Macrophage Control of Herpes Simplex Virus
Type 1 Replication in the Peripheral Nervous
System
This information is current as
of June 17, 2017.
Padma Kodukula, Ting Liu, Nico Van Rooijen, Martine J.
Jager and Robert L. Hendricks
J Immunol 1999; 162:2895-2905; ;
http://www.jimmunol.org/content/162/5/2895
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Copyright © 1999 by The American Association of
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References
Macrophage Control of Herpes Simplex Virus Type 1
Replication in the Peripheral Nervous System
Padma Kodukula,* Ting Liu,† Nico Van Rooijen,§ Martine J. Jager,¶ and
Robert L. Hendricks2*†‡
H
erpes simplex virus type 1 (HSV-1)3 can enter the body
by infecting epidermal cells or epithelial cells of mucosal surfaces. The virus replicates in and destroys these
cells and in the process gains access to the termini of local sensory
neurons. Retrograde axonal transport carries the virus to the neuronal cell bodies in the sensory ganglia within 2 days of the primary infection (1). After HSV-1 corneal infection in mice, the
virus replicates briefly in the trigeminal ganglion (TG), reaching
peak titers by 3–5 days after infection. By 7–10 days after corneal
infection, HSV-1 replication in the TG has ceased, but a portion of
the neurons retains the viral genome in a latent state. The recurrent
nature of herpetic disease appears to be due to periodic reactivation
of latent HSV-1 and axonal transmission to peripheral sites served
by the infected sensory neurons. Recurrent herpetic disease is responsible for the vast majority of the human suffering, loss of
productivity, and visual impairment associated with HSV-1
*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
Received for publication June 22, 1998. Accepted for publication November
30, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grants EY05945, EY10359, and 5 P30 EY08098 from
the National Institutes of Health and by an unrestricted grant from Research to Prevent Blindness, New York, NY. R.L.H. is a Research to Prevent Blindness Senior
Scientific Investigator.
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: [email protected]
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.
Copyright © 1999 by The American Association of Immunologists
infections. A key to preventing recurrent herpetic disease would
seem to lie in preventing or limiting the initial colonization of
sensory neurons after primary infection or in preventing or limiting
the subsequent reactivation of HSV-1 from latency.
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– 4). Macrophages, gd TCR1 T lymphocytes, and TCR-ab1 T cells of both the CD41 and CD81
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 CD81 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 gd TCR1 T cells can be
seen surrounding infected neurons. By 7–12 days after corneal
infection, CD81 T cells are also preferentially drawn to the infected neurons in the ophthalmic branch of the TG (3).
Depletion of gd TCR1 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 TCRab1 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
CD81 T cells or compromise of CD81 T cell function also significantly augments HSV-1 neurovirulence (6, 7). Thus, gd TCR1
T cells represent an important early line of defense against HSV-1
replication in the sensory neurons, whereas CD81 T cells are required to completely shut down HSV-1 replication in the TG and
prevent neurologic damage.
The mechanism by which gd TCR1 T cells control virus replication in sensory neurons is not known. IFN-g 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-g has direct antiviral
0022-1767/99/$02.00
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After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion
(TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as
early as 2 days after corneal infection, reaches peak titers by 3–5 days after infection, and is undetectable by 7–10 days. During
the period of HSV-1 replication, macrophages and gd TCR1 T lymphocytes infiltrate the TG, and TNF-a, IFN-g, the inducible
nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-a, IFN-g, and the iNOS product nitric oxide (NO) all inhibit
HSV-1 replication in vitro. Macrophage and gd TCR1 T cell depletion studies demonstrated that macrophages are the main
source of TNF-a and iNOS, whereas gd TCR1 T cells produce IFN-g. Macrophage depletion, aminoguanidine inhibition of iNOS,
and neutralization of TNF-a or IFN-g all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal
infection. Moreover, individually depleting macrophages or neutralizing TNF-a or IFN-g markedly reduced the accumulation of
both macrophages and gd TCR1 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 gd TCR1 T lymphocytes through their production of antiviral
molecules TNF-a, NO, and IFN-g. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG. The Journal of Immunology, 1999, 162: 2895–2905.
