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1 From the Department of Ophthalmology, LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana; and the 2 Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio.
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Abstract |
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METHODS. HSV-1 (strain McKrae) was applied bilaterally to unscarified corneas of RNase L–null mice and congenic controls. To evaluate the severity of herpetic keratitis, slit lamp examinations (SLE) were performed every other day for 14 days. To study corneal histology and apoptosis, HSV-1–inoculated RNase-L-null and congenic control mice, as well as mock-inoculated mice (apoptosis negative control), were killed at 6 and 18 hours postinoculation (PI). Uninoculated mice that underwent corneal scarification (apoptosis positive control) were killed 2 hours after scarification. Eyes were dissected and the corneas processed for light and transmission electron microscopy and the TUNEL assay.
RESULTS. In comparison with the congenic control mice, RNase L–null mice showed significantly more severe herpetic keratitis (PI day 8, SLE score, mean ± SEM: 3.27 ± 0.10 vs. 2.34 ± 0.06; P < 0.001) and significantly higher mortality (PI day 14, 70% vs. 20%; P < 0.001). Few apoptotic cells were seen in HSV-1–infected RNase L–null mice, although DNA fragmentation consistent with apoptosis was detected in the corneas of congenic control mice 6 and 18 hours after HSV-1 inoculation and in uninfected mice with scarified corneas. Signs of apoptosis were not present in the mock-infected corneas. Electron microscopic evidence of keratocytic apoptosis was detected only in the uninfected scarified corneas and the HSV-1–infected congenic control corneas.
CONCLUSIONS. The increased severity of ocular disease and increased mortality in the RNase L–null mice provides evidence, for the first time, that the 2–5A system contributes to protection during ocular herpetic infection. The reduced frequency of apoptosis in these mice suggests that one possible mechanism for this protective effect could be the induction of apoptosis in corneal cells as a means of reducing the spread of infectious virus.
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Introduction |
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Levels of IFN-ß and IFN-
have been shown to be elevated in the
cornea after viral infection.2
18
19
20
Involvement of the
2–5A system in the IFN-initiated antiviral scenario has been well
elucidated.3
4
7
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11
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31
In brief, IFN signals cells,
leading to an increase in levels of the family of 2',5'-oligoadenylate
synthetases (OAS). Double-stranded RNA derived from viral replication
intermediates and viral transcription activates OAS. Activated OAS
converts ATP into unusual, short 2'-5' linked oligoadenylates called
2–5A. The 2–5A binds to and activates RNase L, cleaving
single-stranded RNA with moderate specificity for sites 3' of UpUp and
UpAp sequences. RNase L rapidly degrades both viral and cellular RNA,
thus crippling the cell’s ability to support viral replication.
Involvement of the 2–5A system in the antiviral effect of IFN by means
of induction of apoptosis has been reported in cell culture experiments
as well as in an animal model containing a targeted disruption in the
gene encoding RNase L (RNase L–null
mouse).8
9
11
24
28
30
31
However, in vivo evidence for the antiviral function of the 2–5A system is limited to studies of encephalomyocarditis virus.24 To our knowledge, there has been no report showing that the 2–5A system mediates an antiviral effect by induction of apoptosis in response to HSV-1 infection in any animal model. In this study, we used RNase L–null mice and congenic controls to investigate the possibility that the 2–5A system plays a role in mediating an antiviral effect in HSV-1 infection in the mouse eye.
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Methods |
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Cells and Virus
HSV-1 strain McKrae was propagated on primary rabbit kidney cell
monolayers in minimal essential medium (GIBCO, Gaithersburg, MD) with
5% fetal bovine serum and titered on African green monkey kidney
cells.
Mouse Inoculation
Each RNase L–null mouse and congenic control mouse was
anesthetized with 150 µl of a mixture of ketamine (10 mg/ml) and
xylazine (5 mg/ml). Each eye received 5 µl of a viral suspension
containing either 2.5 x 105 pfu or 2.5 x 107 pfu of HSV-1 strain McKrae. The cornea was
not scarified. Eyelids were closed and massaged gently for 20 seconds
with care taken to avoid leakage of the inoculum. Mock inoculation was
done by inoculating the cornea with cell culture medium in the same
manner.
