MURINE ACUTE RETINAL NECROSIS* BY C. Stephen Foster, MD, Manfred Zierhut, MD (BY INVITATION), Helen Wu, MD (BY INVITATION), Richard Tamesis, MD (BY INVITATION), AND (BY INVITATION) Nada Jabbur, MD INTRODUCTION

A MURINE COUNTERPART TO HUMAN ACUTE RETINAL NECROSIS CAN BE

created by adapting the von Szily model of herpes simplex virus (HSV) retinitis in rabbits.1 This model, first adapted to mice by Whittum and associates,2 is produced by inoculation of HSV into the anterior chamber of one eye of a susceptible mouse strain, such as BALB/c. Severe ipsilateral iridocyclitis rapidly develops in the injected eye. Interestingly, a destructive retinitis develops 10 days later in the contralateral eye. Histopathologic analysis of the eyes typically shows relative sparing of the ipsilateral retina from the destructive inflammation. The exact mechanisms behind the sparing of the ipsilateral retina and the destruction of the contralateral retina are unknown. Both viral and host strain characteristics exert a profound influence on this interesting model, and the virologic and immunologic contributions to the contralateral necrotizing retinitis and to the protection against necrotizing retinitis in the ipsilateral eye, and even in the contralateral eye in murine strains resistant to this disease model, are currently under intense investigation. We have previously shown that at least one factor influencing susceptibility to the development of destructive contralateral retinitis in this model is the host genetic characteristics at a limited deoxyribonucleic acid segment on chromosome 12 at or around the Igh-1 locus. BALB/c (Igh-la) mice are extremely susceptible to development of the retinitis, whereas Igh-lb congenic CB17 mice are very resistant.3 In athymic BALB/c (nude) mice bilateral necrotizing retinitis develops after unilateral anterior chamber injection with HSV. In an effort to better understand the relative contributions of helper/ inducer (CD4) and cytotoxic/suppressor (CD8) T lymphocytes in the *From the Hilles Immunology Laboratory, Massachusetts Eye and Ear Infirmary, Boston. Supported by grant EY-06008 from the National Institutes of Health and by the Susan Morse Hilles Fund. TR. AM. OPHTH. Soc. vol. LXXXIX, 1991

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protection from or contribu-tion to both ipsilateral and contralateral retinitis in the von Szily model, we have employed in vivo T cell subset depletion techniques in the susceptible BALB/c and in the resistant Igh-1 congenic CB17 mouse, as well as purified T cell subset adoptive transfers into the athymic BALB/c (nude) mouse. The results of these experiments indicate that CD8 (suppressor/cytotoxic) cells are essential for protection against the devastating contralateral retinitis that can develop in certain murine strains after anterior chamber inoculation with HSV, and that both CD4 and CD8 cells are essential participants in the protection enjoyed by the ipsilateral eye in this model. METHODS AND MATERIALS

ANIMALS

Six- to 8-week-old female inbred mice were bred from stock breeders obtained from Jackson Laboratory (Bar Harbor, ME). Mice were housed in micro-isolators, mounted in a ventilated animal rack (VR-1 Lab Products, Inc, BioMedic Corp, Rochelle Park, NJ). The use of animals in this investigation conforms to National Institutes of Health guidelines and to the resolutions on animal care of the Association for Research in Vision and Ophthalmology. The panel of mice employed in our experiments was derived from the BALB/c ByJ breeding colony at Jackson Laboratories. The original construction of the Igh-1 congenic CB17 mouse was performed at Jackson Laboratories, and our breeding stock of CB17 mice was also obtained from this source. The BALB/c (nu/nu) athymic, nude mouse was also obtained, with the appropriate heterozygote breeders, from Jackson Laboratories. Breeding pairs were maintained in our animal colony, and the animals used for these experiments were obtained from the offspring of these breeders at our facility. vIRUS

Stock of HSV type 1 strain KOS were obtained from Dr David Knipe (Harvard Medical School, Boston) and were passed in Vero cells (American Type Tissue Collection, CCL 81, Rockville, MD) in our laboratory. Vero cell monolayers were maintained in 75-cm flasks (Falcon Plastics, Fisher Scientific, Inc, Pittsburgh) using Minimum Essential Media (MEM) with Earle's salts containing 10% fetal bovine serum (FBS) (Grand Island Biological Co, Grand Island, NY), 0.58 mg/ml L-glutamine (GIBCO), 25 ,ug/ml Fungizone (Flow Laboratories, McLean, VA), 100 ,u/ml penicillin, and 100 ,g/ml streptomycin (GIBCO). Confluent Vero cell monolayers were trypsinized, sonicated, centrifuged, and virus harvested in the supernate.

