The role of natural killer cells in murine cytomegalovirus eye infection.

PURPOSE: To study the role of natural killer cells in the dissemination of virus to the eye and virus growth as well as the production of lesions in the eye during primary infection of murine cytomegalovirus. METHODS: Virus activity and lesion production by murine cytomegalovirus in various organs of natural killer cell-depleted BALB/c strain of inbred mice were compared with those of immunocompetent normal BALB/c mice. RESULTS: In mice injected intraperitoneally with murine cytomegalovirus, virus could be isolated from 50% of eyes in the natural killer cell-depleted group whereas no virus was detected from any eye in the control group. In natural killer cell-depleted mice with positive virus isolation from eyes, no murine cytomegalovirus was isolated from either optic nerves or trigeminal ganglia whereas virus was detected from blood lymphocytes, macrophages, granulocytes, and plasma. After intravitreal injection of murine cytomegalovirus, virus titers in eyes of natural killer cell-depleted mice were significantly higher than those in the control group. Derangement of retinal cell layer and inflammatory cells as well as cytomegalic cells in iris and ciliary body were noted in natural killer cell-depleted group, whereas no such changes were observed in the eyes of the control mice. CONCLUSION: Natural killer cell depletion enhances the dissemination of murine cytomegalovirus to the eye through the hematogenous route, and increases virus multiplication as well as lesion production in the eye.

Protective effect of anti-glycoprotein D antibody on herpetic chorioretinitis in newborn rabbits.

Subcutaneous injection of type 2 herpes simplex virus (HSV-2) (10(3) PFU) to newborn rabbits produced severe skin lesions and wide dissemination of the virus to various organs including the eye. Ocular lesions were characterized by retinal folds and choroiditis. HSV could be isolated from mononuclear cells (MNCs) of infected animal blood. Newborn rabbits treated with monoclonal antibody (MAb) against glycoprotein D (gD) of HSV on days 0, 2 and 4 postinfection had little or no skin lesions (0.0-2.3 mm) compared to controls (2.8-13.0 mm). In addition, the MAb treatment significantly suppressed dissemination of the virus to the eye (0% in MAb-treated vs 83% in control) and other organs and reduced the rate of chorioretinitis (0% in MAb-treated vs 50% in control). The treatment of HSV-infected MNCs with MAb resulted in 91-100% reduction in infectivity of the cells. The results suggest that anti-gD MAb protects newborn rabbits from HSV-2 eye infection by neutralizing the virus in skin and inactivating HSV-infected MNCs in blood.

In vivo reactivation of latent murine cytomegalovirus in the eye by immunosuppressive treatment.

Intravitreal inoculation of 10(3) pfu of murine cytomegalovirus (MCMV) to 3-week-old BALB/c mice resulted in virus isolation from eye homogenates for 2 weeks and from co-cultured specimens of the same eye up to 5 weeks after inoculation, indicating that MCMV in the eye became latent 2 weeks after the virus inoculation. Immunosuppressive treatment with daily intramuscular injections of cyclosporine (40 micrograms/g) and cortisone acetate (125 micrograms/g) 9 weeks after intravitreal MCMV inoculation resulted in isolation of infectious virus from ten of 44 eye homogenates (10 of 22 mice) during a 3-week period, indicating in vivo reactivation of latent ocular MCMV. No virus was isolated from eye homogenates of similarly infected mice with sham immunosuppression (daily intramuscular saline injections), nor was any virus isolated from uninfected eyes with immunosuppressive treatment. Three weeks of daily cyclosporine and cortisone injections depleted L3T4+ cells to 6.0%, Lyt-2+ cells to 20% and anti-MCMV antibody to 10% of untreated mice. The results suggest that the eye can serve as a site of latent MCMV that can be reactivated by immunosuppressive means.

Induction of Fc and C3b receptors on rabbit corneal cells by herpes simplex virus.

