Abstract
purpose. Corneal endotheliitis may cause permanent visual loss due to
endothelial decompensation. The pathogenesis underlying this distinct
clinical entity is not known. In the current study, a rabbit herpetic
corneal endotheliitis model was made of induced anterior
chamber-associated immune deviation (ACAID).
methods. One group of rabbits received left-eye intracameral inoculation of
UV-inactivated herpes simplex virus (HSV)-1 (strain McKrae). The second
group received cell medium in the same manner as the first group. The
third group subcutaneously received the same inoculum as the first
group. Seven days later, all right eyes were intracamerally infected
with 2.5 × 104 plaque-forming units of infectious
HSV-1. Eyes were evaluated by slit lamp examination. Two weeks after
infection, rabbits were killed, and right eyes were examined by
immunohistochemical staining and electron microscopy. Aqueous humor was
detected for HSV-1 DNA and antibody.
results. Nonspecific inflammation occurred in the anterior segments of the eyes
from the second and third groups. In contrast, at 14 days after
infection, the first group of rabbits showed a specific pattern of
inflammation that greatly resembled clinical features of corneal
endotheliitis. Viral antigen was detected only in the endothelial
layer. Electron microscopy revealed enlarged intercellular gaps and
infiltration of inflammatory cells that are characteristic of
endothelial defects. HSV-1 DNA was detected at a significantly higher
number in the aqueous humor aspirates from endotheliitis rabbits. In
addition, ACAID was shown to be induced in the rabbits with corneal
endotheliitis.
conclusions. HSV-1 infection can induce corneal endotheliitis and ACAID may play the
pivotal role in this entity.
Herpes simplex virus (HSV) infection is the leading cause of
infectious corneal disease in the world. It has been generally accepted
that herpetic epithelial keratitis results directly from viral
replication in epithelial cells, and stromal keratitis is assumed to
result from a host immune response to viral antigen present in the
stroma. Another clinical entity, in which the target of inflammation is
thought to be the endothelium, has been recognized and recently
classified as corneal endotheliitis.
1 2
Clinical manifestation of corneal endotheliitis varies markedly. Since
Khodadoust et al.
3 reported a “presumed autoimmune
corneal endotheliopathy,” in which they assumed that the disease
resulted from an autoimmune reaction in the corneal endothelium,
clinicians have noticed various forms of corneal endotheliitis. A
variety of names have been given to this distinct clinical entity,
including progressive herpetic corneal endotheliitis,
4 herpetic endothelial keratitis,
5 idiopathic corneal
endotheliopathy,
6 and sporadic diffuse corneal
endotheliitis.
7 Many patients with HSV disease may have
corneal stromal edema that develops without stromal infiltrate. These
patients all share the following findings: keratic precipitates (KPs),
stromal and epithelial edema, and mild iritis. The KPs may be difficult
to see because of extensive corneal edema but usually become evident as
the stromal edema resolves. Many clinicians diagnose this clinical
entity as stromal keratitis; however, it may more properly be
considered to an inflammatory reaction of the endothelium, with only
secondary stromal and epithelial edema.
1 2
Although the exact pathogenesis of corneal endotheliitis is not known,
accumulating evidence has indicated that viral origin is a strong
possibility.
1 2 4 8 9 10 11 12 13 14 15 The role of an infectious virus as
the cause of endothelial infection is supported first by the detection
of HSV-1 antigen, DNA, or viral particles in corneal endothelial
cells.
4 8 9 10 11 12 13 Second, endothelial lesions can rapidly
resolve only with the addition of topically applied and systemic
antiviral treatment.
1 2 4 13 14 15
To date, most of the publications regarding corneal endotheliitis are
clinical observations and case reports. To the best of our knowledge,
there has been no report of an animal model of experimental corneal
endotheliitis. The current experiments were undertaken to test our
hypothesis that HSV-1 infection is one cause of corneal endotheliitis
and that anterior chamber associated immune deviation (ACAID) is the
underlying pathogenic mechanism for this entity. We established
herpetic corneal endotheliitis in a rabbit model in the setting of
induced ACAID.
Materials and Methods
Animals
Normal Japanese albino rabbits (2.5–3.0 kg) were used. All animal
procedures were performed using a combination of ketamine (70 mg/kg)
and xylazine (10 mg/kg) as the anesthetic agent. In managing the
rabbits we adhered strictly to the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research.
Virus
HSV-1 strain McKrae was used in these experiments. Viral stocks
were propagated in primary rabbit kidney (PRK) cells grown in
Dulbecco’s modified Eagle’s medium containing 5% heat-inactivated
fetal bovine serum and antibiotics (Gibco, Grand Island, NY). An
aliquot of passaged virus was titered by plaque assay on African green
monkey kidney cell monolayers. Viral stocks were frozen in small
amounts and stored at −80°C until use.
