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*
Department of Microbiology, University of Tennessee, Knoxville, TN 37996;
Department of Medicine/Arthritis, Northwestern University, Chicago, IL 60611
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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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Among many unsolved issues regarding the pathogenesis of HSK is the identity of Ags responsible for driving the immunopathologic process and subsequently its resolution. One might expect that virus-derived Ags should be primarily involved and that the CD4+ T cells that recognize such Ags in the corneal stroma recruit and guide the inflammatory process. This notion, however, is confounded by the observation that neither replicating virus, viral mRNA or viral proteins, is usually detectable during the clinically apparent progressive stage of HSK (10). Viral DNA can be demonstrated throughout the disease process, but this DNA does not appear to be expressed (11). Viral peptides could conceivably persist in the cornea for long periods in association with Langerhans cells, but no direct evidence supports this possibility.
An alternative source of Ag to drive the HSK could derive from corneal tissue perhaps being unmasked and altered as a consequence of HSV replication. This idea received support from the observations of Avery et al., which showed that HSK could be transferred to virus-infected athymic mice with a cell line reactive with an autoantigen expressed by an Ig isotype (12). We have also shown adoptive transfer of HSK with cell lines of uncharacterized autoreactivity, but which do not additionally express detectable HSV reactivity (13). Unfortunately, in the adoptive transfer models, recipient mice are immunocompromised, and virus replicates for longer periods, including the time during HSK expression. Thus, viral Ags are present and cross-reactivity with such Ags or bystander activation by virus-derived superantigens might cause infiltrating T cells to emanate inflammatory cytokines.
In the present report, we have approached the question of the role of
nonviral Ags in HSK using animals susceptible to HSV but unable to
develop immune responses against HSV proteins. For such studies, we
selected TCR-
ß OVA-transgenic mice that were backcrossed
to SCID and shown to possess CD4+ T cells that were almost
exclusively ß OVA-TCR positive. Moreover the mice were
unable to develop detectable T cell responses to HSV even when
repeatedly immunized with a live but replication-defective mutant HSV.
Such mice still developed HSK upon ocular infection with HSV. We
interpret our results to indicate that the CD4+ T cells,
shown present in lesions, must be mediating inflammation either by
recognizing nonviral autoantigens in the cornea or perhaps more likely
because of nonspecific bystander activation by virus- or host-derived
components.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four- to five-week-old BALB/c mice (Harlan Sprague Dawley, Indianapolis, IN), OVA-TCR-transgenic mice (DO11.10 mice, kindly provided by Dr. Dennis Loh, Washington University School of Medicine, St. Louis, MO and Dr. Casey Weaver, University of Alabama, Birmingham, AL) were used. Transgenic mice were crossed to SCID mice for two generations, and, with brother-sister mating, it was possible to obtain Tg-SCID mice (as screened by PCR-tail DNA and serum IgM-ELISA). Tg-SCID mice thus obtained had only CD4+ T cells and few or no CD8+ T cells, as tested by FACS analysis, and were without any B cells, as screened by serum IgM ELISA. Also, almost all (>98%) of the CD4+ T cells were KJ1-26.1 (anti-OVA-TCR Ab)-positive T cells. Transgenic mice (DO11.10) and HSK-susceptible BALB/c groups of mice were used as the control animals. All food, water, bedding, and instruments were autoclaved or disinfected, and all manipulations were done in a laminar flow hood. To prevent bacterial superinfections, all Tg-SCID and SCID mice received prophylactic treatment of sulfatrim pediatric suspensions (Barre-National, Baltimore, MD) at the rate of 5 ml per 200 ml of drinking water. Antibiotic treatment was started 1 day before the beginning of the experiments. All experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources, Commission Life Sciences, National Research Council. The facilities used were accredited by the American Association for Accreditation of Laboratory Animal Care. All ocular experimental procedures were conducted according to the Association for Research in Vision and Ophthalmology (ARVO) resolution on the use and care of laboratory animals.
