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Departments of
*
Microbiology and
Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78284
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CMV retinitis is the most common ophthalmic infection in AIDS patients; up to 40% of AIDS patients develop CMV retinitis during the course of the disease (5, 6). CMV retinitis is a focal, but progressive, necrotizing infection of the retina, which causes reduction in visual acuity and, if left untreated, blindness (5, 6). Classically, CMV retinitis was a complication of end-stage AIDS, but as treatments for AIDS-associated infections have allowed patients to live longer, CMV retinitis has become more challenging to control therapeutically. Antiviral therapies for ocular CMV infections are initially effective, but disease progression occurs frequently due to the emergence of drug-resistant virus. Additionally, therapies are limited because of patient compliance and drug toxicity (7, 8).
Since members of the cytomegalovirus family are extremely species restricted, the pathogenesis of CMV infection of the retina has been studied using murine cytomegalovirus (MCMV)3-infected BALB/c mice (9, 10, 11, 12, 13, 14). Injection of a low dose (5.0 x 102-1.0 x 103 plaque-forming units (pfu)) of MCMV into the supraciliary space of one eye of an immunosuppressed BALB/c mouse causes a focal, necrotizing retinal infection that resembles CMV retinitis in human (10, 11). This mouse model has been used to study the pathogenesis of ocular CMV infections (10, 11).
One component of the primary immune response to most virus infections
is NK cells. NK cells, a subset of large, granular lymphocytes, are
activated by the cytokines IFN-
, IFN-ß, IFN-
, and IL-12 and
can, upon activation, secrete antiviral cytokines such as IFN- and
TNF- (13, 14). Additionally, NK cells lyse virus-infected cells upon
conjugate recognition of those cells by an "MHC-unrestricted"
mechanism (15, reviewed in Ref. 16). However, the eye constitutively
expresses NK-suppressing cytokines, including TGF-ß (17, 18, 19) and a
15-kDa protein (20), both of which have been shown to diminish NK
cytotoxicity in vitro. In mice, NK cells have been shown to modulate
MCMV replication in many tissues, including lung, liver, spleen, and
salivary gland (9, 13, 14, 21, 22). In humans, NK responses to ocular
CMV infections have not been reported; however, NK activity has been
shown to be elevated during active CMV disease in kidney transplant
patients (23). Importantly, stage IV AIDS patients have been reported
to have lost NK cytolytic activity in in vitro assays (24, 25). The
purpose of the studies reported herein was to examine the role of NK
cells in MCMV retinal infection. The results of these studies show that
NK cells are critical for preventing MCMV retinitis in immunocompetent
mice and that NK cells can also protect T cell-depleted mice from
retinitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female euthymic BALB/c mice, 6- to 8-wk old, were obtained from Taconic (Germantown, NY). Animals were housed on a 12-h light/dark cycle and given unrestricted access to food and water. Animals were housed in accordance with National Institutes of Health guidelines, and all procedures in this study conformed to the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Vision Research.
Virus and virus titrations
Stocks of MCMV (Smith strain) were prepared from salivary gland homogenates from MCMV-infected BALB/c mice as previously described (10). Virus stocks were titered in duplicate on Swiss Brown mouse embryo fibroblasts.
Thymectomy and T cell depletion
Thymectomies were performed using a modification of a protocol by Chin (26). Thymectomized mice were rested for 1 wk before T cell depletion. T cell depletion was accomplished by i.v. injection of 500 µg of anti-CD4 (GK1.5) and 150 µg of anti-CD8 (2.43) (American Type Culture Collection, Manassas, VA). The non-cross-reactive Abs PE-anti-Ly-3.2 (clone 53-5.8; PharMingen, San Diego, CA) and FITC-anti-L3T4 (clone RM4-4; PharMingen) recognizing CD8 and CD4, respectively, were used to determine the efficiency of T cell depletion.
