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,
*
Department of Pathology, University of Illinois, Chicago, IL 60154; Departments of
Ophthalmology and
Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213;
§
Department of Cell Biology and Immunology, Free University, Amsterdam, The Netherlands; and
¶
Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR+ T
lymphocytes infiltrate the TG, and TNF-
, IFN-, the inducible
nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-,
IFN-, and the iNOS product nitric oxide (NO) all inhibit HSV-1
replication in vitro. Macrophage and TCR+ T cell
depletion studies demonstrated that macrophages are the main source of
TNF- and iNOS, whereas TCR+ T cells produce
IFN-. Macrophage depletion, aminoguanidine inhibition of iNOS, and
neutralization of TNF- or IFN- all individually and
synergistically increased HSV-1 titers in the TG after HSV-1 corneal
infection. Moreover, individually depleting macrophages or neutralizing
TNF- or IFN- markedly reduced the accumulation of both
macrophages and TCR+ T cells in the TG. Our findings
establish that after primary HSV-1 infection, the bulk of virus
replication in the sensory ganglia is controlled by macrophages and
TCR+ T lymphocytes through their production of
antiviral molecules TNF-, NO, and IFN-. Our findings also
strongly suggest that cross-regulation between these two cell types is
necessary for their accumulation and function in the infected
TG. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of recent studies using the mouse model of HSV-1 corneal
infection have established that transmission of the virus to the TG is
associated with leukocytic infiltration and cytokine production within
the ganglion 2, 3, 4 . Macrophages, TCR+ T
lymphocytes, and TCR-ß+ T cells of both the
CD4+ and CD8+ subpopulations infiltrate the TG
during this period of active virus replication. Macrophages are the
predominant infiltrating cell in the TG 3–7 days after infection,
whereas CD8+ T cells preferentially accumulate and dominate
the TG infiltrate 7–12 days after infection 3 . During the early
stages of HSV-1 replication, 3–5 days after infection, macrophages and
TCR+ T cells can be seen surrounding infected
neurons. By 7–12 days after corneal infection, CD8+ T
cells are also preferentially drawn to the infected neurons in the
ophthalmic branch of the TG 3 .
Depletion of TCR+ T cells dramatically increases
HSV-1 titers in the TG but does not influence the duration of HSV-1
replication in the ganglion 5 . In contrast, the absence of
TCR-ß+ T cells does not increase HSV-1 titers in the
TG but does result in prolonged, low level HSV-1 replication in the TG,
transmission to the brain, and lethal viral encephalitis 5 . Depletion
of CD8+ T cells or compromise of CD8+ T cell
function also significantly augments HSV-1 neurovirulence 6, 7 . Thus,
TCR+ T cells represent an important early line of
defense against HSV-1 replication in the sensory neurons, whereas
CD8+ T cells are required to completely shut down HSV-1
replication in the TG and prevent neurologic damage.
The mechanism by which TCR+ T cells control virus
replication in sensory neurons is not known. IFN- is produced in the
ganglion early after infection during the acute phase of virus
replication, and the absence of this molecule results in increased
virus replication in the ganglion 8 . Although IFN- has direct
antiviral activity, it also plays an important role in activating
macrophages 9 . In fact, in certain infectious disease models,
macrophage production of TNF- and nitric oxide (NO) is regulated by
TCR+ cells through their production of
IFN- 10, 11 . In addition, HSV infection has been shown to augment
NO synthesis by IFN--treated macrophages 12 . Both TNF- and NO
are potent inhibitors of HSV-1 replication in vitro 11, 13, 14, 15, 16 .
We hypothesized that an interaction between macrophages and
TCR+ T cells might lead to their production of antiviral
cytokines and control of virus replication in sensory neurons. Our
current findings support this hypothesis and demonstrate an important
protective role for the cytokines TNF- and IFN- and the reactive
nitrogen intermediate NO.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (Frederick Cancer Research Center, Frederick, MD), 6–8-wk-old, were anesthetized by i.m. injection of 2 mg of ketamine hydrochloride (Vetalar; Parke-Davis, Morris Plains, NJ) and 0.04 mg of acepromazine maleate (Aveco, Fort Dodge, IA) in 0.1 ml of HBSS into the left hind leg.
Virus
The RE strain of HSV-1 was grown in Vero cells, and intact virions were purified on Percoll (Pharmacia, Piscataway, NJ) as previously described 17 .
Corneal infection
Topical corneal infection of anesthetized mice was achieved by superficially scratching the central cornea 15 times with a 30-gauge needle in a crisscross pattern. A 3-µl HSV-1 suspension (105 plaque-forming units (PFU)) was applied topically to the scarified cornea and rubbed in with the eyelids. All experimental procedures conformed to the Association for Research in Vision and Ophthalmology resolution on the use of animals in research.