UV-Inactivated Virus
UV-inactivated HSV-1 (UV-HSV-1) was made by irradiating HSV-1
strain McKrae with UVB light at a distance of 12 cm for 16 minutes. The
viral titer before irradiation was 2.5 ×
107 plaque-forming units (PFU)/ml. After
irradiation the virus showed no cytopathic effect on the PRK cells
without compromised antigenicity, verified by immunocytochemical study.
Experiment 1: Herpetic Corneal Endotheliitis Model
Experimental Design.
Eighteen rabbits were randomly separated into three groups in equal
number. One group received left-eye intracameral inoculation of
UV-HSV-1 (3.8 × 106 PFU in 150 μl
medium). The second group received PRK cell medium in the same manner
as the first group. The third group received the same inoculum
subcutaneously (SC) as the first group. Seven days later, the right
eyes of all animals were intracamerally infected with 2.5 ×
104 PFU of infectious HSV-1 contained in 100 μl
medium.
After infection all rabbit eyes were monitored in a double-blind
fashion by slit lamp examination every other day for 2 weeks. Blood
samples were collected on postinfection (PI) days 0, 7, and 14. Samples
from each group were pooled and detected for HSV-1–neutralizing
antibodies.
At the conclusion of this experiment (PI day 14) rabbits were killed.
The anterior segment of the eye including cornea, iris, and ciliary
body was dissected and separated randomly into two parts. One part was
examined with immunohistochemical staining. The other part of the
cornea was examined with either scanning electron microscopy (SEM) or
transmission electron microscopy (TEM). Aqueous humor was also
aspirated for detection of HSV-1 DNA and neutralizing antibody.
All experimental procedures were repeated three times, and
HSV-1–neutralizing antibody and PCR samples were analyzed in
triplicate.
Slit Lamp Examination.
Rabbits were examined for the presence of inflammation in the anterior
segment including corneal edema, KPs, and iritis. Each category was
graded with a 0 (normal) to 4 (most severe) scoring system. Each
clinical sign was evaluated by specific criteria. Corneal edema was
scored as follows: 1, detectable thickening; 2, iris detail visible
through edematous cornea; 3, iris detail not visible through edematous
cornea; and 4, severe, whole cornea edema, peripheral iris not visible.
KP was scored as follows: 1, detectable; 2, sporadic, small size; 3,
diffuse, middle size; and 4, clumped, large size. Iritis was scored as
follows: 1, sectorial redness; 2, redness of the whole iris, detectable
flare cell in anterior chamber; 3, swelling and exudation of fiber
protein, mild flare cell; and 4, frank hemorrhage and severe flare
cell. The mean disease scores for corneal edema, KPs, or iritis were
calculated for each group of rabbits on each slit lamp examination.
Detection of HSV-1 Antigen.
Halves of the anterior segments of the eyes were fixed in
periodate-lysine-paraformaldehyde fixative and immersed in a graded
(10%, 20%, and 30%) sucrose series. After embedding in optimal
cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA),
specimens were snap frozen in liquid nitrogen and serially sectioned at
7-μm thickness by cryotome (Cryocut 3000; Leica, Wetzlar, Germany).
Sections were stained with monoclonal anti-HSV-1 antibody in a dilution
of 1:100 (Quartett, Berlin, Germany). A peroxidase-standard kit (PK
4010; Vectastain; Vector, Burlingame, CA) was used, and the staining
procedures followed the manufacturer’s instructions.
Electron Microscopy.
For SEM, corneas were fixed in 3% glutaraldehyde, postfixed in 2%
osmium tetroxide, mounted on stubs, sputter coated with gold-palladium,
and observed (H-800; Hitachi, Tokyo, Japan). For TEM, specimens were
fixed in 3% glutaraldehyde for 3 hours and postfixed in 2%
glutaraldehyde for 2 hours. Specimens were dehydrated with a graded
ethanol series and embedded in Epon 812. Ultrathin sections (70 nm)
were cut, stained with 2% uranyl acetate and lead citrate solutions,
and examined with TEM (H-800, Hitachi, Tokyo, Japan) at 100 kV.
PCR Analysis.
Isogen reagent (Nippon Gene, Tokyo, Japan) was used to extract DNA from
the aqueous humor samples according to the manufacturer’s instruction.
DNA extracted from infected rabbit corneal cell suspension was used as
the positive control. DNA extracted from normal rabbit cornea
homogenate was used as the negative control.