Ocular inoculation
Mice were anesthetized by i.m. injection of a mixture containing 0.02 ml of Rompun and 0.03 ml of Ketamine per 25 g of body mass. The left eyes of mice were injected with 9.0 x 102 pfu of MCMV in a volume of 2 µl via the supraciliary route as previously described (10). Briefly, a superficial trans-scleral entry wound was made parallel and just posterior to the limbus by introducing the bevel of a sharp 30-gauge needle into the supraciliary space. Two microliters of virus (or diluted salivary gland homogenate) followed by 3 µl of air were then injected. The injection was judged successful if indirect ophthalmic observation showed a chorioretinal detachment associated with the appearance of air in the supraciliary space immediately after injection.
Flow cytometry
Animals were deeply anesthetized, perfused with PBS, and spleens and eyes were harvested. Eyes were cleaned of all muscle and connective tissue, leaving only the globe with some conjunctival tissue and approximately 1 mm of optic nerve. The cornea and lens were removed, and a single cell suspension of the remaining ocular tissue was made by pressing it through a 70-µm nylon mesh. Cells were washed three times in HBSS, RBC were lysed by ammonium chloride lysis buffer (ACK) treatment, and cells were washed three times in HBSS and resuspended in FACS buffer (PBS with 3% FBS). Samples were incubated with anti-CD32/CD16 (Fc receptor block) (PharMingen) according to the manufacturer’s recommendations for 15 min. Cells were then resuspended in: FITC-anti-L3T4 (CD4) (PharMingen); PE-anti-Ly-3.2 (CD8) (PharMingen); or FITC-anti-mouse pan-NK (DX-5) (PharMingen). After 15 min, cells were washed three times in FACS buffer and resuspended in FACS buffer for analysis. Large granular cells (predominately lymphocytes and macrophages) were included in the gate, and cell debris and small cells were excluded. By this method, 77.6% of the cells from a spleen, 8.9% of the cells from a normal eye, and 14.3% of the cells from an infected eye were included in the gate. Percent positive cells = % positive stained cells minus % positive cells from the same unstained sample.
Retinitis scoring
Eyes were fixed in buffered formalin, embedded in paraffin, and sectioned at six levels, 200 µm apart. The sections were then stained with hematoxylin and eosin. Changes in the posterior segment of each section were evaluated microscopically as follows: 0 = normal or injection artifact; 1/2 = mild atypical retinopathy: absence of cytomegaly plus retinal folds involving less than three-quarters of the retinal section; 1 = moderate atypical retinopathy: absence of cytomegaly plus retinal folds involving more than three-quarters of the retinal section plus photoreceptor atrophy or retinal infiltration by leukocytes involving more than one-quarter of the retina; 2 = retinal infection: cytomegaly of retinal cells plus partial-thickness retinal necrosis or full-thickness necrosis extending from the ciliary body, but not beyond a one-quarter retinal section from the ciliary body; 3 = necrotizing retinitis: cytomegaly plus full-thickness retinal necrosis existing further than one-quarter of a retinal section from the ciliary body or full-thickness retinal necrosis extending from the ciliary body through one quarter of the section; 4 = severe necrotizing retinitis: cytomegaly with full-thickness necrosis involving the entire retinal section.
A score of 3 or higher was considered positive for retinitis. The highest posterior segment score for each eye was the retinal score and was used to determine the mean retinal score. The average score for all retinal sections of each group was also determined. Differences between groups were determined using the Mann-Whitney U test (27).