In vivo macrophage depletion
Dichloromethylene diphosphonate (clodronic-acid disodium salt tetrahydrate; Cl2MDP) was a gift from Boehringer Mannheim (Mannheim, Germany). Preparation of liposomes containing Cl2MDP or PBS as a control was prepared as described previously 18 . For in vivo macrophage depletion, mice were injected i.v. with 0.2 ml of Cl2MDP liposomes or PBS liposomes as a control (mock depletion) on days 1, 3, and 5 after HSV-1 corneal infection.
In vitro enrichment of TG macrophages
TG were excised from 15 mice, 5 or 7 days after infection and incubated with collagenase (3 mg/ml) for 1 h at 37°C. The TG tissue was then triturated and passed through a 40-µm filter. The resulting single cell suspension was incubated for 2 h at 37°C on the plastic surface of a petri dish (Falcon 3001; Becton Dickinson, Franklin Lakes, NJ). The nonadherent cells were removed by vigorous washing of the petri dish, and the adherent and nonadherent populations were dissolved in lysis buffer before total RNA extraction (RNeasy kit; Qiagen, Santa Clarita, CA) and analysis of mRNA in an RNase protection assay (RPA).
Aminoguanidine treatment
Mice received three daily i.p. injections of aminoguanidine in PBS (total dose 400 mg/kg/day) or three injections of PBS as a control starting 1 day after infection.
In vivo cytokine neutralization
Rat anti-mouse IFN- mAb (R46A2) and rat anti-mouse
TNF- mAb (MP6-T22.11) were generated from hybridomas obtained from
the American Type Culture Collection (Manassas, VA). For cytokine
neutralization, mice received i.p. injections of 0.5 mg of each mAb
alone or a combination of 0.5 mg of both mAb on alternate days starting
1 day before HSV-1 corneal infection. Similar injections of a control
rat mAb (anti-HLA-BW6; American Type Culture Collection) were given
to control for possible nonspecific mAb effects.
In vivo depletion of TCR+ T lymphocytes
Groups of mice received i.p. injections of 0.5 mg of the GL3 mAb
that is specific for the TCR. Injections were initiated 1 day
before HSV-1 corneal infection and were repeated 1 and 3 days after
infection. Controls received similar injections of rat anti-HLA-BW6
mAb. On days 3 and 5 after infection, the TG were removed and total RNA
was extracted and analyzed in an RPA assay.
Virus titration
On days 3, 4, 5, and 7 after HSV-1 corneal infection, mice were sacrificed and the ipsilateral TG was excised and frozen in 0.5 ml of RPMI 1640. The TG were homogenized, subjected to three freeze-thaw cycles, and the suspension was sonicated and centrifuged at 6000 rpm for 10 min. The titer of infectious HSV-1 in the supernatant fluids was determined in a standard viral plaque assay on Vero cell monolayers. The results are expressed as the number PFU per TG.
Immunohistochemical and immunofluorescent staining
Frozen sections of TG were prepared and stained using
immunohistochemical and immunofluorescent staining procedures that were
previously described 3 . Briefly, TG were excised, embedded in OCT
(optimal cryogenic temperature; Tissue Tek; Miles, Naperville,
IL), snap frozen, and 6-µm frozen sections were cut. The
sections were fixed and stained as follows: for HSV-1 Ags using
peroxidase-conjugated rabbit anti-HSV type 1 (Dako, Carpinteria,
CA) followed by diaminobenzidine (DAB) substrate (peroxidase substrate
kit DAB SK04100; Vector Laboratories, Burlingame, CA); for
inducible nitric oxide synthase (iNOS) using polyclonal rabbit
anti-iNOS (Accurate Chemical & Scientific, Westbury, NY) followed
by biotin-conjugated goat anti-rabbit Ig (Zymed Laboratories, San
Fransisco, CA) and then FITC-Avidin (PharMingen, San Diego, CA); for
TNF- by sequential treatment with rat anti-TNF- (MP6-T22.11;
American Type Culture Collection), biotinylated goat anti-rat Ig
(Jackson ImmunoResearch Laboratories, West Grove, PA), ABC
reagent (Vectastain ABC kit; Vector Laboratories), and DAB substrate;
and for IFN- using FITC-conjugated rat mAb to mouse IFN- (R4-6A2;
American Type Culture Collection).
RNase protection assay
Total RNA was extracted from pools of 4 TG using an RNeasy kit
according to the manufacturer’s instructions. A 2-µg aliquot of the
RNA was stored for a semiquantitative RT-PCR analysis. The remaining
RNA was analyzed in a multiprobe RPA assay (PharMingen). The template
set included probes specific for the following mRNA: TCR, IL-12
(IL-12 p-40), TNF-, CD4, CD8, IL-2, F4/80, IFN-, and the
housekeeping genes L32 and glyceraldehyde phosphate dehydrogenase
(GAPDH). The RPA was performed according to the manufacturer’s
instructions, the resulting bands were visualized on a PhosphorImager
(PSI-PC; Molecular Dynamics, Sunnyvale, CA), and the results
were analyzed using ImageQuaNT software.