Each PCR reaction contained 0.1 μg extracted DNA, 0.5 μM of primers
specific for HSV-1 ribonucleotide reductase gene (sense: 5′-ATG CCA GAC
CTG TTT TTC AA-3′ and antisense: 5′-GTC TTT GAA CAT GAC GAA GG-3′; this
primer pair generates a 243-bp product), 1.5 mM
MgCl2, 0.2 mM of each dNTP, 2.5 U of Taq polymerase (AmpliTaq; Perkin–Elmer, Foster
City, CA) and 1× PCR buffer in a 50 μl final volume. Cycling
reactions were performed in a thermal cycler (MJ Research, Waltham,
MA). Each cycle included denaturation at 94°C for 30 seconds,
annealing at 58oC for 30 seconds, and extension
at 72°C for 60 seconds. The final cycle was terminated with a
10-minute extension period at 72°C. Samples underwent 45 cycles of
amplification. The amplified samples were electrophoresed on 2%
agarose gels, stained with ethidium bromide, and visualized using a
digital imaging system (Eagle Eye; Stratagene, Seattle, WA).
HSV-1–Neutralizing Antibody Assay.
Blood samples were centrifuged to harvest sera. Sera and aqueous humor
samples were millipore sterilized with filters containing 0.2-μm
pores (Corning, Westbury, NY). Microneutralization assays were
performed according to the method described by Whittum et
al.
16
Experiment 2: Verification of ACAID Induction after Intracameral
Inoculation of UV-HSV-1
Four groups of rabbits were treated as follows: group A,
subconjunctival inoculation of 3.8 × 106 PFU UV-HSV-1 (150 μl); group B, no treatment; group C,
intracameral inoculation of 3.8 × 106 PFU UV-HSV-1
(150 μl); and group D, intracameral inoculation of 150 μl PRK cell
medium.
Seven days later, all groups except group D received an immunizing dose
of UV-HSV-1 (3.8 × 106 PFU) emulsified 1:1 in CFA
(0.5 mg mycobacterium per milliliter, Gibco, Grand Island, NY). Two
milliliters of this mixture was distributed SC in equal amounts at four
body sites of each rabbit.
Seven days after immunization, delayed type hypersensitivity (DTH) was
detected. The preinjected thickness of each rabbit ear (time zero) was
measured using an engineer’s micrometer (Mitutoyo, Tokyo, Japan). DTH
response was assayed by injecting 150 μl of 3.8 ×
106 PFU erstwhile UV-HSV-1 into the ventral aspect of the
right ear using a 250-μl Hamilton syringe (Wilmad, Reno, NV) equipped
with a 30-gauge needle; the left ear served as the control. The amount
of ear swelling was measured at 24 and 48 hours after challenge.
Results are expressed as the amount of ear swelling at each time point
by calculating the change in thickness of each ear from its respective
time zero measurement and then subtracting the value of the control ear
from that of the viral antigen–tested ear. All experimental procedures
were performed three times.
Statistical Analysis
Statistical comparisons of the scores of the anterior segment
inflammation between groups in experiment 1 and the comparisons of
ear-swelling data between groups in experiment 2 were performed by
conducting an initial one-way analysis of variance (ANOVA) on the
inflammation score or the ear-swelling data, respectively. If the ANOVA
results were significant (
P < 0.05), comparisons were
conducted using the overall ANOVA estimate of the relevant group mean
error to conduct protected pair-wise
t-tests.
17 Comparisons of the frequency of HSV-1 DNA detection in aqueous
humor samples were conducted by χ
2 test. Comparisons of
the HSV-1–neutralizing antibody titers in blood and aqueous humor
between groups were conducted by likelihood ratio test.
18 P < 0.05 defines significance. All analyses were
performed with computer software (Statistical Analysis System; SAS
Institute, Cary, NC).
Results
Clinical Findings
In experiment 1, rabbit eyes were evaluated by slit lamp
examination for 14 days after right eye infection. Experiments were
performed three times, each of which produced very similar results. In
general, all left eyes showed no signs of evident inflammation and no
difference between groups. Examination of right eyes revealed striking
findings. A representative result of slit lamp examination on the right
eyes is depicted in
Figure 1 .