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INOCULATION

Animals were anesthetized with ether. Aqueous humor was drained from the right anterior chamber by paracentesis with a glass needle. The anterior chamber of the right eye was then inoculated with HSV using a 33-gauge needle on a 50-pul Hamilton syringe under binocular microscopy. Each eye was inoculated with 2 x 104 plaque forming units (PFU) of HSV in 5 ,ul of MEM. CLINICAL SCORING

Inoculated mice were examined every 2 days for 10 days using binocular microscopy. Mice were scored for ipsilateral and contralateral intraocular inflammation. Ipsilateral intraocular inflammation was graded on a scale of 1 to 4 depending on anterior chamber cellular reaction, pupillary reflex, iris vascular dilatation, pupillary dilatation, and presence of cataract. Contralateral intraocular inflammation was graded on a scale of 1 to 4 according to degree of vitreous haze, pupillary reflex, dilatation, and iris vascular dilatation. PREPARATION OF TISSUE FOR HISTOPATHOLOGY

Mice were killed 10 or 12 days after anterior chamber inoculation using ether. Both eyes were enucleated, fixed in Karnovsky's fixative (1% paraformaldehyde, 1.25% glutaraldehyde in 0.2 mol/1 sodium cacodylate buffer) for 24 hours at 4°C before rinsing in buffer and dehydrating through ascending concentrations of ethanol. The globes were then infiltrated with glycomethacrylate solution overnight and then embedded in LKB Historesin (LKB Produker AB Bromma, Sweden). Two-micron thick sections were cut using a JB-4 microtome (Sorval, E. I. DuPont, Wilmington, DL) and were stained with hematoxylin and eosin for histopathologic study. Ipsilateral chorioretinitis was defined as the presence of diffuse or focal chorioretinal destruction. Contralateral chorioretinitis was defined as the presence of any combination of chorioretinal necrosis (focal or diffuse), vitreous cellular infiltration, and retinal edema. NIMUNIZATION FOR PRODUCTION OF DONOR T CELLS

BALB/c ByJ mice were immunized 14 and 7 days prior to sacrifice with 2.5 x 105 PFU HSV-1 KOS strain subcutaneously. One day prior to sacrifice, all donor mice were depleted of NK cells with a single intraperitoneal injection of 40 ,ul of anti-asialo GM1 antibody (Wako, Dallas).

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T CELL SUBSET PREPARATION

T cell separation columns (Biotex, Alberta, Canada) were washed with Hanks balanced salt solution (HBSS) with 2% FBS. The columns were activated with the diluted goat antimouse IgG for 1 hour. The donor mice were sacrificed by cervical dislocation, and the spleens were removed and crushed between glass slides. The spleen cells were centrifuged at 130 g, and osmotic red blood cell lysis was achieved with the addition of 9 ml of sterile water with 1 ml of 10 x HBSS. The sample was strained and centrifuged, and the cells were counted with a hemocytometer. One hundred fifty million spleen cells in 1.5 ml of HBSS were added to each column after the columns were washed with 20 ml of HBSS. The sample was eluted with 15 ml of HBSS. The cells were again counted, and the samples were divided into fractions for the CD4 + and CD8 + samples. The anti-CD4 and anti-CD8 monoclonal antibodies (Becton Dickinson, Mountain View, CA) were added to the samples at a concentration of 4 ,ll per million cells targeted and incubated at 0°C for 30 minutes. The excess antibody was removed with three successive washings with HBSS. Sheep anti-rat IgG magnetic Dynabeds (Dynal, Great Neck, NY) were added to each sample at a concentration of 20 million beads to 1 million CD8 + cells for selection of CD4 + cells, and 40 million beads to 1 million CD4 + cells for isolation of CD8 + cells. The mixtures were incubated at 0°C for 30 minutes on a mixer. The magnet was then applied for several minutes to separate the beads from the mixture. The remaining supernatant containing the desired T cell subset was removed for injection into the recipient nude mice. FLOW CYTOMETRIC ANALYSIS

Precolumn and postcolumn samples, as well as post Dynabead fractions, were obtained. One million cells per 100 ,lI were stained with fluorescein- and phycoerythrin-labelled F(ab')2 portions of IgG and IgM (Tago, Burlingame, CA) as controls, and with anti-Thy 1.2, anti-Lyt-2 (CD8), and anti-L3T4 (CD4) antibodies (Becton Dickinson) for 20 minutes at 4°C. The cells were washed twice with phosphate buffered saline with 1% azide and fixed with 2% paraformaldehyde. The samples were analyzed for cell type(s) and degree of purity using a Becton Dickinson FACScan using two-color analysis. CD4 AND CD8 CELL SUBSET ADOPTIVE TRANSFERS