Semiquantitative RT-PCR
A total of 2 µg of RNA from TG of each treatment group was used to synthesize first strand cDNA using the Promega Reverse Transcription kit (Stratagene, La Jolla, CA), and PCR was performed using 1% of the cDNA obtained as template. The following primer sequences were used: iNOS sense, 5'-TTT GCT TCC ATG CTA ATG CGA AAG-3'; iNOS anti-sense, 5'-GCT CTG TTG AGG TCT AAA GGC TCC G-3'; hypoxanthine-guanine phosphoribosyl transferase (HPRT) sense, 5' CTC GAA GTG TTG GAT ACA GGC-3'; and HPRT anti-sense, 5'-GAT AAG CGA CAA TCT ACC AGA G-3'. iNOS cDNA was amplified for 28 cycles (denature, 94°C for 40 s; annealing, 60°C for 20 s; and extension, 72°C for 40 s) and HPRT was amplified for 20 cycles. Preliminary studies determined that these cycle numbers were in the linear range of amplification for each primer set. PCR products were separated by agarose gel electrophoresis and blotted onto nylon membrane (Zeta Probe; Bio-Rad Laboratories, Richmond CA). The iNOS and HPRT cDNA were detected by hybridization with a 32P-labeled 4.1-kb NotI and SfiI fragments of plasmid pPQRS that contains cloned fragments of mouse iNOS and HPRT gene sequences (gifts from Dr. Steve Reiner, University of Chicago, Chicago, IL). The hybridized bands were visualized on a PhosphorImager and the results analyzed using ImageQuaNT software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Groups of HSV-1-infected mice were treated with Cl2MDP
liposomes to deplete macrophages. HSV-1-infected control mice were mock
depleted with PBS liposomes. At 3, 5, and 7 days after infection, the
infected TG were removed and total RNA was extracted from pools of four
ganglia from each treatment group. Leukocytic infiltration of the TG
was monitored using a multiprobe RPA assay to analyze the expression of
mRNA for various leukocyte subpopulation markers. The experiment was
repeated four times, and an identical pattern emerged from each
experiment. The results of two representative experiments are shown in
Fig. 1
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A, lane 2) on days 3 and
5 after infection. Our previous immunohistochemical studies established
that the TG is infiltrated with leukocytes within 3 days after HSV-1
corneal infection 3 . Our current findings are in good agreement with
our previous results (Fig. 1). Three days after HSV-1 corneal
infection, mRNA for the macrophage marker F4/80 was prominent in the TG
and continued to increase through day 7 after infection. The mRNA for T
lymphocyte subpopulation markers including the TCR, CD4, and CD8
were also present but expressed at a much lower level.
Macrophage depletion markedly reduced the level of F4/80 mRNA during
the period of virus replication in the ganglion (Fig. 1). The level of
expression of the T cell subpopulation markers was low at days 3 and 5
after infection and was not influenced by macrophage depletion.
However, by day 7 after infection, macrophage depletion did cause a
marked reduction in mRNA for TCR, CD4, and CD8. Thus,
macrophages regulate the infiltration and/or retention of
TCR+ and TCR-ß+ T lymphocytes in the
infected TG.
Cytokine production in the infected TG
RNA transcripts specific for the cytokines IL-12, TNF-, and
IFN- were readily detectable in the TG by 3 days after HSV-1 corneal
infection (Fig. 1). Macrophage depletion dramatically reduced the level
of expression of mRNA for TNF- on days 3, 5, and 7 after corneal
infection. The messages for IL-12 and IFN- were also reduced,
although the reduction was not consistently seen until day 7 after
infection. The reduction of cytokine message was associated with a
significant reduction of cells expressing IFN- and TNF- protein
in the HSV-1-infected TG of macrophage-depleted mice (Table I).
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but not of
IFN-. Therefore, we proposed that macrophage depletion directly
removed the source of IL-12 and TNF- but indirectly reduced IFN-
production by another cell, such as a TCR+ T cell.
Support for this hypothesis came from two types of experiments. First,
single cell suspensions of TG obtained 7 days after corneal infection
were divided into plastic adherent and nonadherent populations. Total
RNA from these populations was subjected to RPA. The plastic adherent
population was enriched for mRNA specific for the macrophage marker
F4/80 and for the cytokines IL-12 and TNF- (Fig. 2). There was a very weak band for TCR
mRNA and for IFN- mRNA in the nonadherent population and no
discernible bands for these mRNA species in the adherent population
(Fig. 2).
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TCR+ T cells to the
IFN- message in the infected TG, mice were depleted of
TCR+ T cells by injection of GL3 mAb 1 day before and on
alternate days after HSV-1 corneal infection. On days 3 and 5 after
infection, the TG were excised and IFN- mRNA was quantified by RPA.
Depletion of TCR+ T cells caused an 89 and 60%
reduction in IFN- mRNA in the TG on days 3 and 5 after infection,
respectively (Fig. 3). These findings provide
strong support for the notion that macrophages are the main source of
IL-12 and TNF- and TCR+ T cells are the main
source of IFN- during the peak of HSV-1 replication in the TG.