Chromium release assay
One million NK-sensitive YAC-1 target cells were labeled with 100 µCi of 51Cr for 1 h in 500 µl of RPMI 1640 with 5% FBS (RPMI-5). The target cells were then washed three times and seeded into 96-well plates at 104 cells/100 µl/well. Effector cells were prepared by grinding the spleens or lymph nodes between frosted slides, followed by aspiration through a 22-gauge needle to make a single cell suspension. Cells were washed three times in HBSS, RBC were lysed by ACK treatment, and cells were washed three times in HBSS and resuspended RPMI-5. Cells were counted and 100 µl/well of the dilution to give the appropriate effector:target (E/T) ratio was plated in triplicate. For spontaneous or maximum release, 100 µl of media or 100 µl of 10% Triton X-100 (Sigma, St. Louis, MO) was added, respectively, instead of effector cells. Plates were centrifuged for 30 s at 100 x g and incubated for 4 h at 37°C. One hundred microliters of each supernatant were counted in a Wizard 1470 gamma counter (Wallac, Turku, Finland). Each set of triplicates was averaged, and specific lysis was determined. Percent specific lysis = (experimental release - spontaneous release)/(maximum release - spontaneous release) x 100. Spontaneous release in all experiments was less than 10% of the maximum release.
Depletion of NK cytotoxic activity
The protocol for in vivo depletion of NK cytotoxic activity was optimized using the chromium release assay. A single i.v. injection of 10 µg of anti-asialo GM1 (Wako Chemicals, Richmond, VA) reduced poly(I-C)-activated splenic NK activity to undetectable levels through day 3. Since NK activity was detectable again by day 5 (not shown), mice were injected on day -1 and day +4 with anti-asialo GM1 (Wako Chemicals) to maintain NK cell depletion. This regimen had no effect on the splenic T cell populations as determined by flow cytometry. Control animals were mock depleted with i.v. injections of the same volume of PBS. Later experiments showed no difference in retinal pathology 8 days after supraciliary infection with 9.0 x 102 pfu of MCMV between mice injected with PBS or 10 µg of normal rabbit serum.
Poly(I-C) enhancement of NK activity
Two hundred micrograms of poly(I-C) enhanced splenic NK cell activity, which peaked on day 3 as shown by chromium release (not shown). Therefore, for experiments involving enhancement of NK activity in T cell-depleted animals, 200 µg of poly(I-C) in 200 µl of PBS was injected i.p. on days -1, +2, and +5. Control animals were injected i.p. with PBS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Supraciliary injection of MCMV into immunocompromised mice causes,
in addition to retinitis, a marked infection of the iris, which forms
part of the posterior border with the anterior chamber (10, 11).
Because anterior chamber infections of the eye may depress delayed type
hypersensitivity responses, resulting in anterior chamber-associated
immune deviation (19, 28), and because the aqueous humor and vitreous
humor of the eye contain multiple substances that depress NK activity
(17, 18, 19, 20), it was necessary to determine whether supraciliary infection
with MCMV resulted in suppression of NK activity. To define the extent
of NK cell response, mice were injected via the supraciliary route with
9.0 x 102 pfu of MCMV 1, 2, 3, 5, or 7 days
before assay. The superficial cervical and mandibular lymph nodes
ipsilateral to the site of injection were harvested, pooled, and lymph
node cells were assayed for cytolytic activity against NK-sensitive
targets (YAC-1). In the lymph nodes, cytolytic activity was detectable
on day 2 postinfection (p.i.), peaked on day 5 p.i. (Fig. 1
A), and returned to
baseline levels after day 9 p.i. (not shown). Importantly,
cytolytic activity in the contralateral lymph nodes did not increase
from baseline until day 7 p.i. (not shown), suggesting that the NK
activity observed in the ipsilateral lymph node was due to the MCMV
infection in the eye and did not result from systemic or salivary gland
infection.
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B), and returned to baseline after day 9 p.i.