Virus
HSV-1 RE strain used was propagated on vero cells and stored as infectious cell preparations at -70°C. Replication-defective virus AN-1 was kindly provided by Dr. Sandra K. Weller, University of Connecticut Health Center, Farmington, CT.
Corneal infections, immunization, and passive transfer
In each experiment, control eyes were scarified and treated with 4 µl of PBS or tissue culture extracts. The corneal surfaces of deeply anesthetized mice (methoxy flurane; Pittman-Moore, Mondelein, IL) were scarified with a sterile 27-gauge needle, and 5 x 105 TCID-50 HSV-1 RE strain was applied in a 4.0-µl volume and gently massaged onto the eye lids. For immunization with AN-1 virus or UV-inactivated HSV-1, anesthetized mice were injected with 5 x 106 TCID-50 in 50 µl volume on day 0 and day 14 through the footpad route (25 µl per foot pad) of injection. To assess the role of replication in lesion development, mice were infected with virus on the cornea and 24 h later given 300 µl of anti-HSV serum (40 µg/ml HSV-specific IgG as determined by ELISA). In such experiments, controls were infected and given normal mouse serum.
Clinical evaluation
Mice were scored according to their clinical severity by a person who was blinded to the experimental design using a slit lamp biomicroscope (Keeler Instruments, Broomall, PA) as follows; score 0, normal cornea; score 1, neovascularization at periphery, iris visible; score 2, partial opacity, iris not visible; score 3, neovascularization at center, opacity; score 4, bleb formation; score 5, necrosis. The data were plotted as the mean daily clinical score for all animals in a particular treatment group.
Ig ELISA
Serum collected was analyzed for HSV-specific total IgG, using standard ELISA as described previously (14). Basically, ELISA plates were coated with 100 µl of HSV Ag in carbonate buffer (pH 9.8), and, after overnight incubation at 40°C, the plates were washed three times in PBS containing 0.05% Tween 20, pH 7.2 (PBST), and then blocked using PBS (pH 7.2) with 3% dehydrated milk for 2 h at 37°C. A total of 200 µl of serially diluted serum samples (prediluted in PBST) were added in duplicate, and washed wells coated with goat anti-mouse IgG (0.25 µg/ml) were treated with serially diluted standard mouse IgG. Plates were incubated for 2 h at 37°C. After three washes, 100 µl of goat anti-mouse IgG horseradish peroxidase was added. After three washes, 2,2-azino-bis 3-ethyl benz-thiazoline-6-sulfonic acid substrate (Sigma, St. Louis, MO; No. A1888) was added. The concentration of the Abs in the serum samples was determined from the standard curve.
Cytokine assay
For cytokine (IFN-
) assay, splenocytes from mice were
suspended in 10% RPMI 1640, and 106 cells in 1 ml were
stimulated in vitro with 1.5 MOI (multiplicity of infection, before
inactivation) of UV-inactivated HSV-1 (KOS strain). Similar number of
cells were Con A stimulated (5 µg/106 cells/ml) in
12-well culture plates. Plates were incubated at 37°C for 72 h.
The supernatant fluid was collected and stored at -20°C until use.
These supernatants were screened for the presence of IFN- by ELISA
as described previously (14).
Histopathology
Eleven days after infection, sections of eye were prepared for histopathology according to standard procedures. Briefly, at the termination of experiments, whole eyes were fixed in 10% buffered neutral formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. Sections were observed for thickness of cornea, presence of inflammatory infiltrates, neovascularization, epithelial erosions, superficial or deep ulcers, and corneal perforation.
Proliferation assay
Splenocytes (responders) at day 11 p.i. were collected and restimulated in vitro with HSV-1 KOS (MOI of 1.5 before UV irradiation) or OVA peptide (Research Genetics, Huntsville, AL). The HSV-1-specific lymphoproliferation was measured as described previously (15). Briefly, responders:stimulator ratios tested ranged from 10:1 to 0.625:1. Responders plus stimulator mixtures were incubated at 37°C for 4 days. [3H]TdR (1.0 µCi/well) was added, harvested 18 h later, and read using a beta scintillation counter (Trace 96, Inotech, Lansing, MI). The results were expressed as mean plus SD.