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, iNOS mRNA was readily detectable in
infected TG of control mice by 3 days after infection and began to
decline by day 7 after infection. The level of expression of iNOS mRNA
was markedly reduced in the ganglia of macrophage-depleted mice. In
separate experiments, frozen sections of infected TG that were obtained
7 days after HSV-1 corneal infection were stained for iNOS. Numerous
iNOS+ cells were found in direct apposition to neuron cell
bodies within the ophthalmic branch of the TG (Fig. 5). Thus, HSV-1 corneal infection leads to a
rapid infiltration of iNOS+ macrophages into the ophthalmic
branch of the TG.
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Because activated macrophages are present in the infected TG and produce cytokines that inhibit HSV-1 replication in vitro, it was reasonable to propose that macrophages might contribute to the control of HSV-1 replication in the TG after corneal infection. To test this possibility, groups of five to six mice received corneal infections with HSV-1 followed by macrophage depletion with Cl2MDP liposomes or mock depletion with PBS liposomes. At various times after infection the corneas and TG were removed, homogenized, and infectious HSV-1 titers were determined in a standard virus plaque assay.
Macrophage depletion did not significantly enhance or prolong HSV-1
replication in the infected corneas as determined either by corneal
examination or by viral plaque assay of corneal extracts (data not
shown). In the TG of control mice, replicating virus was detectable by
day 3, reached peak titers by day 4, and was no longer detectable by
day 7 after corneal infection (Fig. 6). The
kinetics of HSV-1 replication in the TG was not markedly altered by
macrophage depletion, although very low levels of replicating virus
were routinely detected in the TG of macrophage-depleted mice on day 7,
when replicating virus was no longer detectable in the TG of control
mice. However, macrophage depletion did significantly increase HSV-1
titers throughout the course of virus replication in the TG (Fig. 6).
The increased virus load in the TG of macrophage-depleted mice was
associated with increased HSV-1 dissemination within the ganglion.
Thus, the number of HSV-1 Ag-positive neurons in the TG of
macrophage-depleted mice (59.1 ± 8.73 and 13.7 ± 2.65 on
days 5 and 7 after infection, respectively) was significantly higher
(p < 0.01) than the number in mock-depleted
mice (28.2 ± 3.65 and 2.2 ± 0.13 on days 5 and 7,
respectively) as illustrated in Fig. 7. Our
findings establish that macrophages play an important role in
controlling HSV-1 replication and dissemination within the TG after
corneal infection.
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, TNF-, and NO contribute to the control of HSV-1
replication in the TG
Because mRNA for the cytokines, TNF and IFN-, and for iNOS
was readily detectable in the infected TG, we determined that
the corresponding proteins contributed to the control of virus
replication. Groups of mice received in vivo treatment with
neutralizing Abs to IFN-, TNF-, or a combination of both Abs. At
various times after HSV-1 corneal infection, the TG were excised and
HSV-1 titers in individual ganglia were determined in a virus plaque
assay. Individual neutralization of IFN- or TNF- caused a
significant increase in HSV-1 titers on days 3–7 after infection (Fig. 8). Simultaneous neutralization of both
cytokines had a synergistic effect on virus replication in the
ganglion.
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. Inhibition of iNOS significantly increased virus replication,
particularly when combined with TNF- neutralization (Fig. 9). Thus, TNF-, IFN-, and NO all
contribute directly or indirectly to the control of HSV-1 replication
in the TG.
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and TNF- control leukocyte accumulation in the TG
Groups of four mice received in vivo treatment with neutralizing
Abs to IFN-, TNF-, or a combination of both mAbs. At various
times after HSV-1 corneal infection, the TG were excised and pooled,
and total RNA was extracted and subjected to RPA analysis. The results
of two representative experiments are shown in Fig. 10. Individual
neutralization of TNF- or IFN- reduced leukocytic infiltration of
the ganglia as illustrated by a marked reduction in the levels of mRNA
for TCR, CD4, CD8, and F4/80. Simultaneous neutralization of
both TNF- and IFN- had a synergistic inhibitory effect on
leukocytic infiltration of the TG. The reduction in the accumulation of
leukocytes in the TG was associated with a corresponding decrease in
cytokine mRNA. Thus, neutralization of TNF- and IFN- individually
and synergistically decreased the levels of mRNA for IL-12, TNF-,
and IFN-. The experiment was repeated four times with an identical
pattern emerging from each experiment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies used a multiprobe RPA assay to screen for cytokine and leukocyte subpopulation marker gene expression in the infected TG. This assay permits screening for a large number of transcripts in a single sample. However, there are unique problems associated with the use of this or other molecular biological screening assays at sites of inflammation, especially those induced by HSV-1. Most RPA assays use housekeeping genes to standardize for RNA quantity and hybridization efficiency. However, at an inflammatory site, particularly in nervous tissue, a significant proportion of the housekeeping gene transcripts is contributed by infiltrating inflammatory cells. Moreover, an HSV-1 virion protein, referred to as virus host shutoff, has been shown to destabilize and degrade host mRNA.