As shown in
Figures 1B and 1C , rabbits intracamerally inoculated with
PRK cell medium or SC inoculated with UV-HSV-1 1 week before the
anterior chamber infection developed intense, nonspecific inflammation
in the anterior segments of the eyes. Viral infection was manifested by
moderate to severe ciliary injection, KPs, diffused corneal edema and
severe iritis. The inflammatory reaction peaked on PI days 6 to 8 and
gradually subsided by PI day 14. On PI day 6, clinical scores were
2.5 ± 0.41, 2.25 ± 0.42, and 3.33 ± 0.26 for corneal
edema, KPs, and iritis, respectively, in rabbits receiving left-eye
intracameral inoculation of PRK cell medium 1 week before right-eye
infection. Scores were 2.67 ± 0.44, 2.42 ± 0.38, and
3.08 ± 0.38 for corneal edema, KPs, and iritis, respectively, in
rabbits receiving SC inoculation of UV-HSV-1 before infection. There
were no statistical differences in the severity of corneal edema
(
P = 0.24), KPs (
P = 0.22), or iritis
(
P = 0.1) between these two groups.
In sharp contrast, rabbits that were intracamerally inoculated with
UV-HSV-1 1 week before infection showed profoundly different ocular
inflammatory patterns (
Fig. 1A ). During the first week after
infection, the infected eyes showed development of relatively mild
inflammation in the anterior segments. Inflammatory reactions were
mainly noticed on the cornea, which displayed edema to the same extent
as the other two groups. A relatively “silent” anterior chamber
with very little flare cell reaction and mild iritis was the sign of
the ocular infection in this group. On PI day 6, clinical score was
0.43 ± 0.2 for iritis. Compared with the scores for the other two
groups on the same observation day, the difference was significant
(
P < 0.001). During the second week of infection and
by PI day 14, a distinct inflammatory pattern developed in the anterior
segment that very much resembled the clinical features in some patients
with corneal endotheliitis. The inflamed eye had a large, semicircular
area of stromal edema overlying KPs in the lower part of the
paracentral cornea. The ground-glass appearance of the corneal edema
presented a distinct clinical picture with a definite focal pattern and
definite demarcation between involved and uninvolved cornea. There was
very mild inflammation in the anterior chamber (
Fig. 2A ). Several clumps of the characteristic KPs laid underneath the
distribution of stromal edema, best seen by oblique illumination
(Fig. 2B) .
Electron Microscopic Findings
To better observe the details of the corneal endothelial layer, we
performed electron microscopy studies. Corneas from rabbits that
received intracameral inoculation of PRK cell medium or SC inoculation
of UV-HSV-1 showed morphologically normal endothelia with very few
visible defects. In marked contrast, corneas with herpetic
endotheliitis appeared to possess many inflammatory cells in the
endothelial layer. Enlarged intercellular gaps and infiltration of
inflammatory cells characterized the numerous endothelial defects as
shown by SEM (
Fig. 3A ). Interestingly, some spotty “holes” were distinguishable in the
endothelial layer
(Fig. 3B) . TEM results showed that the infiltrating
cells had the morphologic features of lymphocytes, plasma cells, or
polymorphonuclear cells that incorporated into the destroyed
endothelial layer of the endotheliitis-affected cornea
(Fig. 3C) .
Viral Antigen Detection
Anterior segments of the infected right eyes were investigated to
determine the existence and distribution of HSV-1 antigen. In a typical
experiment, for rabbits receiving intracameral inoculation of UV-HSV-1,
viral antigen was detected only in the endothelial layer of all six
samples. One exception was that, in addition to the endothelial layer,
one eye also had viral antigen detectable in the anterior part of the
ciliary body. In sharp contrast, all samples from rabbits that received
intracameral inoculation of PRK cell medium (six samples) or SC
inoculation of UV-HSV-1 (six samples) showed negative results in all
parts of the anterior segments examined. A representative result of the
HSV-1 antigen detection using immunohistochemical staining from a
cornea with endotheliitis is shown in
Figure 4 .
Detection of HSV-1 DNA in Aqueous Humor
Aqueous humor aspirates harvested on PI day 14 were examined for
HSV-1 DNA. Combining the data from three separate experiments, viral
DNA was detected in 14 of 18 (77.8%) samples from rabbits that
intracamerally received viral antigen 1 week before infection. Four of
18 (22.2%) samples from rabbits that intracamerally received PRK cell
medium had detectable viral DNA. Only 2 of 18 (11.1%) samples from
rabbits that received UV-HSV-1 SC were positive. Aχ
2 test verified that there were significant
differences in the frequency of HSV-1 DNA detection between rabbits
that received viral antigen intracamerally and the other two groups
(with cell medium,
P = 0.003 and with SC UV-HSV-1,
P = 0.001). A representative result of the PCR analysis
is shown in
Figure 5 .