Slit Lamp Examination
Slit lamp examinations (SLE) with 0.1% fluorescein staining
were performed in a masked manner before inoculation and every other
day for 14 days postinoculation (PI). Epithelial lesions and stromal
involvement were graded on a scale of 0 to 4, as follows: 0, normal
cornea; 0.5, punctate lesions; 1.0, dendritic lesions; 1.5, geographic
lesions with stromal edema; 2.0, stromal involvement, pupillary iris
visible; 3.0, stromal involvement, pupillary iris invisible; and 4.0,
severe stromal keratitis, peripheral iris invisible. Mean disease
scores (± SEM) were calculated for each group on each observation day
(PI days 2, 4, 6, 8, 10, 12, and 14).
Experimental Design
To study the clinical manifestations, groups of age- and
sex-matched RNase L–null and congenic control mice were inoculated
bilaterally with HSV-1 strain McKrae. The severity of herpetic corneal
lesions was monitored by SLE, and mortality was recorded.
To analyze apoptosis, six each of RNase L–null and congenic control mice were inoculated bilaterally with HSV-1 (2.5 x 105 pfu per eye), without corneal scarification. Six and 18 hours later, mice were killed, the eyes dissected, and the corneas processed for DNA fragmentation detection (TUNEL assay) and light and transmission electron microscopy (TEM). Corneas of two congenic control mice scarified with a sterile 30-gauge needle in a 5 x 5 line crosshatch pattern served as apoptotic signal positive controls. Both mice were killed 2 hours after corneal scarification because this time point has been reported to be the peak of occurrence of keratocytic apoptosis after corneal wounding.32 Two additional congenic control mice received cell culture medium (mock inoculation). One mock-infected mouse was killed at 6 hours after inoculation and one at 18 hours, along with the HSV-1-infected mice, to serve as apoptotic signal negative controls.
DNA Fragmentation Detection
In situ DNA fragmentation detection was performed by the TUNEL
assay. Whole mouse eyes were removed; all right eyes were immediately
embedded in optimal cutting temperature (OCT) compound (Miles
Laboratories, Elkhart, IN), and frozen in liquid nitrogen. Twenty
7-µm thick sections (every fifth section of approximately 100
sections from each eye) that extended transversely across the central
cornea of the mouse eye were collected and stained with the
peroxidase-based TUNEL method, according to the manufacturer’s
instructions (ApopTag Plus Peroxidase In Situ Apoptosis Detection
Kit; Intergen, Purchase, NY). Twenty sections were stained from each
corneal specimen; 60 sections were stained for each group at each time
point (6 hours and 18 hours). In general, the average numbers of
corneal cells, as evidenced by counterstaining of the nuclei, were
approximately the same on each section of cornea for the two groups
(data not shown). The TUNEL-positive cells were readily recognized by
dark brown labeling. The numbers of apoptotic cells in 10
nonoverlapping, full corneal thickness, randomly selected columns were
counted in a masked fashion for each specimen.13
The
diameter of each column was the 400x field of the microscope.
Light and Transmission Electron Microscopy
All left eyes from mice killed as described previously were
fixed in Karnovsky fixative (3% gluteraldehyde, 1% paraformaldehyde)
overnight. The corneas were then dissected, fixed again in Karnovsky
fixative for an additional 24 hours, postfixed in osmium tetroxide,
dehydrated with ethanol and propylene oxide, and embedded in a mixture
of Araldite 502 and Embed 812 (EMS, Fort Washington, PA). Semithin
sections (0.5 µm) were initially stained with 1% toluidine blue and
examined by light microscopy. Finally, thin sections (70 nm) were cut,
stained with 2% uranyl acetate and lead citrate solutions, and
examined by transmission electron microscopy (Zeiss TEM 10C/CR;
Oberkochen, Germany) at 100 kV.
Statistical Analysis
Mortality and slit lamp scores for the RNase L–null and
congenic control mice were analyzed separately. Mortality was analyzed
with Kaplan–Meier analysis; differences between the two curves were
compared using the Mantel–Haenszel procedure.33
Differences between the slit lamp scores were evaluated using the
Student’s t-test on the scores for the RNase L–null and
congenic control mice. P < 0.05 defined significance.
All data analyses were performed with computer software (Statistical
Analysis System; SAS Institute, Cary, NC).
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Results |
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This set of experiments consisted of three separate experiments with 10 mice in each group in each experiment, for a total of 30 RNase L–null mice and 30 congenic control mice inoculated. All three of the experiments produced similar results, and the data from all three experiments were combined. In contrast to the results of the first experiment, the mean severity of the corneal lesions in the surviving animals on PI day 8 (the peak of corneal disease) was significantly greater in the RNase L–null mice (N = 12/30) than in the congenic controls (N = 26/30) (mean ± SEM SLE scores: 3.27 ± 0.10 vs. 2.36 ± 0.06; P < 0.001; Table 1 , Fig. 1A ).