The expression of receptors for Fc portion (FcR) of immunoglobulin G (IgG) and for a C3b component of complement (C3bR) by herpes simplex virus (HSV) was studied in primary cultures of rabbit corneal cells. Monolayer cultures of epithelial, stromal and endothelial cells of the rabbit cornea were infected with three strains each of type 1 (HSV-1) and type 2 HSV (HSV-2). Rosette methods were used to detect receptors by means of sheep erythrocytes sensitized with rabbit IgG for FcR and C3b-coated sheep erythrocytes for C3bR. The FcR were expressed regularly on epithelial, stromal and endothelial cells by all three strains of both HSV-1 and HSV-2. The C3bR, however, were expressed only by HSV-1 on epithelial and stromal cells. Little or no C3bR activities could be detected on endothelial cells infected with any strain of HSV-1 or HSV-2. The FcR and C3bR expressed on corneal cells were induced by HSV and were blocked by monoclonal antibody to HSV-1 glycoprotein E(gE) or glycoprotein C(gC) respectively, confirming findings of other investigators that gE acts as FcR and gC as C3bR.

Effect of neuraminidase on Fc and C3b receptors on rabbit corneal cells infected with herpes simplex virus.

The effect of neuraminidase on Fc receptors (FcR) and C3b receptors (C3bR) was studied in epithelial, stromal and endothelial cells of the rabbit cornea infected with type 1 (HSV-1) and type 2 herpes simplex virus (HSV-2) in vitro. FcR were induced on epithelial, stromal and endothelial cells of the rabbit cornea by both HSV-1 and HSV-2, but their activities were not enhanced by neuraminidase. On the other hand, the treatment of HSV-infected corneal cells with neuraminidase resulted in the enhancement of C3bR activities on epithelial, stromal and endothelial cells infected with HSV-1, and the enhancing effect of neuraminidase was more pronounced on corneal endothelial cells. A similar neuraminidase treatment had no significant effect on C3bR activities on the corneal cells infected with HSV-2.

Protective effect of passive immunization on herpetic retinitis of newborn rabbits.

We studied the protective effects of passive immunization with virus specific antibody in newborn rabbits inoculated subcutaneously with type 2 herpes simplex virus (HSV-2). Newborn rabbits given anti-HSV-2 antibody intraperitoneally (IP) on days 0, 2 and 4 post infection had smaller herpetic skin lesions and reduced mortality when compared to controls. In addition, the IP treatment using this schedule reduced virus growth in the skin lesions and virus dissemination, so that it decreased the frequency of herpetic retinitis. When the IP antibody administration was started at 24 hours post virus inoculation, according to the schedule days 1, 3 and 5, there was less protection; larger skin lesions, higher mortality, and greater evidence of virus dissemination. Also HSV-infected mononuclear cells (MNCs) treated with anti-HSV serum resulted in a significant reduction in the number of infected MNCs. The results of these studies suggest that anti-HSV-2 antibody contributes to protection against HSV-2 infection of skin as well as eyes, probably by inactivation of the virus locally at the skin inoculation site, and by combating the hematogenous spread of HSV-infected MNCs as well as free virus to various organs including the eye.

Role of virus-infected mononuclear leukocytes in herpetic chorioretinitis of newborn rabbits.

The role of virus-infected mononuclear leukocytes (MNLs) in the pathogenesis of neonatal herpetic chorioretinitis in newborn rabbits was investigated. As early as 2 days after inoculating the animals' skins with type 2 herpes simplex virus (HSV-2), infectious MNLs in the infected animals' peripheral blood were found. The virus was associated, for the most part, with MNLs that belonged to phagocytic and adherent cell fractions. Observations by electron microscopy indicated that HSV-2 was actively replicating in the MNLs. It was also found that as few as 80 virus-infected MNLs injected via the right common carotoid artery were capable of inducing the chorioretinal lesions in 50% of the eyes, but that as many as 10(3) Pfu of free virus were required to produce the same lesions in the same percentage of eyes. This result clearly indicated that virus-infected MNLs were far more efficient in producing chorioretinitis than free virus, and may thus play a crucial role in the pathogenesis of herpetic chorioretinitis in newborn rabbits. When 111In-labeled virus-infected or uninfected MNLs were injected into normal rabbits via the right common carotid artery, the virus-infected MNLs localized more readily in the eye than the uninfected MNLs. The virus-infected MNLs also attached to the cultured vascular endothelial cells significantly more often than the uninfected MNLs. These results suggested that virus-infected MNLs might be easily trapped in the circulation of the eye and, in this way, produce the ocular lesions.