HSV-1 Antibody Detection
Three separate experiments were performed, and in each experiment
all samples were analyzed in triplicate. A representative result from
one assay of peripheral blood is shown in
Table 1 . Results of the HSV-1–neutralizing antibody detection in
aqueous humor samples (PI day 14) are shown in
Table 2 . Rabbits intracamerally inoculated with UV-HSV-1 were able to produce
HSV-1 antibodies in peripheral blood at the time of anterior chamber
infection (1:32), in comparison with less than 1:1 for rabbits
intracamerally inoculated with PRK cell medium and 1:4 for rabbits
primed SC with viral antigen
(Table 1) . A likelihood ratio test showed
that the differences in HSV-1 antibody titer between rabbits
intracamerally inoculated with viral antigen and the other two groups
were significant (
P < 0.001). Moreover, on PI day
14, the aqueous humor samples from rabbits in which corneal
endotheliitis had developed showed a significantly higher antibody
titer in comparison with the other two groups (
Table 2 , likelihood
ratio test,
P < 0.001). The HSV-1 antibody titer
in aqueous humor samples from rabbits receiving PRK cell medium was not
significantly different from that of rabbits receiving SC inoculation
of viral antigen (likelihood ratio test,
P =
0.258).
ACAID Induction by Intracameral Inoculation of HSV-1
Antigen
To test our hypothesis that ACAID is the underlying mechanism of
this herpetic corneal endotheliitis model, experiments were performed
to verify that anterior chamber inoculation of UV-HSV-1 could result in
an impairment of virus-specific DTH response and an intact humoral
antibody-forming ability, which are hallmarks of ACAID.
Three experiments were performed, each of which produced data similar
to that shown in
Table 3 . A summary of the three experiments is displayed in
Figure 6 . Our pilot experiments also included one group in which rabbits were
treated with anterior chamber inoculation of PRK cell medium followed
by immunization. This group was found to be no different from group B
(no treatment). Rabbits that received intracameral inoculation of
UV-HSV-1 (group C) showed significantly impaired DTH responses to HSV-1
antigen. In three experiments, the ear-swelling responses in group C
were only approximately 40% as intense as the subconjunctivally
inoculated, DTH-positive control animals (group A,
P =
0.006). The ear-swelling data of rabbits that received UV-HSV-1
intracamerally (group C) was not significantly different from the
values obtained for rabbits that received PRK cell medium
intracamerally (group D). The DTH response of rabbits receiving
subconjunctival inoculation of UV-HSV-1 (group A) was not significantly
different from that of rabbits receiving immunization alone (group B,
P = 0.247).
Another important hallmark of ACAID is the ability to produce
antibodies to the inoculated viral antigen, even though systemic DTH to
this antigen is impaired (undisturbed antibody
production).
16 19 20 21 22 23 24 An HSV-1–neutralizing antibody
assay in experiment 1 showed that rabbits intracamerally primed with
HSV-1 antigen were able to produce HSV-1–neutralizing antibodies in
peripheral blood (1:32,
Table 1 ). More important, on PI day 14, rabbits
with corneal endotheliitis had significantly higher titers of HSV-1
antibodies in the aqueous humor than did the other two groups
(Table 2) .
Thus, ACAID induction in rabbits after intracameral inoculation of
UV-HSV-1 was confirmed. The pattern of impairment of DTH reactivity
with concomitant production of neutralizing antibody to the virus is
typical of ACAID.
Failure of Corneal Endotheliitis Induction by Systemic
Immunosuppression
Because the specific immune deviation of ACAID is that the
cell-mediated immune response is selectively impaired, whereas humoral
immune responses are undisturbed or enhanced, we investigated whether
systemic immunosuppression (both cell-mediated and humoral immune
responses) could similarly promote herpetic corneal endotheliitis.
Panels of rabbits were immunosuppressed with both 200 mg
cyclophosphamide given intravenously on days 1, 4, 8, and 12 and 10 mg
triamcinolone administered SC daily. The effect of immunosuppression
was confirmed by white blood cell count in peripheral blood. Seven days
later, rabbits were intracamerally inoculated with 2.5 ×
104 PFU of infectious HSV-1 and monitored by slit
lamp examination.
All infected eyes showed development of very severe viral
endophthalmitis and stromal keratitis. No corneal endotheliitis was
found. Neither peripheral blood nor aqueous humor samples showed an
increase in the titer of HSV-1 antibody throughout this experiment
(data not shown). Therefore, systemic immunosuppression could not
promote herpetic corneal endotheliitis.