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To determine the effect of the differential mortality in the two groups, the SLE scores were analyzed in two ways: all animals (scores from all animals alive at each time point) and surviving animals only (scores from only the animals that survived to the end of the observation period). As seen in Figure 1A , the means for all animals and surviving animals were indistinguishable.
TUNEL Assay
Apoptotic cells were scant to nonexistent in the corneas of RNase
L–null mice infected with HSV-1. TUNEL-positive cells were not found
in the corneal sections 6 hours after inoculation (Fig. 2A
) and only a few TUNEL-positive epithelial cells (0.8 ± 0.6 per
400x field) were detected in a small number of sections (4 of 60) at
18 hours (Fig. 2B)
. Apoptotic keratocytes were not seen. In contrast,
the HSV-1-infected congenic controls showed 2.9 ± 1.3
TUNEL-positive cells per 400x field, mostly in the epithelial cell
layer, at 6 hours (Fig. 2C)
and 5.8 ± 1.2 apoptotic cells per
field, located in both the epithelium and anterior stroma, at 18 hours
after inoculation (Fig. 2D)
. The differences in the counts of
TUNEL-positive cells between the RNase L–null and congenic control
mice were significant at 6 (P = 0.004) and 18 hours
(P < 0.001) after inoculation. At 18 hours, the
percentage of apoptotic cells in the congenic controls ranged from
approximately 15% to 30%, based on each field containing 20 to 40
corneal cells. The apoptotic cells were located, for the most part, in
the immediate perimeter around or directly under the infected corneal
surface, indicating the possible protective function of apoptosis.
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Histology
Microscopic evidence of epithelial damage indicative of HSV-1
replication was not found in either the RNase L–null corneal sections
or the congenic control sections obtained 6 hours after inoculation. By
18 hours, microscopic epithelial damage was recognized in 8 of 60
(13.3%) RNase L–null sections and 2 of 60 (3.3%) congenic control
sections (P = 0.034). Epithelial damage was not found
in negative control (mock inoculation) corneas. All positive control
(corneal scarification) corneas showed epithelial layer damage caused
by needle scarification (data not shown).
Areas of sections with evidence of epithelial injury detected by light microscopy (if no epithelial injury, randomly selected areas) were examined by TEM for evidence of apoptosis in underlying stromal keratocytes. Signs of apoptosis were not seen in the mock-infected corneas (Fig. 3A ). Keratocytes in the HSV-1-infected congenic control corneas (18 hours) and the uninfected scarified control corneas (2 hours) showed apoptotic changes mostly in the anterior stroma (Figs. 3B and 3C) . Approximately 5% of the keratocytes underwent changes characteristic of apoptosis, which include chromatic condensation, chromatic fragmentation, and cellular blebbing with formation of membrane-bound cell fragments. No apoptotic keratocytes were seen in the HSV-1-infected RNase L–null mouse corneas (Fig. 3D) .
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Discussion |
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Our clinical observations revealed that in comparison with the congenic controls, mice lacking the RNase L gene had exacerbated herpetic corneal lesions and increased mortality after HSV-1 infection. In the first set of experiments, we used a high viral titer (2.5 x 107 pfu per eye) to inoculate the mice. Although the mortality for RNase L–null mice was higher than that of congenic controls about halfway through the observation period, there was no significant difference in the severity of herpetic corneal lesions between the two groups at that time, possibly because of the small number of survivors available for evaluation. Also, the high dose of HSV-1 can perhaps override the 2–5A system in the congenic controls, that is, the virus may have gained a substantial foothold before enough cells were able to establish an inhibitory state. When a 100-fold lower viral titer was used in the second set of experiments, the severity of corneal disease and mortality were significantly greater in the RNase L–null mice, compared with the congenic controls. Also, the RNase L–null mice developed more severe conjunctivitis, blepharitis, and viral endophthalmitis in comparison with the congenic control mice (data not shown). These findings demonstrate that the RNase L–null mice had significantly increased susceptibility to HSV-1, indicating that the absence of RNase L directly or indirectly increased viral pathogenesis and mortality during acute infection.