Role of lymphocyte and antibody in the pathogenesis of immune-mediated herpetic uveitis.

We investigated the role of various immune components in the pathogenesis of immune-mediated experimental herpetic uveitis. Inbred III/J strain of rabbits were sensitized with an intravitreal injection of 10(3) PFU of type 1 herpes simplex virus (HSV), and sensitized cervical lymph node (LN) cells were obtained on postinfection day 12. Intravitreal injection to the normal III/J rabbit eye of HSV antigen with either sensitized LN cells or anti-HSV serum failed to induce uveitis, whereas intravitreal injection of HSV-antigen with both sensitized LN cells and anti-HSV serum produced severe uveitis within six hours. The combination of sensitized LN cells, HSV-antigen and normal rabbit serum, or that of normal LN cells, HSV antigen and anti-HSV serum, did not induce uveitis. Further studies using B lymphocyte and T lymphocyte fractions from sensitized LN showed that only the combination of sensitized T lymphocytes, HSV antigen and anti-HSV serum regularly produced uveitis following intravitreal injection. These results indicate that the interaction of HSV antigen, sensitized T lymphocytes and anti-HSV antibody may play a role in the pathogenesis of immune-mediated herpetic uveitis.

Suppression of secondary herpes simplex uveitis by cyclosporine.

The authors studied the effect of an immunosuppressive agent, cyclosporine (CyA), on experimental secondary herpes simplex (HS) uveitis. Secondary HS uveitis was induced in a rabbit eye that had recovered from primary HS uveitis by challenging it with an intravitreal injection of herpes simplex virus (HSV) antigen. Daily intramuscular injections of CyA (25 mg/kg body weight) for 7 days prior to the intravitreal challenge with HSV antigen significantly suppressed the induction of secondary HS uveitis, but daily injections of CyA after the challenge with HSV antigen was ineffective. Intravitreal injections of CyA (5 mg) 7 days and 3 days prior to the HSV challenge were less effective, but the combined treatment with seven daily intramuscular CyA and two intravitreal CyA injections prior to the HSV challenge was most effective in the prevention of the uveitis. The daily intramuscular treatment with CyA resulted in a marked reduction of cell-mediated immunity while leaving the level of circulating HSV specific antibody high. No reactivation of latent HSV was detected in trigeminal and superior cervical ganglia of CyA-treated rabbits.

Immunology of viral infections.

Immune responses to nonenveloped and enveloped viruses are different (see Table 1). The immune reactions to nonenveloped viruses are predominantly humoral responses against extracellular viruses, because nonenveloped viruses usually are released rapidly, a process that results in lysis and death of the infected cell, and because viral antigens are not present on the cell membrane. In contrast, cell-mediated immunity, together with humoral immune response, plays a major role in the defense against enveloped viral infections, since the release of enveloped viruses from the infected cells occurs slowly and the cell membrane contains viral antigens that become targets for effector cells of cell-mediated immunity.

Biologic properties of the thymocyte-activating factor (CETAF) produced by a rabbit corneal cell line (SIRC).

