10 PI. ¹⁴ During the acute phase of HSV-1 infection, virus can trigger inflammation in a variety of ways, including direct cell destruction due to virus infection, release of viral products from infected cells, and production of inflammatory molecules such as cytokines by virus infected and/or nearby cells. ^{16 17 18} The studies presented herein revealed that after HSV-1 infection of the anterior chamber of one eye, mRNA and protein levels of TNFα, IFNγ, and IL-4 cytokines were upregulated in the retina of the uninoculated eye during the evolution of retinitis. 

TNFα is predominantly a Th1 and proinflammatory cytokine ¹⁹ and it was upregulated early in the disease process (beginning at day 6 PI). Many of the important functions mediated by TNFα are related to its inflammation-promoting activity, and, for example, TNFα can enhance IFNγ responses, upregulate major histocompatibility complex (MHC)-I molecules, and induce adhesion molecules on vascular endothelial cells. ^{19 20} IL-4 mRNA and protein were upregulated later in the disease process and remained elevated at day 14 PI. IL-4 plays a role, not only in the differentiation of Th2 cells, but also as a key compensatory cytokine for all aspects of the Th2 response. ^{20 21 22} Because IL-4 inhibits CD4⁺ Th1 and the clearance of virus by limiting cytotoxic CD8⁺ T cell function, ^{23 24} the elevated level of IL-4 in the later phase of the disease suggests that this cytokine may play a role in limiting T-cell–mediated cytotoxicity during HSV-1 retinitis. In contrast, because IL-4 is a well-characterized cytokine known to be produced by naïve and activated T cells and to affect B-cell differentiation as well as proliferation and the promotion of B-cell production of antibodies, ^{19 25} the slow upregulation of IL-4 during the process of ARN may help to explain the presence of B cells in the inflamed retina, as shown in this study and in a previous study of this model. ²⁶  

During corneal or systemic HSV-1 infection, IFNγ is regarded as a proinflammatory cytokine. ^{20 27 28} Results of these studies indicate that IFNγ is upregulated throughout the disease process, suggesting that this cytokine plays a role in all phases of the disease. However, because IFNγ is a cytokine with multiple functions, ¹⁹ additional studies are needed to determine whether different effects of this cytokine are exhibited during different phases of HSV-1 retinitis. Although elevation of mRNA for several proinflammatory and anti-inflammatory cytokines has been reported during HSV-1 retinitis, ²⁹ the current study extended these findings by assessment of cytokine protein levels and by localization of cytokines within the retina during the disease. Together, the results of studies of cytokine mRNA and protein during HSV-1 retinitis support the idea that development of fulminant ARN requires the contribution of one or more cytokines. 

In addition to the interactions between cytokines and their receptors, interactions between viral products and the host immune system are also essential. Results of studies of this mouse model suggest that both CD4⁺ and CD8⁺ T cells are crucial in preventing viral infection of the retina of the injected eye by preventing spread of virus from sites that are synaptically connected to the optic nerve and the retina of the injected eye. ^{30 31 32} T cells have also been shown to play a role in exacerbation of retinitis. In addition, B cells and macrophages appear to participate in the disease process. ²⁶ Results of our current studies provide additional information about the location of infiltrating cells; about production of cytokine by these cells; and, by extrapolation, about the contributions of these cells and their products to ARN after uniocular anterior chamber inoculation of HSV-1. Not surprisingly, immunohistochemical staining showed that CD4 T cells, macrophages, and polymorphonuclear cells were the major sources of TNFα, IFNγ, and IL-4 at the peak of the clinical disease. The results revealed that B cells were also a cellular source of TNFα, IFNγ, and IL-4. Approximately one third of the RPE cells produced TNFα, IL-4, or IFNγ at day 9 PI. A smaller number (2%–14%) of GFAP⁺ cells produced TNFα, IL-4, or IFNγ at day 9 PI. 

Taken together, the findings in this study provide additional support for the idea that besides T cells and virus, immunomodulatory factors (cytokines) are actively involved in HSV-1 retinitis. These results also provide additional support for our previous suggestion that B cells and/or macrophages may play a role in the pathogenesis of HSV-1 retinitis. ²⁶ An unexpected finding was that RPE cells that had migrated into the retina produced cytokines, and thus these cells may also contribute to the pathogenesis of retinal destruction, along with previously described immune effector cells. ^{30 31 32} Although activated Müller cells were widely distributed in the infected retina, only a small percentage of TNFα, IL-4, and IFNγ was produced by these cells. This observation suggests that the role of activated Müller cells in HSV-1–induced retinitis may be limited. Further studies using B cell or cytokine knockout mice on a BALB/c background may provide additional insight into the role of specific cells or cytokines in the pathogenesis of ARN. Elucidation of the interactions between and among infiltrating cells, retinal resident cells and virus, and cytokines and virus may provide strategies for the design of therapies specifically targeted toward control of viral infection in the eye that would limit or even prevent damage to the retina and preserve vision. 

 Supported by National Eye Institute Grant EY006012.

 Submitted for publication November 2, 2004; revised December 17, 2004; accepted January 4, 2005.

 Disclosure: M. Zheng, 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.

 

Figure 1.