Our studies clearly demonstrate an inverse correlation between the degree of leukocytic infiltration into the TG and the amount of HSV-1 replication. Thus, any treatment that reduces inflammation would not only reduce the leukocytic contribution to the pool of housekeeping gene mRNA, but would also result in increased virus-induced destabilization of this mRNA pool. For this reason, standardization of these assays is virtually impossible. In our assays, no attempt was made to adjust the quantity of RNA or housekeeping gene message in each sample. Thus, the results reflect the total amount of mRNA for a particular protein in a constant amount of tissue (pool of four TG) for the various treatments. In four separate TG pools from each treatment group, the pattern of mRNA expression was identical, suggesting that the differences observed were not artifactual. Therefore, we believe that the use of a multiprobe RPA assay as a screening device is very useful even in situations that defy normal approaches to standardization. Moreover, the levels of mRNA for leukocyte Ags and cytokines detected by RPA are in close agreement with levels of the corresponding proteins as determined immunohistochemically in this and previous studies 3 .
Our results establish that IFN-, TNF-, IL-12, and iNOS are
expressed in the TG within 3 days after HSV-1 corneal infection. At
this time, TCR+ T cells and macrophages are readily
detectable and surround neurons in the ophthalmic branch of the TG 3 .
In contrast, TCR-ß+ T cells are barely detectable in
the ganglion and are not localized to the neuron cell bodies at this
time. At sites of infection, macrophages are often a major source of
IL-12, TNF-, and iNOS. The TCR+ subpopulation of
T cells is an important source of IFN- in certain infections and
regulates macrophage function with this molecule 10 . Several
observations in the current study point to macrophages as the primary
source of TNF-, IL-12, and iNOS in the HSV-1-infected TG. First, the
temporal pattern of expression of mRNA for TNF- and IL-12 was very
similar to the pattern of expression of mRNA for the macrophage marker
F4/80. Second, in vivo depletion of macrophages resulted in a dramatic
decrease in mRNA for IL-12, TNF-, and iNOS. Third, plastic adherent
cells derived from the HSV-1-infected TG were highly enriched for mRNA
for the macrophage marker F4/80 and for IL-12 and TNF-. Our findings
also suggest that TCR+ T cells are the main source
of IFN- in the infected TG. There was a very similar pattern of
expression of mRNA for TCR and for IFN- in the infected TG.
The plastic nonadherent TG cells were enriched for mRNA for TCR
and for IFN-. In addition, in vivo depletion of
TCR+ T cells with GL3 mAb dramatically reduced mRNA for
IFN- 3 and 5 days after HSV-1 corneal infection.
Our findings also suggest that TCR+ T cells and
macrophages cross-regulate their accumulation and activation in the
infected TG. Depletion of macrophages markedly diminished
TCR+ T cell accumulation and IFN- mRNA and protein
production in the infected TG. Although macrophage depletion
significantly reduced the number of IFN-+ cells in the
TG on days 5 and 7 after infection, the effect was proportionately
greater on day 5 (Table I). This may simply reflect variation in the
efficiency of macrophage depletion. As can be seen in Fig. 1, B and C, more efficient macrophage depletion
tends to be associated with a greater reduction in mRNA for TCR
and IFN-. Alternatively, TCR-ß+ T cells may
contribute to the IFN- production by day 7 and be less dependent on
macrophage regulation. Macrophage depletion also markedly reduced the
mRNA for IL-12, a potent regulator of IFN- production by
TCR+ T cells 20 .
Neutralization of IFN-, which appears to be produced primarily
by TCR+ T cells, reduces the accumulation of
macrophages and their expression of TNF- in the infected TG during
the period of HSV-1 replication. This finding is consistent with the
established capacity of TCR+ T cells to induce
TNF- and iNOS production by macrophages 21 through IFN-.
Moreover, neutralization of TNF- and IFN- individually and
synergistically reduced macrophage and TCR+ T cell
accumulation in the HSV-1-infected TG. This result is consistent with
the observation that TNF- and IFN- can individually and
synergistically induce vascular endothelial cells to produce the
chemokine RANTES 22 , a chemoattractant for both monocytes and
TCR+ T cells 23 . Therefore, it appears that
TCR+ T cells and macrophages reciprocally regulate each
other’s accumulation and activation in the HSV-1-infected TG. The TG
of noninfected mice exhibited a low level of mRNA for F4/80 and no
detectable mRNA for T cell markers or cytokines. Thus, the reciprocal
activation and accumulation of macrophages and TCR+
T cells may be initiated by resident macrophages after early HSV-1
replication in the TG.