2896
CONTROL OF HSV-1 IN THE PERIPHERAL NERVOUS SYSTEM
activity, it also plays an important role in activating macrophages
(9). In fact, in certain infectious disease models, macrophage production of TNF-a and nitric oxide (NO) is regulated by gd TCR1
cells through their production of IFN-g (10, 11). In addition, HSV
infection has been shown to augment NO synthesis by IFN-gtreated macrophages (12). Both TNF-a and NO are potent inhibitors of HSV-1 replication in vitro (11, 13–16).
We hypothesized that an interaction between macrophages and
gd TCR1 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-a and IFN-g and the reactive
nitrogen intermediate NO.
Materials and Methods
In vivo depletion of gd TCR1 T lymphocytes
Groups of mice received i.p. injections of 0.5 mg of the GL3 mAb that is
specific for the gd 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.
Animals
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-ml 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-mm 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-g mAb (R46A2) and rat anti-mouse TNF-a 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.
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-mm 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-a by
sequential treatment with rat anti-TNF-a (MP6-T22.11; American Type
Culture Collection), biotinylated goat anti-rat Ig (Jackson Immuno
Research Laboratories, West Grove, PA), ABC reagent (Vectastain ABC
kit; Vector Laboratories), and DAB substrate; and for IFN-g using FITCconjugated rat mAb to mouse IFN-g (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-mg 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: gd TCR, IL-12 (IL-12
p-40), TNF-a, CD4, CD8, IL-2, F4/80, IFN-g, 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 mg 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, 59-TTT GCT TCC ATG CTA ATG CGA AAG-39; iNOS anti-sense,
59-GCT CTG TTG AGG TCT AAA GGC TCC G-39; hypoxanthineguanine phosphoribosyl transferase (HPRT) sense, 59 CTC GAA GTG
TTG GAT ACA GGC-39; and HPRT anti-sense, 59-GAT AAG CGA CAA
TCT ACC AGA G-39. 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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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.
The Journal of Immunology
Results
Macrophages regulate leukocytic accumulation in the infected
TG
Cytokine production in the infected TG
RNA transcripts specific for the cytokines IL-12, TNF-a, and
IFN-g 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-a on days 3, 5,
and 7 after corneal infection. The messages for IL-12 and IFN-g
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-g and TNF-a protein in the HSV-1-infected TG of macrophagedepleted mice (Table I).
Macrophages are an important source of IL-12 and TNF-a but
not of IFN-g. Therefore, we proposed that macrophage depletion
directly removed the source of IL-12 and TNF-a but indirectly
reduced IFN-g production by another cell, such as a gd TCR1 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-a (Fig. 2). There was a very weak band for gd
TCR mRNA and for IFN-g mRNA in the nonadherent population
and no discernible bands for these mRNA species in the adherent
population (Fig. 2).
To determine the contribution of gd TCR1 T cells to the IFN-g
message in the infected TG, mice were depleted of gd TCR1 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-g mRNA was quantified by RPA. Depletion of gd TCR1 T cells caused an 89 and 60% reduction in
IFN-g 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-a and gd
TCR1 T cells are the main source of IFN-g during the peak of
HSV-1 replication in the TG.
Activated macrophages also produce NO through the activity of
the enzyme iNOS. NO can strongly inhibit HSV-1 replication in
vitro. To determine whether iNOS mRNA is present in the infected
TG, and if it is produced by macrophages, the level of iNOS
mRNA expression in the TG of macrophage-depleted and mockdepleted mice was determined by a semiquantitative RT-PCR assay. As shown in Fig. 4, 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 iNOS1 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 iNOS1 macrophages into the ophthalmic branch of
the TG.
Macrophages control HSV-1 replication in the infected TG
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 6 8.73 and 13.7 6 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 6 3.65 and 2.2 6 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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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.
The TG of noninfected mice contained a low level of mRNA for
F4/80, suggesting that small numbers of macrophages are present
in the normal TG (data not shown). No mRNA for T cell subpopulation markers or cytokines was detectable in the normal TG.
The level of expression of the housekeeping gene transcripts L32
and GAPDH in normal TG was roughly equivalent to that in the
TG of the macrophage-depleted group (Fig. 1A, 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 gd 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 gd
TCR, CD4, and CD8. Thus, macrophages regulate the infiltration
and/or retention of gd TCR1 and TCR-ab1 T lymphocytes in the
infected TG.