Rabbit corneal epithelial cell cultures produce a cytokine (CETAF) that greatly enhances the proliferation of C3H/HeJ mouse thymocytes. The rabbit corneal cell line SIRC was used to generate CETAF activity in the culture supernatant. CETAF was then partially purified by Sephacryl S-200 gel filtration, where peaks of activity eluted in a molecular weight range of 95,000-55,000 (CETAF I) and 30,000-15,000 (CETAF II). Similar to the epidermal cell-derived thymocyte-activating factor (ETAF), CETAF (I and II) stimulated the growth of a human dermal fibroblast line (CRL 1445) in a dose-dependent manner, but failed to enhance the proliferation of an Interleukin 2 (IL 2)-dependent T-cell line (CT 6). Although CETAF did not exhibit any IL 2 activity, it clearly enhanced the IL 2 production by C3H/HeJ mouse splenocytes stimulated with suboptimal doses of lectins. Crude SIRC supernatants as well as the partially purified CETAF preparations showed a marked inhibition of polymorphonuclear neutrophil migration at high concentrations, but were significantly chemotactic when diluted samples were tested. CETAF release by SIRC cells was increased by stimulation with mitomycin C, phorbolmyristate acetate, hydroxyurea, silica, lipopolysaccaride B, and when the cells were cultured under serum-free conditions. These observations suggest that corneal epithelial cells may not only interact with the immune system in a way similar to keratinocytes, but may also stimulate corneal stromal cell through the production of CETAF.

Ocular lesions associated with dissemination of type 2 herpes simplex virus from skin infection in newborn rabbits.

The subcutaneous inoculation of the backs of New Zealand white rabbits 17 to 34 hr old with 10(3) 50% tissue culture infection dose (TCID50) of type 2 herpes simplex virus (HSV-2) induced cutaneous lesions within 24 hr, foci of disseminated infection in many organs (including the eye) on day 3 and thereafter, and the death of the animals on day 5 with infection of the central nervous system. Infectious HSV-2 could be isolated from the mononuclear cells and plasma of the peripheral blood, indicating the active role of both elements in the dissemination of the virus. Infectious HSV was also recovered from the corresponding sensory ganglia of the skin lesion (the cervicothoracic ganglia) as early as 2 days after the subcutaneous inoculation of the virus. About 40% of the animals developed ocular consisting of retinal folds with or without degenerative changes, Iritis and choroiditis also developed in some eyes. Infectious HSV-2 could be isolated from 33% of the eyes on days 4 and 5. Thus the newborn rabbit may serve as a suitable experimental animal for the study of HSV-2-induced chorioretinitis in the human newborn.

Acyclovir therapy of neonatal herpes simplex virus type 2 infections in rabbits.

New Zealand white rabbits less than 30 h old were inoculated subcutaneously with 10(3) 50% tissue culture infectious doses of type 2 herpes simplex virus. The animals were randomly assigned to a treatment schedule of daily intraperitoneal injections of acyclovir, beginning on the day of virus inoculation for 6 or 12 days, on post-inoculation day 1 for 6 days, or on post-inoculation day 2 for 6 days. The acyclovir was given in doses of 50 mg/kg of body weight per day. Similarly infected animals receiving daily intraperitoneal injection of Eagle minimum essential medium served as controls. All of the control animals died on day 4 or 5 after inoculation. At death they exhibited severe skin lesions, viremia, and dissemination of virus in various visceral organs and spinal as well as trigeminal ganglia. In contrast, animals treated with acyclovir failed to develop significant skin lesions, and death did not occur while treatment continued. Termination of treatment after 6 days resulted in late-onset fatal disease and virus isolation from the brain in many rabbits regardless of the treatment schedule. No such late fatality was observed and no virus could be detected from the brain when treatment was initiated on the day of virus inoculation and continued for 12 consecutive days. With respect to all of the variables studied, treatment for 12 days beginning on the day of virus inoculation was most effective.

Different susceptibilities of skin to type 1 and type 2 herpes simplex viruses in newborn rabbits.

Skin infections with type 1 herpes simplex virus (HSV-1) were compared with skin infections with type 2 virus (HSV-2). Five strains each of HSV-1 and HSV-2 were tested by injecting 10(3) 50% tissue culture infective doses of each strain subcutaneously into 1-day-old New Zealand white rabbits. All five strains of HSV- 2 produced severe skin lesions that resulted in wide dissemination of the infection to many organs, paralysis of the hind legs, and finally death. The virus could be isolated frequently from skin lesions, from various organs (liver, lungs, adrenal glands, brain, and eyes), and from circulating leukocytes and plasma. In contrast, all five strains of HSV-1 failed to produce significant skin lesions or dissemination of virus, only half of the skin lesions yielded HSV, and no virus could be isolated from the blood. These results indicate that HSV-1 dose not grow well in the skin of newborn rabbits and fails to disseminate, whereas HSV-2 is dermatotropic and disseminates readily to many organs by hematogenous routes.