(not shown). The trends in splenic cytolysis were the same if the
percent specific lysis for each point was normalized either to splenic
mass or number of spleen cells. The number of cells recovered from
ocular tissues was too small to perform cytolytic assays on YAC-1
targets. To show that NK cells infiltrate ocular tissues during MCMV
infection, eyes and spleens were harvested 5 days after supraciliary
inoculation of 9.0 x 102 pfu of MCMV and analyzed by
flow cytometry for the presence of NK cells (Fig. 2). Mock-infected (not shown) and
uninfected animals were used as controls. Spleens from uninfected and
mock-infected animals contained an average percent (±SEM) of 5.8
± 0.4 and 5.7 ± 0.1 NK cells (DX-5+), respectively,
while the average percent of NK cells in spleens from infected mice was
8.1 ± 0.3. There was a small population of DX-5+
cells (12.5 ± 0.4) in uninjected eyes and in mock-infected eyes
(14.9 ± 4.6), which increased in MCMV-infected eyes to 19.4
± 2.3. A small percentage of DX-5+ splenocytes are
CD3+ (information provided by PharMingen). In normal
spleens, a population of CD4+, DX-5+ cells
(0.5 ± 0.2 above background) was detected; however, this
population did not increase in infected animals and was not detected in
infected or uninfected eyes. Additionally, on day 5 p.i.,
CD8+ T cells were not detected in infected eyes by flow
cytometry (not shown).
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To determine whether the NK response following supraciliary inoculation of MCMV mediates protection from MCMV retinitis, mice were treated with rabbit anti-asialo GM1 serum to deplete NK cells (see Materials and Methods). The supraciliary space of the left eye of each mouse was infected with 9.0 x 102 pfu of MCMV on day 0; mice were harvested on day 8. Eyes were fixed, sectioned, stained, and scored for retinitis.
As shown in Figure 3A, the
retinas of NK-depleted animals had retinal necrosis with loss of
retinal layer integrity. Cytomegalic cells were present in the outer
and inner nuclear layers of the retina as well as in the retinal
pigment epithelium. Additionally, the eyes of NK-depleted mice had
marked choroiditis as well as shortening of the photoreceptors and
atypical folding of the retinal layers. The choroid of infected eyes
from mock-depleted, MCMV-infected mice (as well as mock-infected eyes
from NK-depleted mice, not shown) appeared to be normal, and only minor
atypical retinal folding resulting from the injection was observed
(Fig. 3B). Eight days following supraciliary
injection of MCMV, 70% of the NK-depleted mice developed retinitis
compared with only 20% of the control mice (Table I). The average retinal score as well as
the average section score of eyes of NK-depleted mice were
significantly higher than those of control mice (Table I).
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Previous studies have shown that T cell-depleted mice infected with MCMV via the supraciliary route develop retinitis (10, 11). Therefore, to determine whether NK cells can prevent or modulate MCMV retinitis in the absence of T cells, mice were thymectomized and T cell depleted by injection of mAbs to CD4 and CD8. Mice were rested for 2 wk following T cell depletion and then injected with the NK cell activator poly(I-C) or PBS (control mice) on day -1, +2, and +5. All mice were injected with 9.0 x 102 pfu of MCMV on day 0. On day 8 p.i., mice were sacrificed; eyes were sectioned for histopathologic examination, and spleens were removed to verify T cell depletion. Flow cytometric analysis of splenocytes from these mice on day 8 p.i. (using Abs non-cross-reactive to GK1.5 and 2.43) showed 94.5 ± 1.7% of the CD4+ cells and 99.9% of the CD8+ cells were depleted.
Infected eyes from mice receiving poly(I-C) had significantly less
ocular pathology than non-poly(I-C)-treated, T cell-depleted mice (Fig. 4A, Table II). The mean retinal score, the average
score of all eye sections, and the percentage of eyes with retinitis in
T cell-depleted mice treated with poly(I-C) and depleted of NK cells by
anti-asialo GM1 treatment were not significantly
different from the eyes of T cell-depleted mice treated with PBS only
(Table II), suggesting that protection is mediated by NK cells and not
by antiviral effects of poly(I-C) or by the IFNs induced by poly(I-C)
(29, 30, 31). The eyes of T cell-depleted, poly(I-C)-treated mice injected
with salivary gland homogenate had no microscopic evidence of ocular
pathology.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Importantly, flow cytometry of DX-5-FITC-stained cells detected NK cells in ocular tissues 5 days following MCMV infection. The percentage of ocular cells falling into the gated forward and side scatter light parameters increased in MCMV-infected eyes, reflecting the infiltration of inflammatory cells. A higher percentage of gated cells recovered from MCMV-infected ocular tissues were DX5+ compared with mock-infected and uninjected controls. Surprisingly, DX-5+ cells were detected in normal eyes of perfused mice, a finding that suggests that there is a resident population of DX-5+ cells in the eye. It remains to be determined whether this DX-5+ population is truly an NK population and if so, whether NK cells are continually cycling in and out of the eye or are strictly local. The increase of NK activity (and of DX5+ cells, Bigger and Atherton, unpublished observation) in the draining lymph nodes following infection suggests that the increase in the DX-5+ population in ocular tissues results, at least in part, from migration of these cells into the eyes, and not just from an expansion of resident cell populations.