RNA isolation and RT-PCR
On day 11 p.i., splenocytes and cornea were collected and
transferred to Tri-reagent (Molecular Biology, Cincinnati, OH). The
corneas were teased with a sterile 21-gauge needle and titrated with a
sterile 1-ml syringe plunger to expose the cells to lytic action of the
Tri-reagent. The total cellular RNA was isolated from the Tri-reagent
cellular lysate by adding chloroform, and centrifugation followed by
ethanol/isopropyl ethanol precipitation of the aqueous RNA solution
according to the manufacturer’s instructions. The RNA thus obtained
was reverse transcribed using oligo(dT) primers and super script (Life
Technologies, Bethesda, MD) according to standard protocol (16). The
cDNA thus obtained was used as a template in subsequent qualitative and
quantitative PCRs. Primers for different V,C and Vß,Cß were
used to check various segments of TCR (17). For semiquantitation of the
leaky V-chain of TCR, equal amounts of cDNA samples were amplified
with V1, V2, V5, V8, V11 primers and compared with
V13.1 expression in Tg-SCID and BALB/c samples.
Flow cytometric analysis
Isolated cervical lymph node cell populations were analyzed for
cell surface markers by flow cytometry. Viable cells were blocked with
heat-inactivated FBS and washed three times with FACS buffer (1x PBS
with 1% BSA and 0.05% NaN3). For Tg-SCID mice
characterization, cells from cervical lymph nodes were stained with
anti-CD4-FITC and anti-CD8-PE. For transgenic TCR-positive T
cells, KJ1-26.1 (anti-OVA TCR Ab, a kind gift from Dr.
Phillipa Marrack, National Jewish Hospital, Denver, CO) Ab was used and
detected by adding goat anti-mouse IgG2a-FITC Ab. To check for
activation markers on CD4+ T cells, lymph node cells were
blocked with FBS and double stained with FITC or PE-labeled
anti-CD4 and anti-CD45 RB-PE or anti-L-selectin-PE. Dual
V TCR analysis was done using TCR V 2-PE and KJ1-26.1 Ab. Events
were collected and analyzed using a Becton Dickinson (Mountain View,
CA) FACScan analyzer.
Immunohistochemical staining
At the termination of experiments, eyes were nucleated and snap frozen in OCT compound (Miles, Elkhart. IN). Eight-micron-thick sections were cut, air dried, and fixed in cold acetone for 5 min. The sections were then blocked with heat-inactivated goat serum and stained with biotinylated anti-CD4, anti-Thy1.2 (PharMingen, San Diego, CA) or biotinylated KJ1-26.1 Ab (biotinylated anti-OVA TCR, a kind gift from Dr. Jerold Woodward, University of Kentucky, Lexington, KY). Sections were then treated with horseradish peroxidase-conjugated streptavidin (1:1000) and 3,3'-diaminobenzidine (Vector, Burlingame, CA) and counterstained with hematoxylin. For OVA-TCR (KJ1-26.1) staining, sections were also treated with a tyramide signal amplification kit (TSA Indirect, Dupont NEN, Boston, MA) before treatment with diaminobenzidine.
Delayed-type hypersensitivity (DTH) assays
On the day of testing, each mouse was injected with 20 µl of 105 UV-inactivated HSV-1 KOS (106 pfu before inactivation) in the right ear and the same volume of vero cell extract in the left ear. The right and left ear thicknesses of each mouse were measured with a screw gauge Odimeter (Oditest, H. C. Kroeplin, Schluechtern, Germany) and recorded individually. The thickness was measured at 1 h before ear injection, then at 24, 48, and 72 h after injections, and the values were represented as n x mm-2. The mean ear thickness of each ear from each group of animals was calculated, and the mean increase between before and 48 h after injections was compared among BALB/c mice, DO11.10 mice, and Tg-SCID mice.