Purified CD4 and CD8 T cell subsets were adoptively transferred into nude (nu/nu) recipients via intraperitoneal injection. Eight million cells (either CD4 or CD8) were injected into each recipient mouse. Additional

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mice received 24 million whole T cell transfers containing CD4 and CD8 cells depleted of NK cells, B cells, and macrophages. IN VIVO T CELL SUBSET DEPLETIONS

The hybridoma cell lines GK1.5 (anti-CD4) and 2.43 (anti-CD8) were obtained from Dr Hugh Auchincloss, Jr (Boston). The cells were grown with MEM, to which was added RPMI 1640, HEPES buffer, gentamicin, nonessential amino acids, glutamine, 2-mercaptoethanol, and fetal calf serum. B6D2F mice were used for production of ascites containing GK1.5 antibody; DBA/2 mice were used for the 2.43 antibody ascites production. These mice were injected intraperitoneally with 0.5 ml of Pristane (Sigma, St Louis) 5 to 10 days before injection of the hybridoma cells. One day prior to hybridoma injection, the mice were irradiated (500 rads). Approximately 3 x 106 cells were injected intraperitoneally into each mouse destined to produce antibody-rich ascites. Ascites were tapped by paracentesis once a week after hybridoma growth and ascites production reached adequate levels. Anti-CD4 antibodies were administered to 10 BALB/c ByJ mice and anti-CD8 antibodies to another 10. Each mouse was injected with 0.1 ml of the appropriate antibody-rich ascites 3 days and 2 days prior to HSV anterior chamber inoculation, as well as 2 days and 7 days after inoculation. Ten control BALB/c mice were not injected with ascites. The T cell subset depletions were monitored by flow cytometry using spleen cell suspensions at the end of the experiment as described previously. ANTI-HSV ANTIBODY DETERMINATION

Blood was collected from the tail veins or carotid arteries of each mouse on day 12 following anterior chamber inoculation with HSV-1. An indirect enzyme-linked immunosorbent assay (ELISA) was used. Serum samples were prepared in twofold serum dilutions (1:25 to 1:1600) and added to HSV-1-coated and control-coated plates (Whitaker Bioproducts, Walkersville, MD). The ELISA for HSV-1-specific IgG antibodies was performed using a 1:2000 dilution of peroxidase conjugatged F(ab')2 rabbit antimouse anti-IgG (heavy and light chains) (Jackson Immunoresearch Laboratories, West Grove, PA). Thirty minutes after the addition of o-phenylenediamine dihydrochloride substrate, the optical density of all HSV-1-coated and control-coated wells was measured using a Titertek Multiskan spectrophotometer (Flow Labs). Data were plotted as the absorbance at 492 nm against the serum dilution, with the background of nonimmune serum controls subtracted out as described previously.4 The end point of sensitive serum was defined as the reciprocal of the dilution

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that produced a mean absorbance of 0.2 above background in triplicate wells. STATISTICAL ANALYSIS

The significance of the results was assessed by chi-square analysis. RESULTS

FLOW CYTOMETRY OF ADOPTIVELY TRANSFERRED CELLS

After T cell column separation of cells, 98% of lymphocytes did not stain with the phycoerythrin- and fluorescein-conjugated F(ab')2 portions of IgG and IgM controls, indicating little, if any, contamination with B cells. Ninety-seven percent of the cells were Thy 1.2+, and 3.7% stained with donkey antimouse Ig, indicating a highly pure population of T cells (Table I). Seventy-four percent of these T cells were CD4 +, and 22% were CD8+. TABLE I: PURITY OF T CELL SUBSETS BY FLOW CYTOMETRY GROUP

Mixed CD4 CD8

THY 1.2+

LYT 2+

L3T4+

97% 99% 95%

22% 0% 91%-95%

74% 98% 0%

After isolation of CD8 + T cells by incubation of whole T cell populations with anti-L3T4 monoclonal antibody and magnetic bead separation, 95% of cells were Thy 1.2 + and 91% were CD8 + with 8% nonstaining cells. No cells stained with anti-L3T4 (CD4) (Fig 1). Isolation of CD4 + cells revealed complete separation of CD4 + cells as well, with no CD8 + cells and 98% CD4 + cells (Fig 2). CHORIORETINAL AND SKIN DISEASE PATTERNS

Ipsilateral iridocyclitis developed in all mice following anterior chamber inoculation. This evolved over 7 days, with decrease in severity ofanterior chamber inflammation, but posterior synechiae and cataract formation. In 30% of control mice, herpes dermatitis developed on the right side of the head by day 8, and 80% had skin lesions by day 10. All had severe skin dermatitis by day 12. Nude mice reconstituted with CD4 + cells had skin lesions by day 6. Nude mice reconstituted with CD8 + cells had much