Aspirin, cyclophosphamide, and dexamethasone effects on experimental secondary herpes simplex uveitis.

The effects of aspirin, cyclophosphamide, and dexamethasone on secondary herpes simplex uveitis were studied in rabbits. Neither daily treatment with aspirin (rectal suppositories, 650 mg begun 24 hours before challenge) nor cyclophosphamide injections every two days (80 mg begun eight days before challenge) had any effect on the severity of the uveitis, on the rise in intraocular pressure (IOP), or on the host's immune responses. As in the control animals, infectious herpes simplex virus (HSV) could not be isolated from iris tissues of either aspirin- or cyclophosphamide-treated rabbits. On the other hand, twice-daily treatment with topical dexamethasone (0.1% drops begun 24 hours before challenge) lessened the severity of the uveitis appreciably and suppressed the rise in IOP, but iris tissues yielded infectious HSV in two of ten eyes. Although the dexamethasone had no effect on the neutralizing-antibody or macrophage migration inhibition factor, it markedly suppressed the chemotactic activity of the aqueous humor for both polymorphonuclear leukocytes and macrophages.

Herpetic eye disease in rabbits after inoculation of autonomic ganglia.

Since herpes simplex virus (HSV) can cause persistent infection of autonomic ganglia of both humans and experimentally infected animals, we followed the pattern of eye disease and viral growth after HSV inoculation of one superior cervical ganglion in rabbits. Of 27 inoculated animals, eye disease or detectable virus developed in 18. Anterior uveitis was the most common clinical manifestation (94%), but conjunctivitis and dendritic keratitis were also frequent (60%). All 12 uveal-retinal specimens tested and five of seven ipsilateral superior cervical ganglia had detectable virus. If recurrent herpetic iritis in humans is associated with persistent infection of the superior cervical ganglion, autonomic mediators might trigger episodes of virus shedding. In patients with herpetic iritis, then, the use of epinephrine and other adrenergic agonists or antagonists should be avoided.

Chemotactic activity and cellular infiltration of aqueous humor in experimental herpes simplex uveitis.

The degree of chemotactic activity of the aqueous humor for a specific type of inflammatory cell is in direct proportion to the extent of such cells infiltrating the aqueous humor of rabbits with either primary or secondary experimental herpes simplex uveitis. Chemotaxis may thus be a contributing factor in the pathogenesis of herpetic uveitis.

Herpesvirus in sensory and autonomic ganglia after eye infection.

In herpesvirus hominis (HVH) infections, virus harbored in the sensory ganglia is now thought to be the main source of recurrent infection at peripheral sites. Experimental HVH infection of the external eye in rabbits produces an acute infection and then latent infection of the trigeminal ganglion. In this study, acute infection of the automatic ganglia serving the eye (superior cervical and ciliary) as well as trigeminal ganglia occurred after HVH inoculation of rabbits' corneas with a herpes type 1 strain (RE). Latent virus infection was detected in the trigeminal ganglion of one of five animals tested six months after initial infection. Since the superior cervical and other autonomic ganglia serving the eye become infected during acute herpes simplex virus infection of the external eye in rabbits, it is possible that these ganglia are also sources of reinfection in recurrent herpetic disease of the eye. Following the initial eye disease with this virus strain, HVH shedding could not be demonstrated even after induction attempts by topically applied epinephrine or systemic use of cyclophosphamide. Thus, establishment of latent HVH infection in the ganglia and chronic shedding of virus into the external eye is not a constant feature of this animal model, but may depend on the specific strain of herpesvirus used.