Virus recovery
At various time points p.i., eye swabs were taken using sterile swabs soaked in McCoy medium containing 100 IU/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Grand Island, NY). The swabs were then placed in tubes containing 500 µl of the above mentioned transport medium and stored at -80°C. For detection of HSV in swabs, the samples were thawed and vortexed, and 100 µl of each sample from individually marked mice was used for quantification of virus recovery by standard pfu assay on vero cell cultures as described elsewhere (18).
Statistical analysis
Wherever specified, data obtained were analyzed for statistical significance by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As reported previously, infection of the scratched cornea of
BALB/c mice with HSV-1 RE is followed after 8 to 10 days by the
development of a clinically evident inflammatory response in the cornea
(19). Typically, responses are at their peak around 3 wk p.i., and they
may persist for several weeks. A similar response profile is recorded
in Figure 1
. In addition, Figure 1 shows
the response pattern that occurred in DO11.10 mice. It is apparent that
the clinical pattern was much the same. Sample BALB/c and DO11.10 mice
were killed at 14 days p.i., and their eyes were examined
histologically and additionally analyzed by immunohistochemistry for
the presence of CD4+ T cells (Fig. 2). Essentially no differences were
observed between the two animal strains.
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, the DO11.10 mice responded
immunologically to HSV and developed both T cell and Ab responses.
Moreover, the DO11.10 mice recovered normally from infection and did
not succumb to viral encephalitis. The explanation for such patent
anti-HSV responses and protective immunity was presumably
because of the extensive TCR -chain leakage evident in the
DO11.10 mouse (17). Thus, as shown in Figure 3, the DO11.10 mice expressed most of the
TCR -chains expressed by BALB/c at least as detected by RT-PCR for
-chain and ß-chain mRNA. Quantitative comparisons of - and
ß-chain expression in transgenic and BALB/c animals were not made.
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In an attempt to reduce or perhaps eliminate TCR -chain leakage
in the transgenic mice, DO11.10 mice were backcrossed to SCIDs, and the
offspring were selected that were TCR Tg+ and
SCID+ (Tg-SCID). Such mice were shown by FACS analysis to
possess T cells, all of which were CD4+ and expressed the
KJ1-26.1 Id marker (Fig. 4). The mice
possessed no B cells and failed to generate IgM Ab to environmental Ags
(data not shown). These Tg-SCID mice were highly susceptible to HSV
infection and rapidly succumbed to encephalitis following ocular
infection with HSV-1 RE at 4 to 5 wk of age. Animals infected via the
cornea at 7 to 8 wk of age survived for as long as 13 days before dying
from lethal encephalitis. Notably, however, most animals of this age
groups expressed mild ocular lesions (clinical score 3 at day 10) that
were approximately equal to those observed in BALB/c and DO11.10 mice
at the same time frame (Fig. 5). Analysis
of lesions by histopathology as well as for the presence of
CD4+T cells by immunohistochemistry revealed a situation
comparable to that observed in BALB/c mice (Fig. 6). In addition, the T cells in the
cornea were clonotype positive (KJ1-26.1+). Accordingly, we
judge that the Tg-SCID mice had lesions of HSK, although all animals
died of HSV encephalitis within 4 to 5 days of their lesions being
manifest. Control SCIDs lacking T cells failed to express lesions when
examined around day 10 p.i. (Fig. 6) and also died of encephalitis
by 14 days. Analysis of virus recovery from the infected eyes of
Tg-SCID (Table II) revealed that
infectious virus was demonstrable up to day 11 whereas, in the BALB/c
mice, infectious virus was not demonstrable beyond 6 days p.i. In
another set of experiments to determine whether the disease was indeed
a result of HSV replication, Tg-SCID mice were infected with HSV and,
after 24 h, given anti-HSV antiserum. This procedure not only
protected mice from death by encephalitis, but also, as shown
previously by Pepose and colleagues (20), prevented the development of
primary HSK. These data are shown in Figure 7, as are the results in Tg-SCID mice
that received normal mouse serum. The latter animals developed HSK and
died of encephalitis.