2897
2898
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FIGURE 1. Macrophages regulate leukocytic
infiltration and cytokine production in the TG. On
days 1, 3, and 5 after HSV-1 corneal infection,
groups of four mice were depleted of macrophages by i.v. injection of 0.2 ml of Cl2MDP liposomes or were mock depleted with PBS liposomes. At the indicated times, TG from each
treatment group were pooled, total RNA was extracted, and mRNA for leukocyte subpopulation
markers, cytokines, and the housekeeping genes,
L32 and GAPDH, were detected in a multiprobe
RPA. The resulting bands were visualized with a
PhosphorImager (A). The lane designations are:
1, mock depleted and 2, macrophage depleted.
The relative amounts of leukocyte and cytokine
message were analyzed using ImageQuaNT software. The quantitative analysis of the bands in A
is shown graphically in B. Quantitative data from
a similar experiment are shown in C. The crosshatched bars represent TG from mock-depleted
mice, and the solid bars represent TG from macrophage-depleted mice.
CONTROL OF HSV-1 IN THE PERIPHERAL NERVOUS SYSTEM
The Journal of Immunology
2899
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FIGURE 1. (continued)
Table I. Cytokine-producing cells in the infected TG
Day 5a
TNF-a1 cellsc
IFN-g1 cells
Day 7a
Control
Mac. Depl.
p valueb
Control
Mac. Depl.
p valueb
128.2 6 15.1
45.2 6 7.2
95.5 6 6.8
8.2 6 2.5
0.0952
0.0079
139.4 6 11.6
156.0 6 21.7
92.8 6 8.5
73.8 6 10.8
0.0159
0.0159
a
On day 5 or 7 after HSV-1 corneal infection, the TG were removed from animals that were depleted of macrophages (Mac. Depl.) with Cl2MDP liposomes and from control
mice that were mock depleted with PBS-liposomes. Frozen sections of TG were prepared and stained for TNF-a or IFN-g.
b
Mann-Whitney U test.
c
The number of cells that stained for intracellular cytokines were counted in a masked fashion and recorded as the number of positive cells in the ophthalmic branch per
section (n 5 5).
2900
CONTROL OF HSV-1 IN THE PERIPHERAL NERVOUS SYSTEM
FIGURE 2. Cytokine profile of plastic adherent and nonadherent cells
obtained from TG that were excised 7 days after HSV-1 corneal infection.
Single cell suspensions were prepared from 30 infected TG and allowed to
adhere to the plastic surface of a tissue culture flask for 2 h. The nonadherent cells were removed and total RNA was extracted from the plastic
adherent and nonadherent cells. The mRNA for leukocyte subpopulation
markers and cytokines was analyzed in a multiprobe RPA. The relative
amount of message for F4/80, TNF-a, gd TCR, IFN-g, and IL-12 in adherent cells (solid bars) and nonadherent cells (crosshatched bars) is shown
as PhosphorImager units.
Because mRNA for the cytokines, TNFa and IFN-g, 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-g, TNF-a, 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-g or TNF-a 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.
In separate experiments, mice were treated with the iNOS inhibitor, aminoguanidine alone, or in combination with the neutralizing Ab to TNF-a. Inhibition of iNOS significantly increased virus replication, particularly when combined with TNF-a
neutralization (Fig. 9). Thus, TNF-a, IFN-g, and NO all contribute
directly or indirectly to the control of HSV-1 replication in the TG.
FIGURE 3. In vivo depletion of gd TCR1 T cells reduces IFN-g
mRNA in the infected TG. Groups of four mice were depleted of gd TCR1
T cells by injection of the GL3 mAb on days 21, 12, and 14 after HSV-1
corneal infection or they were mock depleted by similar treatment with a
control mAb. The TG from each treatment group were excised on the
designated day after infection, pooled, and total RNA was extracted and
analyzed with the multiprobe RPA. The relative amount of IFN-g mRNA
in TG from mock-depleted control mice (solid bars) and from gd TCR1 T
cell-depleted mice (cross-hatched bars) is shown.
IFN-g and TNF-a control leukocyte accumulation in the TG
Groups of four mice received in vivo treatment with neutralizing
Abs to IFN-g, TNF-a, 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-a or IFN-g reduced leukocytic infiltration of the ganglia as illustrated by a marked reduction in the levels of mRNA for gd TCR, CD4, CD8, and F4/80.
Simultaneous neutralization of both TNF-a and IFN-g 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-a and IFN-g individually and synergistically decreased the levels of mRNA for IL-12, TNF-a, and IFN-g.
The experiment was repeated four times with an identical pattern
emerging from each experiment.