Discussion
Our interest and attention to conduct experiments testing a role
for ACAID in herpetic corneal endotheliitis stems from the common
clinical finding that mild inflammation of the anterior chamber
accompanies corneal endotheliitis in patients. Why, if herpetic
infection exists in these eyes, as suggested by a large number of
reports,
1 2 8 9 10 11 12 13 14 15 is the inflammation in the anterior
chamber so mild and the pathogenic region restricted to the
endothelium? We have reasoned that the answer may lie in a unique
immune response that occurs when antigens enter the anterior chamber.
ACAID, first described by Streilein and
associates
20 21 22 23 24 offers a potential explanation for this.
DTH is a powerful inducer of immunogenic inflammation in which
interferon-γ is a prominent effector cytokine. Because
antigen-specific DTH is selectively suppressed in ACAID, we
hypothesized that the existence of ACAID due to viral antigens may
permit HSV-1 to invade and damage corneal endothelial cells, while
avoiding the antiviral effects of T cells that mediate delayed type
hypersensitivity.
Our data showed that rabbits primed with UV-HSV-1 through anterior
chamber 1 week before infection developed a distinctive pattern of
infection that very much resembled clinical corneal endotheliitis in
patients. Immunohistochemical staining revealed that HSV-1 antigen was
only detected in the destroyed endothelial cell layer. Therefore, the
viral infection appeared to be limited to the corneal endothelium,
implying that keratocytes, corneal epithelial cells, and iris and
ciliary body tissues were protected from viral spread. Inflammatory
cells including lymphocytes, plasma cells, and polymorphonuclear cells
were found to infiltrate the region where endothelial cells were
destroyed. The corneal edema that manifested in the infected eyes could
reflect the destruction or dysfunction of the endothelial cell layer.
It is well known that HSV-1 can be transported in retrograde fashion to
neuronal ganglia, such as the trigeminal ganglion, after primary
infection of mucocutaneous tissues. Within the trigeminal ganglion, the
virus usually establishes a long-term, latent infection that can become
intermittently active.
25 26 Low doses of virus may be
spontaneously shed in the eye. It is likely that HSV-1 particles can be
released into the anterior chamber through the trigeminal nerves that
innervate the trabecular meshwork, the iris, and the ciliary
body.
13 25 26 Some of the viral particles can be captured
by indigenous antigen-presenting cells, which, in turn, generate an
immunogenic signal that induces virus-specific ACAID. We believe that
the anterior chamber inoculation of UV-HSV-1 in our experiments
produced ACAID by a similar mechanism. The reason we used viral
antigen, not infectious virus, is that by injecting inactivated virus,
a much higher concentration of viral antigen could be delivered without
destruction of the anterior segment of the eye. This “silent”
priming is a scenario very similar to that of clinical patients who
have asymptomatic spontaneous viral shedding from low numbers of viral
particles.
The amount of viral shedding during any particular reactivation may
vary considerably. We suspect that when the dose of released virus is
at or near 104 PFU, which is similarly produced in our
rabbit model by intracameral infection of infectious HSV-1, the
reactivating virus may travel into the immunosuppressive
microenvironment of the anterior chamber and cause herpetic corneal
endotheliitis. Actually, in our pilot experiments, we have attempted to
infect with lower (102 PFU) or higher (106 PFU)
doses of infectious HSV-1. But in neither case did we observe typical
clinical manifestations of corneal endotheliitis. In our hands, only
challenging the anterior chamber with 104 PFU of infectious
HSV-1 produced this model, suggesting that only a specific dose of
viral release into the ACAID-predominated eye can induce herpetic
corneal endotheliitis. It is likely that there is a threshold amount of
infectious virus that creates this disease and that the balance of
ACAID and host effector responses plays an important role.
In ACAID, virus-specific DTH is suppressed and virus-specific cytotoxic
T-cell responses are blunted but serum anti-HSV antibodies are present,
or even enhanced.
19 20 21 22 23 24 This spectrum of immune effectors
makes it possible for HSV-1 to avoid lysis by class I-restricted T
cells. We found that high titers of neutralizing antibodies were
present in the eyes with corneal endotheliitis. We postulate that these
antibodies effectively neutralized the extracellular virus, thus
preventing the spread of virus into other cells and tissues within the
anterior chamber. The rather minimal inflammation observed in the
anterior segments of the eyes with endotheliitis supports our
postulation.
There are two points to emphasize. First, the route of ocular priming
is the key to the induction of herpetic corneal disease. Corneal
endotheliitis did not develop in rabbits primed SC with viral antigen.
Moreover, our pilot experiments showed that intrastromal inoculation of
HSV-1 antigen followed by the same-eye intracameral HSV-1 infection
could promote stromal keratitis and keratouveitis, but not corneal
endotheliitis. These findings suggest that the susceptibility of the
cornea to the burden of keratouveitis or endotheliitis during HSV-1
infection is probably dictated by the unique features of the immune
response elicited by antigens presented first to the immune system
through the eye.