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The fact that Tg-SCID mice could express HSK, even though they
failed to survive HSV infection, could mean that they too had
sufficient V-chain leakage and developed anti-HSV
CD4+ T cells that in turn orchestrated the ocular
inflammatory response. Alternatively, it could be that the HSK
responses in the Tg-SCID mice were the consequence of a
nonvirus-specific, perhaps autoreactive, response, set off in the
cornea as a consequence of HSV infection.
Following HSV ocular infection, evidence was sought for lymphoid
expansion in Tg-SCID mice. Mice with HSK had enlarged and more cellular
cervical lymph nodes (Table III) and,
upon analysis by FACS for activation markers, showed a higher
percentage of CD4+ T cells expressing the activated
phenotype, i.e., CD45RB low and L-selectin low, than cells from
uninfected mice (Fig. 8). To assess
whether Tg-SCID mice could develop anti-HSV immunity, animals with
early HSK were killed at day 11 p.i., and their spleens were
tested for HSV-specific lymphoproliferation and cytokine secretion. As
controls, infected BALB/c and DO11.10 splenocytes were tested at the
same time p.i. The results shown in Table IV indicate that the Tg-SCID mice failed
to respond to HSV, but they did respond to OVA stimulation. In
addition, mice were infected in the food pad with UV inactivated HSV-1
KOS and tested 2 wk later for HSV-1-specific DTH response. Such DTH
responses were lacking in Tg-SCID mice but were present in similarly
immunized BALB/c control mice (Table V).
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that the Tg-SCID
mice still failed to develop HSV-specific immunity detectable either in
vivo by DTH or by in vitro Ag stimulation of their splenocytes. Further
attesting to the failure of such AN-1-immunized mice to generate immune
responses to HSV was the fact that the mice still succumbed to
encephalitis upon ocular infection with HSV-1 RE. Before death,
however, the immunized and ocularly infected mice all developed HSK
lesions (Table VII).
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The above results indicate that Tg-SCID mice fail to recognize HSV
Ags, yet such animals still expressed HSK lesions. Virus infection did
result in an increase in T cell activation, but the issue arose as to
how the repertoire-restricted Tg-SCID T cells could mediate HSK. One
possibility was that Tg-SCID T cells showed V or Vß leakage and
that such dual V TCR+ T cells recognized corneal Ags and
mediated HSK. Evidence for V segment leakage was searched for by
RT-PCR using splenic RNA collected from both uninfected and
HSV-infected Tg-SCID mice. As is apparent in Figure 9 and Table VIII, evidence for V (but not Vß)
leakage was present in splenic samples in both groups of animals.
Interestingly, examination of individual animals showed that all of
them expressed V1, V2, V5, and V11 in addition to the
V13.1 transgene. We obtained no evidence that HSV infection changed
the nature of the V leakage, but the expression level appeared
increased in samples from HSV-infected Tg-SCIDs (data not shown). By
semiquantitative PCR, V2 showed the most leakage of the four extra
V segments expressed. Evidence for cells in the draining lymph node
of HSV-infected mice that expressed surface V2 in addition to
V13.1 was sought for by FACS analysis. Such dual V-positive T
cells were present in DO11.1O samples (but the 2.9% value is likely
not significant) but were not detectable in either Tg-SCID or BALB/c
samples (Fig. 10). Furthermore, perhaps
arguing against a pathogenic role for V dual-positive cells in
mediating HSK, we were unable to find evidence for leaked V