To study the underlying pathogenesis of the observed clinical features, we analyzed HSV-1–induced apoptosis in the cornea at very early stages of infection. Six hours after viral inoculation, signs of corneal epithelial lesions were not evident in either the RNase L–null mice or the congenic control mice. However, apoptotic cells could already be detected in the corneas of HSV-1–infected congenic control mice, suggesting that the signaling process mediating cell death precedes the microscopic epithelial damage caused by viral replication. Eighteen hours after inoculation, a few corneal epithelial lesions were noted in the congenic controls, whereas significantly more lesions were seen in the RNase L–null mouse corneas, indicating the early onset of HSV-1 epithelial effects in the vulnerable RNase L–null mice. Nevertheless, the RNase L–null corneas had significantly fewer apoptotic cells than the congenic control corneas, and those few cells were found only in the epithelial layer; in the congenic control corneas, apoptotic cells were detected in both the epithelial layers and the anterior stroma. Although we did not directly determine HSV-1 distribution in the cornea, our TUNEL assay and electron microscopic results showed that most of the apoptotic keratocytes were located beneath the HSV-1–infected epithelial area (as evidenced by epithelial damage), suggesting a protective role for apoptosis in HSV-1–infected corneas.13 As a defense mechanism, corneal cells are signaled to undergo apoptosis to prevent further spread of HSV-1 to the deeper stroma as well as higher levels of replication in the epithelium, followed by retrograde neuronal transport to the brain, causing death. Therefore, the significantly reduced apoptosis in the corneas of RNase L–null mice correlates with a decreased ability to resist HSV-1 infection, leading to increased severity of herpetic keratitis and high mortality.
The anti–HSV-1 potential of the 2–5A system has been studied in vitro. Fujihara et al.34 reported that introduction of 2–5A suppressed HSV-1 syncytium formation in BHK cell cultures and prevented the lethal effect of HSV-2 in infected guinea pigs. Cayley et al.35 showed that HSV can induce RNase L inhibitors (2–5A–related compounds) and that RNase L activation, as determined by rRNA cleavage, was minimal in IFN-treated, HSV-infected Chang cells. However, the possibility exists that early, localized activation of RNase L, not detected by the methods used, could be important. Khabar et al.36 reported that the absence of the PKR gene did not compromise the anti–HSV-1 activity of IFN in mouse embryo fibroblasts. However, when both the PKR gene and the RNase L gene were absent, the anti–HSV-1 activity of IFN was reduced by about fivefold, indicating that PKR and RNase L cooperate in some way to provide the anti–HSV-1 effect of IFN.
Other mechanisms, aside from apoptosis induction by RNase L, cannot be ruled out. For example, the activation of RNase L in infected cells could simply halt the infection without requiring induction of apoptosis. Also, it is possible that both apoptosis and another mechanism are involved. In addition, effects of RNase L on cytokine production and MAPKs that could be responsible for enhanced innate or adaptive immune responses against HSV-1 have been reported by Iordanov et al.37
In another study, intracerebral inoculation of the KOS strain of HSV-1 was lethal for both wild type and RNase L–knockout mice within 8 to 10 days.17 Therefore, the effect of RNase L is no longer observed when the ocular route of infection is bypassed. These results support the idea that the antiviral function of RNase L is to suppress the spread of HSV-1 through apoptosis. However, alternative explanations, including differences in viral doses and virus strains, are also possible.
In summary, our data show that mice lacking RNase L have significantly increased susceptibility to ocular HSV-1 infections. Our study provides the first in vivo evidence that the 2–5A system could be important for controlling viruses other than picornaviruses (such as encephalomyocarditis virus). Alternative possibilities, such as contributions from unknown genetic differences in the RNase L–null mice, cannot be completely ruled out, however. Further experiments, including immunopathologic studies of ocular and brain tissues and determination of local and systemic immune responses in RNase L–null mice after HSV-1 infection, could help to elucidate the activity of the 2–5A system during viral infection.
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Acknowledgements |
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Footnotes |
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Supported in part by US Public Health Service Grants EY06311 (JMH), EY02672 (HEK), and EY02377 (LSU Eye Center Core Grant) from the National Eye Institute, and CA44059 (RHS) from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and an unrestricted departmental grant (LSU Eye Center) from Research to Prevent Blindness, Inc., New York.
Submitted for publication February 7, 2000; revised June 20 and September 5, 2000; accepted September 12, 2000.
Commercial relationships policy: N.
Corresponding author: James M. Hill, LSU Eye Center, 2020 Gravier Street, Suite B, New Orleans, LA 70112-2234. jhill{at}lsuhsc.edu
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References |
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1 34.5-mutants of herpes simplex virus 1 Proc Natl Acad Sci USA 92,10516-10520This article has been cited by other articles:
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