Because the sensory neurons cannot be regenerated, it is
essential that virus replication is controlled without destruction of
the infected neuron. The cytokines, TNF- and IFN-, and the
nitrogen radical NO have all been shown to inhibit HSV-1 replication in
vitro 11, 14, 15, 16 . Our findings clearly establish that in vivo
neutralization of IFN- or TNF- or inhibition of NO production by
iNOS results in a dramatic increase in virus replication in the
ganglion. The elaboration of these cytokines by TCR+
T cells and macrophages that are in direct apposition to the infected
neurons may terminate virus replication without neuronal toxicity.
This and our previous study 5 demonstrate that the virus load
in the TG after HSV-1 corneal infection is markedly increased in the
absence of TCR+ T cells and macrophages. The
increased virus titers were associated with increased numbers of
infected neurons. Thus, one function of macrophages and
TCR+ cells may be to prevent the lateral dissemination of
HSV-1 from one neuron to the next within the ganglion. However, virus
replication in the ganglion was ultimately terminated with similar
kinetics in the presence or absence of either of these inflammatory
cells. It is not yet clear whether termination of virus replication in
neurons requires exogenous help or whether it is determined by factors
that are endogenous to the neurons. If the latter is true, then one may
question the significance of controlling the level of HSV-1 replication
in the neurons. We propose and are currently testing two alternative
hypotheses. The first is that by preventing the lateral spread of HSV-1
from neuron to neuron and by limiting virus replication within each
neuron, TCR+ T cells and macrophages reduce the
number of latently infected neurons and the number of copies of latent
viral genome within each neuron. The second possibility is that a high
level of HSV-1 replication leads to viral destruction of the neuron and
to fewer latently infected neurons. Thus, control of virus replication
by TCR+ T cells and macrophages may represent a
compromise between the virus and the host. Immune protection from viral
destruction of host neurons and the resulting loss of corneal sensation
may increase the number of latently infected neurons, permitting the
virus to be retained in the sensory ganglia for the lifetime of the
host. Although the factors that influence the likelihood of HSV-1
reactivation from latency are poorly defined, there is evidence that
the frequency of latently infected neurons and the number of copies of
viral genome in each neuron could be contributing factors 24 . Thus,
the effectiveness of the innate immune response during acute HSV-1
replication in the sensory ganglia might dramatically influence the
likelihood and frequency of recurrent herpetic disease in later life.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert L. Hendricks, University of Pittsburgh School of Medicine, 915 Eye and Ear Institute, 203 Lothrop Street, Pittsburgh, PA 15213-2588. E-mail address:
3 Abbreviations used in this paper: HSV-1, herpes simplex virus type 1; Cl2MDP, clodronic-acid disodium salt tetrahydrate; TG, trigeminal ganglion; iNOS, inducible nitric oxide synthase; NO, nitric oxide; RPA, RNase protection assay; GAPDH, glyceraldehyde phosphate dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyl transferase; PFU, plaque-forming units.
Received for publication June 22, 1998. Accepted for publication November 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/ cells protect mice from herpes simplex virus type 1-induced lethal encephalitis. J. Exp. Med. 185:1969. interferon expression during acute and latent nervous system infection by herpes simplex virus type 1. J. Virol. 69:4898.[Abstract]
interferon and tumor necrosis factor in macrophage activation during murine cytomegalovirus and herpes simplex virus infections. J. Virol. 69:904.[Abstract]
cell-induced nitric oxide production enhances resistance to mucosal candidiasis. Nat. Med. 1:552.[Medline]
-induced nitric oxide synthase. Science 261:1445. in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumor necrosis factor-. J. Gen. Virol. 78:195.[Abstract]
interferon and tumor necrosis factor exert a synergistic blockade on the replication of herpes simplex virus. J. Virol. 63:1354.. Virology 180:822.[Medline]
T cells for production of IFN- is mediated by bacteria via macrophage-derived cytokines IL-1 and IL- 12. J. Immunol. 154:5832.[Abstract]
/ T cells in priming macrophages to produce tumor necrosis factor-. Eur. J. Immunol. 25:1465.[Medline]
plus TNF- and inhibition by IL-4 and IL-13. J. Immunol. 154:1870.[Abstract]
/ß and / T lymphocytes to chemokines. Eur. J. Immunol. 28:104.[Medline]