Discussion
After primary HSV-1 infection at a peripheral site, the virus is
transmitted to the neuron cell bodies of the sensory ganglia (1).
The virus then replicates in the ganglion for 5– 6 days. A role for
an adaptive immune response in controlling HSV-1 replication in
the sensory ganglia is now well established (6, 7, 19). This study
demonstrates an important role for innate immunity in controlling
early virus replication in the ganglion as well.
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
FIGURE 5. Cells expressing iNOS in the HSV-1-infected TG. Infected
TG were excised 7 days after HSV-1 corneal infection. Frozen sections
were prepared and stained for iNOS using an indirect immunofluorescent
staining technique. The primary anti-iNOS Ab was omitted as a control
(A). Numerous cells exhibiting cytoplasmic staining for iNOS (arrows) are
seen surrounding neuron cell bodies in the ophthalmic branch of the TG (B).
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IFN-g, TNF-a, and NO contribute to the control of HSV-1
replication in the TG
FIGURE 4. Macrophage depletion dramatically reduces the mRNA for
iNOS in the HSV-1-infected TG. The TG were excised on days 3 (lanes 1
and 2), 5 (lanes 3 and 4), and 7 (lanes 5 and 6) from groups of macrophagedepleted (lanes 2, 4, and 6) and mock-depleted (lanes 1, 3, and 5) mice;
total RNA was extracted. A 2-mg aliquot of each RNA preparation was
analyzed for iNOS mRNA and housekeeping gene (HPRT) mRNA by a
semiquantitative RT-PCR assay. The bands were identified by Southern
blot analysis.
The Journal of Immunology
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
FIGURE 7. Macrophage depletion results in increased HSV-1 dissemination in the TG. On days 1, and 3, after HSV-1 corneal infection, groups
of six mice were depleted of macrophages by i.v. injection of 0.2 ml of
Cl2MDP liposomes or were mock depleted with PBS-liposomes. On day 5
and day 7 after infection, the TG were excised, frozen sections were prepared, and HSV Ag-positive neurons were identified by immunohistochemical staining. On day 5 (A and B) and day 7 (C and D), there were
more HSV Ag-positive neurons in TG from macrophage-depleted mice (B
and D) than in TG from mock-depleted mice (A and C).
FIGURE 8. In vivo neutralization of TNF-a and IFN-g augments
HSV-1 replication in the TG. Groups of six mice received i.p. injections of
neutralizing rat mAb to TNF-a, IFN-g, or a combination of both mAbs
beginning 1 day before HSV-1 corneal infection and continuing on alternate days after infection. Controls were treated with a control rat mAb of
irrelevant specificity. The HSV-1 titers in extracts of individual TG obtained at the indicated time after corneal infection were determined by a
viral plaque assay and recorded as the mean 6 SE number of PFU/TG. The
significance of differences in HSV-1 titers in TG from mice that received
control mAb or cytokine-neutralizing mAb was assessed by a Student’s t
test and indicated as (p, p , 0.05; pp, p , 0.01).
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,
FIGURE 9. In vivo inhibition of iNOS augments HSV-1 replication in
the TG. Groups of six mice received i.p. injections of control mAb of
irrelevant specificity or of neutralizing mAb to TNF-a on alternate days
beginning 1 day before infection; or received daily i.p. injections of the
iNOS inhibitor aminoguanidine (AG); or received a combination of AG
and mAb to TNF-a. The HSV-1 titers in extracts of individual TG obtained
at the indicated time after corneal infection were determined by a viral
plaque assay and were recorded as the mean 6 SE number of PFU/TG. p,
Significant (p , 0.05) difference in HSV-1 titers when compared with mice
treated with control mAb as assessed by Student’s t test.
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FIGURE 6. Macrophage depletion augments HSV-1 replication in the
TG. On days 1, 3, and 5 after HSV-1 corneal infection, groups of six mice
were depleted of macrophages by i.v. injection of 0.2 ml of Cl2MDP liposomes or were mock depleted with PBS liposomes. The HSV-1 titers in
extracts of individual TG obtained at the indicated time after corneal infection of mock-depleted (solid bar) and macrophage-depleted (crosshatched bar) mice were determined by a viral plaque assay and recorded as
the number of PFU/TG. pp, Difference in HSV-1 titers in TG from mockdepleted and macrophage-depleted mice was significant (p , 0.01) by
Student’s t test.