27 Second, the corneal endotheliitis model
results from conditions in which there is an antigen-specific
suppression of cell-mediated immunity and intact humoral immune
response (ACAID). Nonspecific systemic immunosuppression, in which both
cell-mediated and humoral immune responses are suppressed, cannot
promote corneal endotheliitis. Theoretically, any ACAID-like immune
response that leads to a pattern of impairment of cell-mediated
immunity with concomitant production of neutralizing antibody to HSV-1
should be considered in the interpretation of the current data observed
in our studies.
In summary, our experiments revealed that HSV-1 infection could be one
cause of corneal endotheliitis. ACAID may play an important role in
this distinct clinical entity. The strain of HSV-1, viral dose, and
subtle changes in the immune status of the host may conspire to create
the various features of corneal endotheliitis observed in clinics.
Supported in part by Kozuka Ophthalmologic Fellowship, Kozuka Eye
Clinic, Matsuyama; a Fuji International Medical Grant, Ehime University
School of Medicine; and the Japan National Society for the Prevention
of Blindness (1995–1997).
Submitted for publication June 15, 1999; revised September 2, 1999; accepted October 5, 1999.
Commercial relationships policy: N.
Corresponding author: Xiaodong Zheng, LSU Eye Center, 2020 Gravier Street, Suite B, New Orleans, LA 70112-2234.
xzheng@lsumc.edu
Table 1. HSV-1–Neutralizing Antibody Detection in Peripheral Blood
Table 1. HSV-1–Neutralizing Antibody Detection in Peripheral Blood
| Group | Post Infection Day | | |
| 0 | 7 | 14 |
| Naive | <1:1* | <1:1 | <1:1 |
| AC-antigen | 1:32 | 1:64 | 1:64 |
| AC-medium | <1:1 | 1:16 | 1:64 |
| SC-antigen | 1:4 | 1:16 | 1:64 |
Table 2. HSV-1–Neutralizing Antibody Detection in Aqueous Humor at PI Day 14
Table 2. HSV-1–Neutralizing Antibody Detection in Aqueous Humor at PI Day 14
| Experiment | Test | Naive | AC-antigen | AC-medium | SC-antigen |
| 1 | 1 | <1:1 | 1:128 | 1:16 | 1:16 |
| 2 | <1:1 | 1:128 | 1:16 | 1:16 |
| 3 | <1:1 | 1:256 | 1:32 | 1:32 |
| 2 | 4 | <1:1 | 1:128 | 1:16 | 1:16 |
| 5 | 1:2 | 1:256 | 1:32 | 1:32 |
| 6 | <1:1 | 1:128 | 1:16 | 1:16 |
| 3 | 7 | <1:1 | 1:128 | 1:16 | 1:16 |
| 8 | <1:1 | 1:128 | 1:32 | 1:32 |
| 9 | <1:1 | 1:128 | 1:16 | 1:32 |
| P * | | <0.001 | — | <0.001 | <0.001 |
| Experiment* | Group | | | |
| A | B | C | D |
| 1 | 740 ± 78 | 702 ± 64 | 293 ± 26 | 238 ± 24 |
| 2 | 828 ± 101 | 782 ± 95 | 337 ± 42 | 277 ± 35 |
| 3 | 793 ± 85 | 765 ± 80 | 312 ± 33 | 265 ± 31 |
| Total, † | 787 ± 82 | 750 ± 76 | 314 ± 30, ‡ | 260 ± 27, ‡ |
The authors thank J. Wayne Streilein (Schepens Eye Research
Institute) for valuable advice on this project; Mary E.
Marquart (Lousiana State University Eye Center) for critical comments;
and Daizaburo Shimizu and Masachika Syuto (Ehime University School of
Medicine) for expert assistance with electron microscopy.
Liesegang TJ. Classification of herpes simplex virus keratitis and anterior uveitis.
Cornea. 1999;18:127–143.
[CrossRef] [PubMed]Holland EJ, Schwartz GS. Classification of herpes simplex virus keratitis.
Cornea. 1999;18:144–154.
[CrossRef] [PubMed]Khodadoust AA, Attarzadeh A. Presumed autoimmune corneal endotheliopathy.
Am J Ophthalmol. 1982;93:718–722.
[CrossRef] [PubMed]Robin JB, Steigner JB, Kaufman HE. Progressive herpetic corneal endotheliitis.
Am J Ophthalmol. 1985;100:336–337.