expression in corneal samples from Tg-SCID mice with early HSK (Fig. 11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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That HSK represents an immunopathologic lesion in both man, the natural host, and in the murine experimental counterpart is widely accepted (2, 4, 13). However, the identity of Ags involved as targets for the immunopathologic response remain to be defined. Characteristically, virus-induced immunopathologic diseases occur in situations where virus persists in the presence of an ongoing immune response (22, 23). Most commonly, either CD8+ or CD4+ T cells are the cells involved in controlling lesion development (23). In HSK, the primary cell type that orchestrates lesions, are CD4+ T cells of the Th1 cytokine-producing profile (5, 6). However, during disease expression, it is usually not possible to demonstrate either replicating virus or viral gene expression (24). Although it is difficult to exclude the possibility that viral Ag-derived peptides persist in association with corneal APCs and that these drive the inflammatory process, other mechanistic explanations have emerged. Some have advocated that the principal cognate Ags recognized by CD4+ T cells are host Ags unmasked in the normally avascular cornea as a consequence of HSV replication (1, 12), or by inflammatory products released from the rapidly responding neutrophil response (9, 13). The host Ag target hypothesis receives strongest support by studies of Avery et al. (12), who investigated the basis for the marked differences in susceptibility between two congenic mouse strains. Their observations were interpreted to indicate that HSV infection caused the availability of a corneal Ag that cross-reacted with the IgG2a isotype of Ig. Other more indirect lines of evidence supporting a role for autoantigens in HSK include the observance of antiself peptide-reactive T cells following HSV infection and the adoptive transfer of HSK to infected SCIDs with an autoreactive cell line (Ref. 25; S. J. Babu and B. T. Rouse, manuscript in preparation).
Our observation that viral Ag-unreactive Tg-SCID mice still develop HSK
could be explained by their T cells recognizing nonviral Ags, perhaps
even host-derived autoantigens expressed as a consequence of virus
replication in the cornea. Corneal Langerhans cells presenting nonviral
autoantigens that may be cross-reactive to the OVA-specific
TCR may present such a possibility. Although we cannot formally reject
the autoantigen hypothesis, we consider the explanation as unlikely.
Thus, it is difficult to accept the idea that a T cell
repertoire-handicapped mouse would still be able to recognize an
autoantigen but not one or more of the abundant Ags derived from a
virus that encodes more than 70 proteins (26). Alternatively, some
viral or virus-induced self proteins could act as a superantigen
stimulating CD4+ T cells via binding to Vß region of TCR
(27). We, in fact, observed a mild lymphocytosis in mice following HSV
infection and showed that lymphocytes expressing activation markers
were more abundant in infected mice. HSV itself is not known to express
superantigen activity, so the explanation for the lymphocyte
stimulation remains unknown. A possible source of stimulation is host
heat shock proteins (HSP), some of which are up-regulated as a sequel
to HSV infection (28). There is some evidence that host HSPs may act as
cognate Ags in certain autoimmune diseases and that some of them may
act as polyclonal activators (29, 30). Conceivably, T cells expressing
more than a single TCR could exhibit loose recognition characteristics
that include virus-induced HSPs. Indeed some have advocated that T
cells with multiple V expression could be involved in mediating
certain autoimmune diseases (31). In our study, we demonstrated V
leakage in lymphoid tissue in Tg-SCID mice and noted that the extent,
although not the spectrum, of leakage appeared elevated after HSV
infection. However, arguing against the pathogenic role of dual V
cells in HSK was our failure to demonstrate V leakage in corneal
tissue taken from Tg-SCID mice with HSK. In addition, the lymphoid
tissue from such mice did not contain significant number of cells with
dual V expression.