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"4

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FL2 FIGURE 1

Two-color flow cytometry display of numbers of CD4 and CD8 cells in T cell preparation depleted of CD4 cells. Note large numbers of CD8 cells and lack of CD4 cells detected by flow cytometry.

milder dermatitis, with vessicles in only 25% of mice by day 10. Only 1 of the 11 mice (9%) reconstituted with whole T cell populations had HSV dermatitis. By day 8, 30% of control mice, 60% of CD4 + recipients, and 33% of CD8 + recipients (but none of the mixed T cell recipients) had contralateral uveitis with evidence of pupillary dilatation, iris vessel dilatation, and vitreous haze (Table II). By day 10, contralateral uveitis was present in 76% of control mice (Fig 3), 47% of CD8 + recipients, 80% of CD4 + recipients, and 9% of whole T cell population recipients. By day 12, 35% of the control mice had died; the remaining control mice all had contralateral uveitis and severe encephalitis. Eighty percent of the CD4 +

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X

4

1 4S

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FL2 FIGURE 2

Flow cytometry analysis of CD4 and CD8 cells in T cell preparation depleted of CD8 cells. Note large numbers of CD4 cells and total absence of CD8 cells from this preparation.

recipients, 50% of the CD8 + reconstituted mice, and 27% of the whole T cell recipients had contralateral disease (Table II). None of these mice had encephalitis. There was no significant difference in the presence of contralateral uveitis between control mice and CD4 recipients by chi-square analysis. CD8 and mixed T cell recipients, however, had significantly less uveitis than controls (P 0.05 and P 0.001, respectively). Histopathologic evaluation by day 12 confirmed these clinical findings (Table II) with the exception of more microscopic focal contralateral retinitis in the CD8 recipients (69%). The grade of severity of contralateral retinitis in this group, however, was significantly less (Fig 4A and B). Ipsilateral retinitis was present in all groups, with no statistical difference between the groups (Fig 5A and B). -

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FIGURE 3

Contralateral retina. control nude mouse, 10 days following right eye anterior chamber injection with HSV. Note total destruction of retina with large numbers of inflammatory cells present in area of retinal necrosis.

TABLE

GROUP

II: PRESENCE OF RETINITIS (DAY 12)

CLINICAL CONTRALATERAL

Control CD4 CD8 Mixed T cell

100% 80% 50% 27%

HISTOPATHOLOGIC IPSILATERAL CONTRALATERAL

100% 100% 72% 73%

100% 80% 69% 27%

There was significant anti-HSV antibody production in all but one of the whole T cell recipients (Table III). This antibody-deficient mouse was the only mouse with clinically significant contralateral retinitis by day 10. In the CD8+ and control groups there was no significant difference in antibody levels between the highest dilution and the control wells, indicating that antibody production was not responsible for protection from contra or ipsilateral disease in the CD8 reconstituted nude mice.

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FIGURE 4

A: Contralateral retina, nude mouse recipient of adoptively transferred CD4 cells. Note profound retinal necrosis from contralateral necrotizing retinitis 10 days following right eye anterior chamber corneal inoculation with HSV. B: Contralateral retina, nude mouse, 10 days following right eye anterior chamber inoculation with HSV in CD8 T cell adoptive transfer recipient. Note remarkable difference in degree of contralateral retinitis in this CD8 recipient.

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FIGURE 5

A: Ipsilateral retina, nude mouse, CD4 transfer recipient. Note lack of protection from ipsilateral retinitis. B: Ipsilateral retina, 10 days following right eye anterior chamber inoculation with HSV in nude mouse recipient of CD8 adoptively transferred cells prior to inoculation. Note ipsilateral retinitis, indicating lack of protection from ipsilateral retinitis from adoptively transferred CD8 cells.

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TABLE III: ANTI-HSV-1 ANTIBODY DETERMINATION BY ELISA GROUP

ANTI-HSV-1 ANTIBODY TITER

Control CD4 CD8

Mixed T cell

1:15 1:170 1:8 1:630

There was significant anti-HSV antibody production in four of the five CD4 + recipients. CD8 AND CD4 CELL DEPLETION OF CB17 AND BALB/c MICE

CB17 mice, ordinarily HSV retinitis-resistant, were rendered deficient of CD4 or CD8 T cell through monoclonal antibody anti-CD4 or anti-CD8 treatment. The extent of CD4 and CD8 T cell depletion was monitored by flow cytometry analysis of anti-CD4 and anti-CD8 stained aliquots of peripheral blood lymphocytes isolated at the end of the experiment (day 12) (Table IV). The impossibility of obtaining adequate amounts of blood repeatedly, longitudinally, during the course of the study for peripheral blood lymphocyte flow cytometry analysis precluded our ability to analyze the extent of depletion throughout the course of the study. TABLE IV: FLOW CYTOMETRY ANALYSIS OF CD4 AND CD8 DEPLETIONS