[CrossRef] [PubMed]Vannas A, Ahonen R. Herpetic endothelial keratitis. Acta Ophthalmol. 1981;52:296–301.
Ohashi Y, Kinoshita S, Mano T, Kiritoshi A, Ohji M. Idiopathic corneal endotheliopathy: a report of two cases.
Arch Ophthalmol. 1985;103:1666–1668.
[CrossRef] [PubMed]Hakin KN, Dart JK, Sherrard E. Sporadic diffuse corneal endotheliitis.
Am J Ophthalmol. 1989;108:509–515.
[CrossRef] [PubMed]Holbach LM, Font RL, Naumann GO. Herpes simplex stromal and endothelial keratitis: granulomatous cell reactions at the level of Descemet’s membrane, the stroma, and Bowman’s layer.
Ophthalmology. 1990;97:722–728.
[CrossRef] [PubMed]Vannas A, Ahonen R, Makitie J. Corneal endothelium in herpetic keratouveitis.
Arch Ophthalmol. 1983;101:913–915.
[CrossRef] [PubMed]Sundmacher R, Neumann–Haefelin D. Herpes simplex virus isolations from the aqueous humor of patients suffering from focal iritis, endotheliitis, an prolonged disciform keratitis with glaucoma.
Klin Monatsbl Augenheilkd. 1979;175:488–501.
[PubMed]Olsen TW, Hardten DR, Meiusi RS, Holland EJ. Linear endotheliitis.
Am J Ophthalmol. 1994;117:468–474.
[CrossRef] [PubMed]Holbach LM, Asano N, Naumann GO. Infection of the corneal endothelium in herpes simplex.
Am J Ophthalmol. 1998;126:592–594.
[CrossRef] [PubMed]Ohashi Y, Yamamoto S, Nishida K, Okamoto S, Kinoshita S, Hayashi K. Demonstration of herpes simplex virus DNA in idiopathic corneal endotheliopathy.
Am J Ophthalmol. 1991;112:419–423.
[CrossRef] [PubMed]Ohashi Y. Treatment of herpetic keratitis with acyclovir: benefits and problems.
Opthalmologica. 1997;211(suppl 1)29–32.
[CrossRef] Cheng CK, Chang SW, Hu FR. Acyclovir treatment for linear endotheliitis on grafted corneas.
Cornea. 1995;14:311–315.
[CrossRef] [PubMed]Whittum JA, Niederkorn JY, McCulley JP, et al. Intracameral inoculation of herpes simplex virus type 1 induces anterior chamber associated immune deviation. Curr Eye Res. 1983;2:691–697.
Milliken GA, Johnson DE. Analysis of Messy Data. 1984; Van Nostrand–Reinhold New York.
Alan A. Categorical Data Analysis. 1990; John Wiley & Sons New York.
Whittum JA, McCulley JP, Niederkorn JY, Streilein JW. Ocular disease induced in mice by anterior chamber inoculation of herpes simplex virus.
Invest Ophthalmol Vis Sci. 1984;25:1065–1073.
[PubMed]Streilein JW. Ocular immune privilege and the Faustian Dilemma. The Proctor Lecture.
Invest Ophthalmol Vis Sci. 1996;37:1940–1950.
[PubMed]Streilein JW, Wilbanks GA, Talor A, Cousins S. Eye-derived cytokines and the immunosuppressive intraocular microenvironment: a review.
Curr Eye Res. 1992;11:41–47.
[CrossRef] [PubMed]Niederkorn JY, Streilein JW. Analysis of antibody production induced by allogeneic tumor cells inoculated into the anterior chamber of the eye.
Transplantation. 1982;33:573–577.
[CrossRef] [PubMed]Streilein JW. Molecular basis of ACAID.
Ocul Immunol Inflamm. 1997;5:217–218.
[CrossRef] [PubMed]Niederkorn JY, Ferguson TA. Anterior chamber associated immune deviation (ACAID). Pepose JS Holland GN Wilhelmus KR eds. Ocular Infection and Immunity. 1996;96–102. Mosby New York.
Liesegang TJ. Biology and molecular aspects of herpes simplex and varicella-zoster virus infections.
Ophthalmology. 1992;99:781–799.
[CrossRef] [PubMed]Pavan–Langston D. Herpes simplex virus ocular infections: current concept of acute, latent and reactivated disease.
Trans Am Ophthalmol Soc. 1990;88:727–796.
[PubMed]Streilein JW, Dana MR, Ksander BR. Immunity causing blindness: five different paths to herpes stromal keratitis. Immunol Today. 1997;9:443–449.