An alternative mechanism by which CD4+ T cells become
activated to mediate immunoinflammatory lesions in Tg-SCID mice could
involve a process akin to bystander activation. Accordingly, in the
presence of a "cytokine storm," cells with appropriate receptors
may become activated and in turn contribute to the inflammatory
response. This is thought to be a mechanistic event in the disease
Dengue hemorrhagic fever (32). This bystander activation is usually
considered to largely involve mononuclear phagocytes (33),
but it has also clearly been shown to apply in some circumstances
to CD4+ T cells (34, 35). Since we did not observe any
detectable anti-HSV reactivity even after AN-1 immunization, it is
tempting to speculate that CD4 T cells in the cornea are activated into
effector function by mechanisms other than those involving TCR
occupancy. One prominent consequence of HSV replication in the mouse
eye is the expression of many chemokines and cytokines, contributed
by corneal epithelium and perhaps by recruited inflammatory cells (19, 21, 36). Therefore, we speculate that, in the HSV-infected eye, this
proinflammatory microenvironment could activate ingressing CD4 T
cells. Indeed, it has been reported previously that resting naive human
CD4+ T cells could be activated to proliferate by a
cytokine combination consisting of IL-2, TNF-, and IL-6 (34). Such a
combination also caused effector function in resting memory CD4 T
cells, as measured by lymphokine synthesis and providing help for Ig
production by B cells (34). Alternatively, Ag-independent T cell
activation could also occur through accessory molecules on the T cells.
One such mechanism includes CD2-mediated interactions on the T cells
with ligands such as LFA-3 and CD59. Indeed, it has been reported that
LFA-3 and CD59 expressed on rat retinal pigment epithelium could
trigger syngeneic T cells to proliferate and produce IL-2 in the
absence of any exogenous Ag (37). LFA-3 and CD59 are reported to be
expressed by corneal epithelium, stromal keratocytes, and retinal
pigment epithelium (37, 38). While the role of these molecules in
nonspecific T cell activation in the murine cornea is not clear, it is
tempting to speculate that such possibilities could occur in our mouse
model. Accordingly, we propose that CD4 T cells drawn into the
HSV-infected cornea become activated either by the proinflammatory
environment of the cornea and/or by activation through accessory
molecules such as CD2 in a TCR-independent manner. Such nonspecifically
activated CD4 T cells generate effector molecules that could recruit
and activate polymorphonuclear neutrophils and macrophages that further
contribute to the inflammation process in the eye. Additional
experiments are currently underway to test these ideas.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Barry T. Rouse, Department of Microbiology, University of Tennessee, M409 Walters Life Sciences Building, Knoxville, TN 37996. E-mail address:
3 Abbreviations used in this paper: HSV, herpes simplex virus; DTH, delayed-type hypersensitivity; HSK, herpetic stromal keratitis; Tg-SCID, D011.10 mice back-crossed to SCID mice; OVA, OVA peptide (323–339); OVA-TCR, OVA peptide (323–339)-specific TCR; SI, stimulation index; PE, phycoerythrin; p.i., postinfection; HSP, heat shock protein; pfu, plaque-forming unit.
Received for publication March 5, 1998. Accepted for publication June 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 are protective in the skin but pathologic in the corneas of HSV-1-infected mice. J. Immunol. 149:3023.[Abstract]
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S. A. Curran, O. M. FitzGerald, P. J. Costello, J. M. Selby, D. J. Kane, B. Bresnihan, and R. Winchester Nucleotide Sequencing of Psoriatic Arthritis Tissue before and during Methotrexate Administration Reveals a Complex Inflammatory T Cell Infiltrate with Very Few Clones Exhibiting Features That Suggest They Drive the Inflammatory Process by Recognizing Autoantigens J. Immunol., February 1, 2004; 172(3): 1935 - 1944. [Abstract] [Full Text] [PDF]
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P. Lundberg, P. Welander, X. Han, and E. Cantin Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo J. Virol., October 15, 2003; 77(20): 11158 - 11169. [Abstract] [Full Text] [PDF]