However, merely determining that supraciliary inoculation of MCMV induces an NK response does not provide information about the role of such a response during ocular infection. Depletion of NK cells allowed MCMV to infect and subsequently destroy the retina in a significantly higher percentage of mice injected with MCMV via the supraciliary route. Results from the NK depletion studies show the importance of NK cells in protection of the retina in this model and are further supported by the finding that poly(I-C)-induced NK cells protect the retina following MCMV infection in susceptible mice. Although Ikeda et al. (31) showed that delayed mortality in SCID mice infected with MCMV via the i.p. route and treated with poly(I-C) was independent of NK cells, our studies show that NK cells are required for poly(I-C)-mediated protection in the eye. Although high levels of IFNs have been detected in the peritoneal cavity following i.p. injection of poly(I-C), it is unlikely that these IFNs play a significant role in virus replication in the eye, especially since serum levels of such IFNs decrease within 18 h following i.p. injection of poly(I-C) (31).
The observation that poly(I-C) enhancement of NK cells protected the retina in MCMV-infected, T cell-depleted mice suggests that NK cells may modulate CMV retinal disease in humans and that therapeutic augmentation of NK activity might be used to treat CMV retinitis. NK cells may modulate ocular CMV infection in AIDS patients, since approximately 90% of AIDS patients are seropositive for CMV but only about half of these develop CMV retinitis (5, 6). Interestingly, kidney transplant patients with active CMV disease have elevated NK activity compared with patients without active CMV infection, even during posttransplant immunosuppression (23) and, while viral pneumonia is common in these patients, retinitis is not (5, 6).
In humans, CMV retinitis correlates with low CD4+ lymphocyte counts (33), and in BALB/c mice, T cell depletion predisposes to MCMV retinitis following ocular infection (10, 11). While there is a correlation between low CD4+ cell counts and CMV retinitis (33) and a correlation between AIDS progression and loss of NK function (24, 25), studies have not been done to determine whether there is a correlation between NK cell activity and development of CMV retinitis. AIDS patients have been reported to have very low T cell responses to CMV and yet to live for 5 yr or longer before the onset of CMV retinitis (34). There are also reports of patients with high CD4+ lymphocyte counts who have CMV retinitis (5, 6, 33). Loss of NK function, which has been reported in human AIDS patients as they progress to stage IV AIDS (24, 25), could contribute to or be sufficient for retinal infection by CMV. Similarly, a functional NK response could be responsible for protecting T cell-depleted patients from CMV retinitis. The results of these animal studies suggest that further investigation of NK activity in AIDS patients may be warranted.
In conclusion, the results of these studies show that depletion of NK cells in otherwise immunocompetent BALB/c mice predisposed the retina to MCMV infection. Enhancement of NK activity in T cell-depleted mice protected the retina from destructive retinitis. By extrapolation, these results suggest that NK activity may provide protection against CMV retinitis in seropositive human patients and that strategies to boost NK activity may be efficacious in modulating CMV retinitis, even in patients with low T cell counts.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sally S. Atherton, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284. E-mail address:
3 Abbreviations used in this paper: MCMV, murine cytomegalovirus; pfu, plaque-forming unit; p.i., postinfection.
Received for publication November 7, 1997. Accepted for publication February 18, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J. Exp. Med. 182:1045.This article has been cited by other articles:
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