Control BALB/c CD4 depleted BALB/c CD8 depleted BALB/c Control CB17 CD4 depleted CB17 CD8 depleted CB17

CD4

CD8

21% 1.5% 19% 18% 0.8% 19%

11% 18% 0.9% 10% 20% 0.7%

Depression of either the CD8 or the CD4 T cell subset in CB17 mice resulted in the development of pronounced ipsilateral necrotizing retinitis after anterior chamber inoculation of HSV (Table V). Ipsilateral retinitis also developed in BALB/c mice after either CD4 or CD8 depletion (Table V), and, interestingly, a dramatic enhancement in the retinitis in the contralateral eye was also seen in BALB/c mice depleted of CD8 T cells.

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TABLE V: RETINITIS AFTER CD4 OR CD8 T CELL DEPLETION IPSILATERAL

CB17 controls CD4 depleted CB17 CD8 depleted CB17 BALB/c controls CD4 depleted CB17 CD8 depleted CB17

0% 100% 100% 0% 100% 100%

(0/20) (0/20) (0/20) (0/20) (20/20) (20/20)

CONTRALATERAL

0% 0% 0% 40% 30% 90%

DISCUSSION

Von Szily1 first described the phenomenon of ipsilateral anterior uveitis, delayed contralateral necrotizing retinitis, and ipsilateral retinal sparing in rabbits after anterior chamber inoculation with live HSV in 1924. Whittum and associates2 modified this model to BALB/c mice in 1984, and from that point the search for explanations for these two interesting phenomena, the contralateral retinitis and the ipsilateral retinal sparing, has been intensively pursued by several research groups. Several observations have been reproduced by the various groups and form a body of information about this model that is more or less agreed upon by those involved with similar research. Live virus must be present to produce the model. The virus must reach the contralateral retina to produce the contralateral necrotizing retinitis. At least one route (and perhaps the predominant one) through which the virus spreads from the inoculated to the contralateral eye is through neural pathways to the brain, with subsequent migration through the contralateral optic nerve into the contralateral retina.5 Various inbred strains of mice exhibit differential susceptibility to development of the contralateral retinitis.3 At least one genetic marker for susceptibility to the contralateral retinitis is the Igh-1 gene on chromosome 12. Congenic mice differing in less than 1% of the genetic material on chromosome 12 around the Igh locus exhibit extraordinary differences in susceptibility to development of the von Szily model. CB17 (H2d, Igh-lb) mice are extremely resistant to development of contralateral retinitis, but BALB/c (H2d, Igh-la) mice are quite susceptible; CAL-20 (H2d, Igh-1d) mice are intermediate between these two.3 HSV can be isolated and cultured from the contralateral retina, even in the "resistant" mouse strains not exhibiting the contralateral retinitis.6'7 The immunopathology of the retina in mice developing contralateral retinitis is characterized by macrophage, natural killer cell, and T lymphocyte infiltration. The CD4 subset of T cells is more prominent than is the CD8 subset in the inflammatory infiltrate.8

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Bilateral destructive retinitis develops in athymic (nude) mice after anterior chamber inoculation with HSV.8 These mice lack T cells but have abundant macrophages and natural killer cells, and it is these cells that are present in the inflammatory infiltrates of the retina. Taken together, these replicated and confirmed findings indicate that the contralateral retinitis seen in the von Szily model, while requiring the presence of live HSV, does not develop primarily because of HSV cell lysis, but rather because of the animal's inflammatory response to the virus, with macrophages and natural killer cells being the predominant cell types responsible for the retinal destruction in the contralateral eye. But why is the ipsilateral retina spared? And why is the contralateral retina of mice of certain "resistant" strains not destroyed, even though live virus is present in the contralateral retina? Although the ipsilateral retina of all mice (except athymic mice) is architecturally normal in the von Szily model, a relatively large number of T lymphocytes is present in the ipsilateral choroid.7 IgG-bearing B cells may be found in the ipsilateral choroid as early as 2 days after HSV inoculation, and relatively large numbers of helper (CD4) T cells are found in both the anterior chamber and the ciliary body of the ipsilateral eye within 24 to 48 hours after HSV inoculation. This T cell response precedes the appearance of macrophages and suggests a helper T cell response with B cell activation. Ipsilateral local ocular antigen processing and subsequent immunoregulatory events that modulate the generation of an inflammatory response in the ipsilateral retina may account for ipsilateral retinal sparing in this model. Our results, reported herein, suggest this also, and further suggest that both CD4 and CD8 T cell subsets are critical for the protection from destructive inflammation enjoyed by the ipsilateral retina. Absence of either subset renders all mice susceptible to destructive ipsilateral retinitis. Freedom from destructive contralateral retinitis enjoyed by select strains of inbred mice is more difficult to fully understand at this point. Our data, however, give the first indication that CD8 lymphocytes may play some role in protecting the contralateral retina from a destructive inflammatory response to the presence of HSV, independent of anti-HSV antibody. In 1985, Whittum-Hudson and associates9 suggested a possible protective role from contralateral retinitis for CD8 (suppressor) T cells. Their work differed from ours dramatically in that it was based on pharmacologically modified mice, whereas our experiments were performed in adult nude mice or in mice selectively depleted of one T cell subset or another. It is also clear from other experiments that anti-HSV antibody alone is a potent protector of the contralateral retina and ipsilateral retina.