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 D. W. Mullins, R. S. Martins, and K. D. Elgert Tumor-Derived Cytokines Dysregulate Macrophage Interferon-{gamma} Responsiveness and Interferon Regulatory Factor-8 Expression Experimental Biology and Medicine, March 1, 2003; 228(3): 270 - 277. [Abstract] [Full Text] [PDF]
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D. J. J. Carr and S. Noisakran The Antiviral Efficacy of the Murine Alpha-1 Interferon Transgene against Ocular Herpes Simplex Virus Type 1 Requires the Presence of CD4+, {alpha}/{beta} T-Cell Receptor-Positive T Lymphocytes with the Capacity To Produce Gamma Interferon J. Virol., August 12, 2002; 76(18): 9398 - 9406. [Abstract] [Full Text] [PDF]
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 M. Zheng, D. M. Klinman, M. Gierynska, and B. T. Rouse DNA containing CpG motifs induces angiogenesis PNAS, June 25, 2002; 99(13): 8944 - 8949. [Abstract] [Full Text] [PDF]
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 M Halberstadt, M Machens, K-A Gahlenbek, M Bohnke, and J G Garweg The outcome of corneal grafting in patients with stromal keratitis of herpetic and non-herpetic origin Br J Ophthalmol, June 1, 2002; 86(6): 646 - 652. [Abstract] [Full Text] [PDF]
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S. Lassmann, C. Kincaid, V. C. Asensio, and I. L. Campbell Induction of Type 1 Immune Pathology in the Brain Following Immunization Without Central Nervous System Autoantigen in Transgenic Mice With Astrocyte-Targeted Expression of IL-12 J. Immunol., November 1, 2001; 167(9): 5485 - 5493. [Abstract] [Full Text] [PDF]
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M. Zheng, S. Deshpande, S. Lee, N. Ferrara, and B. T. Rouse Contribution of Vascular Endothelial Growth Factor in the Neovascularization Process during the Pathogenesis of Herpetic Stromal Keratitis J. Virol., October 15, 2001; 75(20): 9828 - 9835. [Abstract] [Full Text] [PDF]
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S. Deshpande, M. Zheng, S. Lee, K. Banerjee, S. Gangappa, U. Kumaraguru, and B. T. Rouse Bystander Activation Involving T Lymphocytes in Herpetic Stromal Keratitis J. Immunol., September 1, 2001; 167(5): 2902 - 2910. [Abstract] [Full Text] [PDF]
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 M. Zheng, M. A. Schwarz, S. Lee, U. Kumaraguru, and B. T. Rouse Control of Stromal Keratitis by Inhibition of Neovascularization Am. J. Pathol., September 1, 2001; 159(3): 1021 - 1029. [Abstract] [Full Text] [PDF]
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F. Benencia, M. C. Courrèges, G. Gamba, H. Cavalieri, and E. J. Massouh Effect of Aminoguanidine, a Nitric Oxide Synthase Inhibitor, on Ocular Infection with Herpes Simplex Virus in Balb/c Mice Invest. Ophthalmol. Vis. Sci., May 1, 2001; 42(6): 1277 - 1284. [Abstract] [Full Text]
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D. J.J. Carr, P. Härle, and B. M. Gebhardt The Immune Response to Ocular Herpes Simplex Virus Type 1 Infection Experimental Biology and Medicine, May 1, 2001; 226(5): 353 - 366. [Abstract] [Full Text]
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S. P. Deshpande, S. Lee, M. Zheng, B. Song, D. Knipe, J. A. Kapp, and B. T. Rouse Herpes Simplex Virus-Induced Keratitis: Evaluation of the Role of Molecular Mimicry in Lesion Pathogenesis J. Virol., April 1, 2001; 75(7): 3077 - 3088. [Abstract] [Full Text]
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S. K. Eo, S. Lee, S. Chun, and B. T. Rouse Modulation of Immunity against Herpes Simplex Virus Infection via Mucosal Genetic Transfer of Plasmid DNA Encoding Chemokines J. Virol., January 15, 2001; 75(2): 569 - 578. [Abstract] [Full Text]
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S. P. Deshpande, M. Zheng, M. Daheshia, and B. T. Rouse Pathogenesis of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions in B-Cell-Deficient Mice J. Virol., April 15, 2000; 74(8): 3517 - 3524. [Abstract] [Full Text]
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S. Gangappa, S. P. Deshpande, and B. T. Rouse Bystander Activation of CD4+ T Cells Accounts for Herpetic Ocular Lesions Invest. Ophthalmol. Vis. Sci., February 1, 2000; 41(2): 453 - 459. [Abstract] [Full Text]
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