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FIGURE 10. In vivo neutralization of
TNF-a and IFN-g reduces leukocytic accumulation and cytokine production in the
TG. Groups of four mice received i.p. injections of neutralizing mAb to TNF-a,
IFN-g or a combination of both mAbs beginning 1 day before HSV-1 corneal infection and continuing on alternate days
after infection. Controls were treated with
a rat mAb of irrelevant specificity. On
days 3, 5, and 7 after infection the four TG
of each treatment group were removed,
pooled, and total RNA was extracted. The
mRNA for leukocyte subpopulation markers and cytokines was analyzed in a multiprobe RPA. The resulting bands are
shown (A) for RNA from TG of mice
treated with: control mAb (lane 1), antiTNF-a (lane 2), anti-IFN-g (lane 3), or
anti-TNF-a and anti-IFN-g (lane 4). The
quantitative analysis of the bands in A is
shown graphically in B. Quantitative data
from a similar experiment are shown in C.
CONTROL OF HSV-1 IN THE PERIPHERAL NERVOUS SYSTEM
The Journal of Immunology
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FIGURE 10. (continued)
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 corre-
sponding proteins as determined immunohistochemically in this
and previous studies (3).
Our results establish that IFN-g, TNF-a, IL-12, and iNOS are
expressed in the TG within 3 days after HSV-1 corneal infection.
At this time, gd TCR1 T cells and macrophages are readily detectable and surround neurons in the ophthalmic branch of the TG
(3). In contrast, TCR-ab1 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-a, and iNOS. The gd TCR1 subpopulation of T
cells is an important source of IFN-g in certain infections and
regulates macrophage function with this molecule (10). Several
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CONTROL OF HSV-1 IN THE PERIPHERAL NERVOUS SYSTEM
creased in the absence of gd TCR1 T cells and macrophages.
The increased virus titers were associated with increased numbers of infected neurons. Thus, one function of macrophages
and gd TCR1 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, gd TCR1 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 gd TCR1 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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observations in the current study point to macrophages as the primary source of TNF-a, IL-12, and iNOS in the HSV-1-infected
TG. First, the temporal pattern of expression of mRNA for TNF-a
and IL-12 was very similar to the pattern of expression of mRNA
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macrophages resulted in a dramatic decrease in mRNA for IL-12,
TNF-a, 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-a. Our findings also
suggest that gd TCR1 T cells are the main source of IFN-g in the
infected TG. There was a very similar pattern of expression of
mRNA for gd TCR and for IFN-g in the infected TG. The plastic
nonadherent TG cells were enriched for mRNA for gd TCR and
for IFN-g. In addition, in vivo depletion of gd TCR1 T cells with
GL3 mAb dramatically reduced mRNA for IFN-g 3 and 5 days
after HSV-1 corneal infection.
Our findings also suggest that gd TCR1 T cells and macrophages cross-regulate their accumulation and activation in the infected TG. Depletion of macrophages markedly diminished gd
TCR1 T cell accumulation and IFN-g mRNA and protein production in the infected TG. Although macrophage depletion significantly reduced the number of IFN-g1 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 gd TCR and IFN-g. Alternatively,
TCR-ab1 T cells may contribute to the IFN-g 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-g production by gd TCR1 T cells (20).
Neutralization of IFN-g, which appears to be produced primarily by gd TCR1 T cells, reduces the accumulation of macrophages and their expression of TNF-a in the infected TG
during the period of HSV-1 replication. This finding is consistent with the established capacity of gd TCR1 T cells to induce
TNF-a and iNOS production by macrophages (21) through
IFN-g. Moreover, neutralization of TNF-a and IFN-g individually and synergistically reduced macrophage and gd TCR1 T
cell accumulation in the HSV-1-infected TG. This result is consistent with the observation that TNF-a and IFN-g can individually and synergistically induce vascular endothelial cells to
produce the chemokine RANTES (22), a chemoattractant for
both monocytes and gd TCR1 T cells (23). Therefore, it appears that gd TCR1 T cells and macrophages reciprocally regulate each other’s accumulation and activation in the HSV-1infected 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 gd TCR1 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-a and IFN-g, and the
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in vivo neutralization of IFN-g or TNF-a or inhibition of NO
production by iNOS results in a dramatic increase in virus replication in the ganglion. The elaboration of these cytokines by
gd TCR1 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 in-
The Journal of Immunology
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