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Acute retinal necrosis in the human is a devastating event, often with a poor visual outcome in spite of apparently successful control of the viral process through high-dose intravenous antiviral therapy. Both human and murine histopathologic studies demonstrate, quite clearly, that it is the individual's inflammatory response to the virus that is responsible for the tissue destruction resulting in blindness. It would seem that one of the most reasonable ways to attempt to change this sad state of affairs is to better understand the immune/inflammatory response to HSV in general and in a model of necrotizing retinitis or acute retinal necrosis in particular. Our results to date suggest that therapeutic strategies aimed at modulating natural killer cells and/or macrophage activity or enhancing CD8 T cell activity and/or anti-HSV antibody availability early in the course of HSV retinitis may have an ameliorating effect on the destructive consequences of HSV replication in the retina. Further studies in our laboratory on the murine von Szily model to test these hypotheses are in progress. REFERENCES 1. von Szily A: Experimentelle endogene Infektions Ubertragung von Bulbus zu Bulbus. Klin Monatsbl Augenheilkd 1924; 72:593-602. 2. Whittum JA, McCulley JP, Niederkorn JY, et al: Ocular disease induced in mice by anterior chamber inoculation of herpes simplex virus. Invest Ophthalmol Vis Sci 1984; 25:1065-1073. 3. Foster CS, Opremcak EM, Rice B, et al: Clinical, pathologic, and immunopathologic characteristics of experimental murine herpes simplex virus stromal keratitis and uveitis is controlled by gene products from the Igh-1 locus on chromosome 12. Trans Am Ophthalmol Soc 1987; 85:293-311. 4. McKendall RR, Woo W: Antibody activity to herpes simplex virus in mouse Ig classes and IgG subclasses. Arch Virol 1988; 98:225-233. 5. Atherton SS, Streilein JW: Two waves of virus following anterior chamber inoculation of HSV-1. Invest Ophthalmol Vis Sci 1987; 28:571-579. 6. Cousins SW, Gonzolez A, Atherton SA: Herpes simplex retinitis in the mouse: Clinicopathologic correlations. Invest Ophthalmol Vis Sci 1989; 30:1485-1494. 7. Hemady R, Tauber J, Ihley TM, et al: Viral isolation and systemic immune responses after intracameral inoculation of herpes simplex virus type 1 in Igh-1-disparate congenic murine strains. Invest Ophthalnol Vis Sci 1990; 31:2335-2341. 8. Zaltas MM, Opremcak EM, Hemady R, et al: Immunopathology of herpes simplex virus chorioretinitis in the von Szily model. Invest Ophthalmol Vis Sci 1991. In press. 9. Whittum-Hudson J, Farazdaghi M, Predergast RA: A role for T lymphocytes in preventing experimental herpes simplex virus type 1-induced retinitis. Invest Ophthalmol Vis Sci 1985; 26:1524-1532.