 

Photomicrographs of the retinas of the uninoculated eyes of BALB/c mice showing the location of CD4⁺ (A, B), Gr-1⁺ (D, E), F4/80⁺ (G, H), CD19⁺ (J, K), RPE65⁺ (M, N), and GFAP⁺ (P, Q) cells 9 (left) and 14 (middle) days after injection of 2 × 10⁴ PFU HSV-1 (KOS) into the anterior chamber of one eye. Photomicrographs of the uninoculated eye of mice injected with an equivalent volume of Vero cell extract with tissue culture medium and stained with CD4 (C), Gr-1 (F), F4/80 (I), CD19 (L), RPE65 (O), and GFAP (R) antibodies are shown for comparison. Uninoculated eyes of experimental virus-inoculated mice or mock-injected control mice were collected, embedded in OCT, sectioned on a cryostat, and reacted with antibodies.

Figure 2.

 

TNFα, IL-4, and IFNγ mRNA in the uninoculated eye of HSV-1–injected mice 6, 8, and 11 days after injection of 2 × 10⁴ PFUs of HSV-1 (KOS) into the anterior chamber of one eye (A). Band intensities of the cytokine messages are normalized to β-actin, which was used as the internal control (B). Samples from mock-injected mice were collected on day 6. RNA was extracted from the posterior segment of experimental and control eyes, reverse transcribed, and amplified by semiquantitative RT-PCR.

Figure 3.

 

TNFα, IFNγ, and IL-4 in the supernatants from cells collected from mice 6, 9, and 14 days after inoculation of 2 × 10⁴ PFU of HSV-1 (KOS) into the anterior chamber of one eye and cultured for 48 hours after collection. The concentration of cytokine in the supernatant from cells from the posterior segment is shown. The levels of cytokine were measured by cytometric bead array. *Significant differences between virus- and mock-injected control mice.

Figure 4.

 

Photomicrographs of the retina of the uninoculated eye showing the location of CD4⁺, Gr-1⁺, F4/80⁺, CD19⁺, and RPE⁺ cells and TNFα 9 days after uniocular anterior chamber injection of 2 × 10⁴ PFU HSV-1 (KOS). (A, D, G, J, M, P) Sections stained with antibody to TNFα. (B, E, H, K, N, Q) Sections stained for CD4, Gr-1, F4/80, CD19, RPE65, and GFAP, respectively. (C, F, I, L, O, R) Merged images of the two micrographs to the left in each group.

Table 1.

 

Cells and TNFα, IL-4, and IFNγ in the Posterior Segment during HSV-1 Infection of the Retina

Cell Marker	CD4	Gr-1	F4/80	CD19	RPE65	GFAP
TNF-α
Marker+ cells (n)	90 ± 17	150 ± 31	100 ± 16	110 ± 21	105 ± 21	125 ± 19
Cells double+ for marker and TNFα (n)	72 ± 13	101 ± 23	95 ± 25	85 ± 22	40 ± 12	12 ± 8
Double+ (%)	79–80	65–68	83–103	70–81	33–41	4–14
IL-4
Marker+ cells (n)	65 ± 11	47 ± 11	41 ± 8	45 ± 13	59 ± 9	110 ± 23
Cells double+ for marker and IL-4 (n)	43 ± 13	35 ± 9	30 ± 6	38 ± 16	21 ± 7	7 ± 5
Double+ (%)	55–73	72–75	72–73	68–94	28–41	2–9
IFNγ
Marker+ cells (n)	70 ± 12	107 ± 24	139 ± 26	140 ± 22	80 ± 19	145 ± 31
Cells double+ for marker and IFNγ (n)	55 ± 10	90 ± 23	116 ± 21	120 ± 15	30 ± 14	15 ± 10
Double+ (%)	77–79	80–86	83–84	83–88	26–44	4–14

 On day 9 after uniocular anterior chamber inoculation of 2 × 10⁴ PFU HSV-1 (KOS), the noninjected eye was removed, frozen in OCT, and sectioned on a cryostat. Sections were stained for CD4, Gr-1, F4/80, CD19, RPE65, or GFAP and TNFα, IL-4, or IFNγ.

Figure 5.

 

Photomicrographs of the retina of the uninoculated eye showing the location of CD4⁺, Gr-1⁺, F4/80⁺, CD19⁺, and RPE⁺ cells and IL-4, 9 days after uniocular anterior chamber injection of 2 × 10⁴ PFU HSV-1 (KOS). (A, D, G, J, M, P) Sections stained with antibody to IL-4. (B, E, H, K, N, Q) Sections stained for CD4, Gr-1, F4/80, CD19, RPE65, and GFAP, respectively. (C, F, I, L, O, R) Merged images of the two panels to the left in each group.

Figure 6.

 

Photomicrographs of the retina of the uninoculated eye showing location of CD4⁺, Gr-1⁺, F4/80⁺, CD19⁺, and RPE⁺ cells and IFNγ, 9 days after uniocular anterior chamber injection of 2 × 10⁴ PFU HSV-1 (KOS). (A, D, G, J, M, P) Sections stained with antibody to IFNγ. (B, E, H, K, N, Q) Sections stained for CD4, Gr-1, F4/80, CD19, and RPE65, respectively. (C, F, I, L, O, R) Merged images of the two panels to the left in each group.

The authors thank Jeanene Pihkala (Institute for Molecular Medicine and Genetics, Medical College of Georgia) for assistance with the cytometric bead array analysis and with analysis of the results.