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 G. M. G. M. Verjans, R. Q. Hintzen, J. M. van Dun, A. Poot, J. C. Milikan, J. D. Laman, A. W. Langerak, P. R. Kinchington, and A. D. M. E. Osterhaus Selective retention of herpes simplex virus-specific T cells in latently infected human trigeminal ganglia PNAS, February 27, 2007; 104(9): 3496 - 3501. [Abstract] [Full Text] [PDF]
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P. Lundberg, P. V. Welander, C. K. Edwards III, N. van Rooijen, and E. Cantin Tumor Necrosis Factor (TNF) Protects Resistant C57BL/6 Mice against Herpes Simplex Virus-Induced Encephalitis Independently of Signaling via TNF Receptor 1 or 2 J. Virol., February 1, 2007; 81(3): 1451 - 1460. [Abstract] [Full Text] [PDF]
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 J. Melchjorsen, J. Siren, I. Julkunen, S. R. Paludan, and S. Matikainen Induction of cytokine expression by herpes simplex virus in human monocyte-derived macrophages and dendritic cells is dependent on virus replication and is counteracted by ICP27 targeting NF-{kappa}B and IRF-3. J. Gen. Virol., May 1, 2006; 87(Pt 5): 1099 - 1108. [Abstract] [Full Text] [PDF]
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S. Suvas, A. K. Azkur, and B. T. Rouse Qa-1b and CD94-NKG2a Interaction Regulate Cytolytic Activity of Herpes Simplex Virus-Specific Memory CD8+ T Cells in the Latently Infected Trigeminal Ganglia J. Immunol., February 1, 2006; 176(3): 1703 - 1711. [Abstract] [Full Text] [PDF]
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H. Lauterbach, C. Ried, A. L. Epstein, P. Marconi, and T. Brocker Reduced immune responses after vaccination with a recombinant herpes simplex virus type 1 vector in the presence of antiviral immunity J. Gen. Virol., September 1, 2005; 86(9): 2401 - 2410. [Abstract] [Full Text] [PDF]
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T. H. Mogensen, J. Melchjorsen, L. Malmgaard, A. Casola, and S. R. Paludan Suppression of Proinflammatory Cytokine Expression by Herpes Simplex Virus Type 1 J. Virol., June 1, 2004; 78(11): 5883 - 5890. [Abstract] [Full Text] [PDF]
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S. Vollstedt, S. Arnold, C. Schwerdel, M. Franchini, G. Alber, J. P. Di Santo, M. Ackermann, and M. Suter Interplay between Alpha/Beta and Gamma Interferons with B, T, and Natural Killer Cells in the Defense against Herpes Simplex Virus Type 1 J. Virol., April 15, 2004; 78(8): 3846 - 3850. [Abstract] [Full Text] [PDF]
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T. E. Morrison, A. Mauser, A. Klingelhutz, and S. C. Kenney Epstein-Barr Virus Immediate-Early Protein BZLF1 Inhibits Tumor Necrosis Factor Alpha-Induced Signaling and Apoptosis by Downregulating Tumor Necrosis Factor Receptor 1 J. Virol., January 1, 2004; 78(1): 544 - 549. [Abstract] [Full Text] [PDF]
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P. Lundberg, P. Welander, H. Openshaw, C. Nalbandian, C. Edwards, L. Moldawer, and E. Cantin A Locus on Mouse Chromosome 6 That Determines Resistance to Herpes Simplex Virus Also Influences Reactivation, While an Unlinked Locus Augments Resistance of Female Mice J. Virol., November 1, 2003; 77(21): 11661 - 11673. [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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L. Malmgaard and S. R. Paludan Interferon (IFN)-{alpha}/{beta}, interleukin (IL)-12 and IL-18 coordinately induce production of IFN-{gamma} during infection with herpes simplex virus type 2 J. Gen. Virol., September 1, 2003; 84(9): 2497 - 2500. [Abstract] [Full Text] [PDF]
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 J. Melchjorsen, L. N. Sorensen, and S. R. Paludan Expression and function of chemokines during viral infections: from molecular mechanisms to in vivo function J. Leukoc. Biol., September 1, 2003; 74(3): 331 - 343. [Abstract] [Full Text] [PDF]
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T. H. Mogensen, J. Melchjorsen, P. Hollsberg, and S. R. Paludan Activation of NF-{kappa}B in Virus-Infected Macrophages Is Dependent on Mitochondrial Oxidative Stress and Intracellular Calcium: Downstream Involvement of the Kinases TGF-{beta}-Activated Kinase 1, Mitogen-Activated Kinase/Extracellular Signal-Regulated Kinase Kinase 1, and I{kappa}B Kinase J. Immunol., June 15, 2003; 170(12): 6224 - 6233. [Abstract] [Full Text] [PDF]
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S. A. McClellan, X. Huang, R. P. Barrett, N. van Rooijen, and L. D. Hazlett Macrophages Restrict Pseudomonas aeruginosa Growth, Regulate Polymorphonuclear Neutrophil Influx, and Balance Pro- and Anti-Inflammatory Cytokines in BALB/c Mice J. Immunol., May 15, 2003; 170(10): 5219 - 5227. [Abstract] [Full Text] [PDF]