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DISCUSSION DR LEE M. JAMPOL. Doctor Foster and co-workers have presented further work investigating the mechanisms of retinal necrosis in mice with the von Szily model of herpes simplex retinitis. They relate this murine necrotizing retinitis to human acute retinal necrosis by titling the manuscript "Murine Acute Retinal Necrosis." Before discussing their results, I wish to address whether or not this murine model is relevant to human acute retinal necrosis. The acute retinal necrosis syndrome (Surv Ophthalmol 1991; 35:327-343) is an apparently new disease, characterized by the development of anterior segment inflammation, an occlusive retinal vasculitis (mostly arteritis), and large confluent areas of necrotizing retinitis. Severe vitreous inflammation may be present. The disease usually begins in the peripheral retina. The original description of the disease was in healthy, immunocompetent patients, although a similar retinopathy may be noted in patients with AIDS and other immunocompromised patients. Is murine acute retinal necrosis really the same disease as human acute retinal necrosis? My first comparison is in regard to the viruses involved. The von Szily model utilizes herpes simplex virus (HSV). Interestingly, the majority of cases of human acute retinal necrosis are caused by herpes zoster, although a minority are definitely due to HSV. The evidence implicating other herpes viruses, such as cytomegalovirus, is weak, although some reports have suggested that cytomegalovirus may occasionally be the etiologic agent. The trademark of the von Szily model of HSV is that in immunocompetent animals, the ipsilateral retina is relatively protected following anterior chamber inoculation of the virus whereas the contralateral retina demonstrates severe retinitis with necrosis. I am not aware of any evidence implicating this mechanism in humans. The acute retinal necrosis syndrome in humans may be unilateral or bilateral. Often the disease is first noted as a severe unilateral anterior uveitis. However, in these situations, the necrotizing retinitis first develops ipsilateral to the anterior chamber reaction. The second eye may be simultaneously or subsequently involved. Almost invariably the involvement of the second eye is, at least initially, less severe than that of the first eye. This may reflect a delay in spread of the virus or some immune protection. There may be short or prolonged periods of latency before the second eye becomes involved. What are the mechanisms of retinal necrosis in the murine and human diseases? Foster and others working on the von Szily model have developed strong evidence that the murine immune response is responsible for the majority of tissue necrosis. Live virus is necessary in the involved retina, but histopathologic studies strongly suggest that a cell-mediated immune response to the virus is responsible for a significant proportion of the tissue destruction (Invest Ophthalmol Vis Sci. In press). Current clinical concepts of acute retinal necrosis in humans implicate the occlusive vasculitis, the resultant retinal ischemia, and viral cell damage as the mechanisms of tissue damage. So far, discussions have not implicated the immune response itself as a major mechanism of tissue damage. Histopathologic studies, however, have shown considerable cellular infiltration of the human retina. In

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addition, there is work by Holland and co-workers (Am J Ophthalmol 1989; 108:370-374) suggesting that a specific HLA type, HLA DQW7, may be associated more frequently with acute retinal necrosis. This HLA type is present in only 19% of the population at large, but was present in over half of their patients with acute retinal necrosis. Japanese studies (J Eye [Atarashii Ganka] 1989; 6:107-114) have implicated other HLA types. Thus, immune response may play some role in tissue necrosis in the human as well. The mainstay of therapy in the human disease is antiviral therapy, usually acyclovir. This antiviral therapy definitely promotes healing of the retinal lesions and protects the second eye. The prompt response to antiviral therapy with or without anti-inflammatory therapy suggests to me that the major mechanisms of damage in the human disease may be viral rather than immunologic. However, both factors may be important. Once antiviral therapy has been initiated, systemic or periocular corticosteroids may be helpful in minimizing retinal damage. Corticosteroids have been thought to be effective by modulating inflammation rather than by suppressing an immune response. To my knowledge, there is no evidence that corticosteroid therapy alone or suppression of the immune response alone is of any value in protecting the first or second eye in patients with acute retinal necrosis. Interestingly, in the mice antiviral therapy (acyclovir) is similarly effective, and the combination of antiviral therapy and corticosteroids is apparently beneficial (Invest Ophthalmol Vis Sci [Suppl] 1990; 31:224). The major complication leading to visual loss in the human disease is the development of retinal detachment, which occurs despite effective antiviral therapy. I do not know if detachments occur in the mice. Recent work has attempted to prevent the subsequent development of human retinal detachment with prophylactic laser treatment. Other investigators have concentrated on development of more effective surgical techniques to reattach retinas in these patients. In some patients, prophylactic pars plana vitrectomy, scleral buckling, and even intraocular infusion of acyclovir have been used, but the value of these therapies is uncertain. A second mechanism of visual loss in human acute retinal necrosis is optic nerve swelling. Optic nerve sheath decompression may be of some benefit in this condition. This remains speculative. Despite apparent differences between human and murine acute retinal necrosis, Doctor Foster's work on this fascinating murine disease is important. The present work presents convincing evidence that: The protection of the ipsilateral retina from retinal necrosis depends on the presence of both CD4 and CD8 cells and that CD8 cells are necessary for protection of the contralateral eye from retinitis. Previous work by this group demonstrated that host genetic factors (Trans Am Ophthalmol Soc 1987; 85:293311) and in some situations anti-herpes antibody (In press) are crucial for the protection of the contralateral eye. Humans with AIDS and acute retinal necrosis have depleted CD4 cells, but most patients with acute retinal necrosis have normal CD4 and CD8 cell counts and normal immunoglobulin response and are apparently immunocompetent.

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I want to thank the authors for providing me with their manuscript in advance and presenting this important work on this unique animal model. Additional immunologic, virologic, and histopathologic studies on immunocompetent and immunosuppressed patients with human acute retinal necrosis will provide further knowledge of the mechanisms of disease in these patients. Doctor Foster's work may help as well. DR HUGH R. TAYLOR. I would like to thank Doctor Foster for an interesting presentation. This model not only addresses a very important clinical problem but also asks some very important immunological questions in its own right. I would like to ask Doctor Foster a question about the mechanisms of natural resistance. As I understand it the CJ57 black mice are naturally resistant to developing retinitis, in both the ipsilateral and contralateral eyes. When these mice are immuno-depleted they develop ipsilateral retinitis but still do not develop contralateral retinal disease. This suggests that the protection against retinal infection in the ipsilateral eye is immunologically based, but there must be other nonimmune mechanisms that protect the contralateral eye. I was wondering whether he had information on what these mechanisms might be. For example, whether they affect neuronal uptake or transport, or otherwise relate to the ability of the virus to enter those cells. Such a finding might have relevance to the development of latency and, therefore, immunity against infections. Thank you. DR DEVRON H. CHAR. This is a very elegant model. I just have one query. What we have seen in tumor immunology has been fascinating in that initially we thought the cytotoxic T cells played a major role in cytotoxicity on a direct cell to cell basis. What has become more apparent over the last 18 months is that often those cells, in fact, are producing their effects indirectly through soluble cytokines. I wonder, in this model, what CD8 cells are doing. Do they directly influence the disease on a cellular basis or through a soluble cytokine (lymphokine)? DR C. STEPHEN FOSTER. I would like to thank the discussants very much for their remarks and insights. With respect to Doctor Jampol's comments it is quite clear that this model is not the same as human acute retinal necrosis. We do hope, though, that it will provide some opportunity to gain some insights into therapies that might be more successful than the current therapies that we who are involved in treating our patients are currently having. The mice do not develop retinal detachments. They do develop optic neuritis and one can create something that is slightly more like the human equivalent if one chooses to do the primary inoculation in a different way. I did not want to get into this and I will not spend a lot of time on it, but there is something extremely special about placing antigenic material, viral or otherwise, into the anterior chamber. A complex series of immunologic events have developed and we think that those events explain, at least in part, some of the interesting phenomena that we see in this model. But, in

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fact, if one instead introduces the virus directly into the vitreous cavity one develops a necrotizing retinitis in the ipsilateral eye. There is no ipsilateral retinal protection. And so in that sense it is slightly more like the human equivalent. Vasculitis is not part of the mechanism of retinal destruction but rather it is a cell mediated immune response which creates the destruction. Antiviral agents as Doctor Jampol commented on in this model are effective. The addition of steroids reduces the amount of inflammation and the retina is slightly more preserved when one combines the therapies. Steroids alone make the process considerably worse. In regard to comments made by Doctor Taylor asking a question about natural resistance: his was one of the features that fascinated us the most about this model. That is to say, different inbred mice exhibit extraordinary differential susceptibility to development of this model. His comments had to do with black mice and black mice in general are highly resistant. What explains this differential susceptibility? Well, we tried to get at that question by looking at congenic mice that differ one from the other in a very tightly confined region of their genetic material, specifically, 1% of their deoxyribonucleic acid chromosome 12, in an effort to eliminate as many of the confounding variables as we can eliminate. Our work has led us to the conclusion that at least one profound influence on the susceptibility to this model is in genes that code for immunoglobulin heavy chains. Possibly genes nearby that affect T cell receptors, as well may be involved. It is clear if one talks about other mouse strains, however, black mice compared to A/J, for example, that many other things can be discovered that are different between those mice. Natural killer cell activity is tremendously different between A/J and C57 black mice. Just the ability of cells, retinal and corneal, to support viral replication is dramatically different from one murine strain to another. Antibody and T cell functions in this congenic Igh-1 group are the only things that we can demonstrate so far that are different. Cell support of viral replication and the like are the same. Finally, with respect to Doctor Char's question: how are these CD8 cells in fact providing some protection in the nude mice that received them? Are they doing so by killing virus or virally infected cells? Are they doing so by elaborating cytokines which are having the same effect without cell contact or by having a surrogate effect by recruitment of other cell types that can kill a virus? Perhaps. Frankly, my suspicion is that they are doing so through an entirely different mechanism. My suspicion is that they are doing so through their function of immunoregulation in their function as suppressor T cells rather than an aggressive killing type T cell. Again, many thanks.