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Y. Langelier, S. Bergeron, S. Chabaud, J. Lippens, C. Guilbault, A. M.-J. Sasseville, S. Denis, D. D. Mosser, and B. Massie The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation J. Gen. Virol., November 1, 2002; 83(11): 2779 - 2789. [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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P. Harle, V. Cull, M.-P. Agbaga, R. Silverman, B. R. G. Williams, C. James, and D. J. J. Carr Differential Effect of Murine Alpha/Beta Interferon Transgenes on Antagonization of Herpes Simplex Virus Type 1 Replication J. Virol., June 5, 2002; 76(13): 6558 - 6567. [Abstract] [Full Text] [PDF]
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N. N. Panjwani, L. Popova, and P. K. Srivastava Heat Shock Proteins gp96 and hsp70 Activate the Release of Nitric Oxide by APCs J. Immunol., March 15, 2002; 168(6): 2997 - 3003. [Abstract] [Full Text] [PDF]
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J. Melchjorsen, F. S. Pedersen, S. C. Mogensen, and S. R. Paludan Herpes Simplex Virus Selectively Induces Expression of the CC Chemokine RANTES/CCL5 in Macrophages through a Mechanism Dependent on PKR and ICP0 J. Virol., February 22, 2002; 76(6): 2780 - 2788. [Abstract] [Full Text] [PDF]
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Z. Mikloska and A. L. Cunningham Alpha and Gamma Interferons Inhibit Herpes Simplex Virus Type 1 Infection and Spread in Epidermal Cells after Axonal Transmission J. Virol., December 1, 2001; 75(23): 11821 - 11826. [Abstract] [Full Text]
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S. R. Paludan and S. C. Mogensen Virus-Cell Interactions Regulating Induction of Tumor Necrosis Factor Alpha Production in Macrophages Infected with Herpes Simplex Virus J. Virol., November 1, 2001; 75(21): 10170 - 10178. [Abstract] [Full Text] [PDF]
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S. R. Paludan Requirements for the Induction of Interleukin-6 by Herpes Simplex Virus-Infected Leukocytes J. Virol., September 1, 2001; 75(17): 8008 - 8015. [Abstract] [Full Text] [PDF]
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S. Ellermann-Eriksen and V. Kruys Expression of TNF-{alpha} by Herpes Simplex Virus-Infected Macrophages Is Regulated by a Dual Mechanism: Transcriptional Regulation by NF-{kappa}B and Activating Transcription Factor 2/Jun and Translational Regulation Through the AU-Rich Region of the 3' Untranslated Region J. Immunol., August 15, 2001; 167(4): 2202 - 2208. [Abstract] [Full Text] [PDF]
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S. A. Huber, D. Graveline, W. K. Born, and R. L. O'Brien Cytokine Production by V{gamma}+-T-Cell Subsets Is an Important Factor Determining CD4+-Th-Cell Phenotype and Susceptibility of BALB/c Mice to Coxsackievirus B3-Induced Myocarditis J. Virol., July 1, 2001; 75(13): 5860 - 5869. [Abstract] [Full Text] [PDF]
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M. Franchini, C. Abril, C. Schwerdel, C. Ruedl, M. Ackermann, and M. Suter Protective T-Cell-Based Immunity Induced in Neonatal Mice by a Single Replicative Cycle of Herpes Simplex Virus J. Virol., January 1, 2001; 75(1): 83 - 89. [Abstract] [Full Text]
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S. A. Huber, D. Graveline, M. K. Newell, W. K. Born, and R. L. O'Brien V{gamma}1+ T Cells Suppress and V{gamma}4+ T Cells Promote Susceptibility to Coxsackievirus B3-Induced Myocarditis in Mice J. Immunol., October 15, 2000; 165(8): 4174 - 4181. [Abstract] [Full Text] [PDF]
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H. Tsunobuchi, H. Nishimura, F. Goshima, T. Daikoku, Y. Nishiyama, and Y. Yoshikai Memory-Type CD8+ T Cells Protect IL-2 Receptor {alpha}-Deficient Mice from Systemic Infection with Herpes Simplex Virus Type 2 J. Immunol., October 15, 2000; 165(8): 4552 - 4560. [Abstract] [Full Text] [PDF]
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P. Lundberg and G. A. Splitter gamma delta + T-Lymphocyte Cytotoxicity against Envelope-Expressing Target Cells Is Unique to the Alymphocytic State of Bovine Leukemia Virus Infection in the Natural Host J. Virol., September 15, 2000; 74(18): 8299 - 8306. [Abstract] [Full Text]
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K. Ikeda, H. Wakimoto, T. Ichikawa, S. Jhung, F. H. Hochberg, D. N. Louis, and E. A. Chiocca Complement Depletion Facilitates the Infection of Multiple Brain Tumors by an Intravascular, Replication-Conditional Herpes Simplex Virus Mutant J. Virol., May 15, 2000; 74(10): 4765 - 4775. [Abstract] [Full Text]
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 A. A. Nash T Cells and the Regulation of Herpes Simplex Virus Latency and Reactivation J. Exp. Med., May 1, 2000; 191(9): 1455 - 1458. [Full Text] [PDF]
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T. Liu, K. M. Khanna, X. Chen, D. J. Fink, and R. L. Hendricks Cd8+ T Cells Can Block Herpes Simplex Virus Type 1 (HSV-1) Reactivation from Latency in Sensory Neurons J. Exp. Med., May 1, 2000; 191(9): 1459 - 1466. [Abstract] [Full Text] [PDF]
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