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SystemType 1 Replication in the Peripheral Nervous Macrophage Control of Herpes Simplex Virus

Jager and Robert L. HendricksPadma Kodukula, Ting Liu, Nico Van Rooijen, Martine J.

http://www.jimmunol.org/content/162/5/28951999; 162:2895-2905; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Macrophage Control of Herpes Simplex Virus Type 1Replication in the Peripheral Nervous System

Padma Kodukula,* Ting Liu, † Nico Van Rooijen,§ Martine J. Jager,¶ andRobert L. Hendricks2*†‡

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 asearly as 2 days after corneal infection, reaches peak titers by 3–5 days after infection, and is undetectable by 7–10 days. Duringthe period of HSV-1 replication, macrophages andgd TCR1 T lymphocytes infiltrate the TG, and TNF-a, IFN-g, the induciblenitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-a, IFN-g, and the iNOS product nitric oxide (NO) all inhibitHSV-1 replication in vitro. Macrophage and gd TCR1 T cell depletion studies demonstrated that macrophages are the mainsource of TNF-a and iNOS, whereasgd 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 cornealinfection. Moreover, individually depleting macrophages or neutralizing TNF-a or IFN- g markedly reduced the accumulation ofboth macrophages andgd TCR1 T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virusreplication in the sensory ganglia is controlled by macrophages andgd TCR1 T lymphocytes through their production of antiviralmolecules TNF-a, NO, and IFN-g. Our findings also strongly suggest that cross-regulation between these two cell types is nec-essary for their accumulation and function in the infected TG. The Journal of Immunology,1999, 162: 2895–2905.

H erpes simplex virus type 1 (HSV-1)3 can enter the bodyby infecting epidermal cells or epithelial cells of muco-sal surfaces. The virus replicates in and destroys these

cells and in the process gains access to the termini of local sensoryneurons. Retrograde axonal transport carries the virus to the neu-ronal cell bodies in the sensory ganglia within 2 days of the pri-mary infection (1). After HSV-1 corneal infection in mice, thevirus replicates briefly in the trigeminal ganglion (TG), reachingpeak titers by 3–5 days after infection. By 7–10 days after cornealinfection, HSV-1 replication in the TG has ceased, but a portion ofthe neurons retains the viral genome in a latent state. The recurrentnature of herpetic disease appears to be due to periodic reactivationof latent HSV-1 and axonal transmission to peripheral sites servedby the infected sensory neurons. Recurrent herpetic disease is re-sponsible for the vast majority of the human suffering, loss ofproductivity, and visual impairment associated with HSV-1

infections. A key to preventing recurrent herpetic disease wouldseem to lie in preventing or limiting the initial colonization ofsensory neurons after primary infection or in preventing or limitingthe subsequent reactivation of HSV-1 from latency.

A number of recent studies using the mouse model of HSV-1corneal infection have established that transmission of the virus tothe TG is associated with leukocytic infiltration and cytokine pro-duction within the ganglion (2–4). Macrophages,gd TCR1 T lym-phocytes, and TCR-ab1 T cells of both the CD41 and CD81

subpopulations infiltrate the TG during this period of active virusreplication. Macrophages are the predominant infiltrating cell inthe TG 3–7 days after infection, whereas CD81 T cells preferen-tially accumulate and dominate the TG infiltrate 7–12 days afterinfection (3). During the early stages of HSV-1 replication, 3–5days after infection, macrophages andgd TCR1 T cells can beseen surrounding infected neurons. By 7–12 days after cornealinfection, CD81 T cells are also preferentially drawn to the in-fected neurons in the ophthalmic branch of the TG (3).

Depletion ofgd TCR1 T cells dramatically increases HSV-1titers in the TG but does not influence the duration of HSV-1replication in the ganglion (5). In contrast, the absence of TCR-ab1 T cells does not increase HSV-1 titers in the TG but doesresult in prolonged, low level HSV-1 replication in the TG, trans-mission to the brain, and lethal viral encephalitis (5). Depletion ofCD81 T cells or compromise of CD81 T cell function also sig-nificantly augments HSV-1 neurovirulence (6, 7). Thus,gd TCR1

T cells represent an important early line of defense against HSV-1replication in the sensory neurons, whereas CD81 T cells are re-quired to completely shut down HSV-1 replication in the TG andprevent neurologic damage.

The mechanism by whichgd TCR1 T cells control virus rep-lication in sensory neurons is not known. IFN-g is produced in theganglion early after infection during the acute phase of virus rep-lication, and the absence of this molecule results in increased virusreplication in the ganglion (8). Although IFN-g has direct antiviral

*Department of Pathology, University of Illinois, Chicago, IL 60154; Departments of†Ophthalmology and‡Molecular Genetics and Biochemistry, University of PittsburghSchool of Medicine, Pittsburgh, PA 15213;§Department of Cell Biology and Immu-nology, Free University, Amsterdam, The Netherlands; and¶Department of Ophthal-mology, Leiden University Medical Center, Leiden, The Netherlands

Received for publication June 22, 1998. Accepted for publication November30, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grants EY05945, EY10359, and 5 P30 EY08098 fromthe National Institutes of Health and by an unrestricted grant from Research to Pre-vent Blindness, New York, NY. R.L.H. is a Research to Prevent Blindness SeniorScientific Investigator.2 Address correspondence and reprint requests to Dr. Robert L. Hendricks, Universityof Pittsburgh School of Medicine, 915 Eye and Ear Institute, 203 Lothrop Street,Pittsburgh, PA 15213-2588. E-mail address: hendricksRR@MSX.UPMC.edu3 Abbreviations used in this paper: HSV-1, herpes simplex virus type 1; Cl2MDP,clodronic-acid disodium salt tetrahydrate; TG, trigeminal ganglion; iNOS, induciblenitric oxide synthase; NO, nitric oxide; RPA, RNase protection assay; GAPDH, glyc-eraldehyde phosphate dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; PFU, plaque-forming units.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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activity, it also plays an important role in activating macrophages(9). In fact, in certain infectious disease models, macrophage pro-duction of TNF-a and nitric oxide (NO) is regulated bygd TCR1

cells through their production of IFN-g (10, 11). In addition, HSVinfection has been shown to augment NO synthesis by IFN-g-treated macrophages (12). Both TNF-a and NO are potent inhib-itors of HSV-1 replication in vitro (11, 13–16).

We hypothesized that an interaction between macrophages andgd TCR1 T cells might lead to their production of antiviral cyto-kines and control of virus replication in sensory neurons. Our cur-rent findings support this hypothesis and demonstrate an importantprotective role for the cytokines TNF-a and IFN-g and the reactivenitrogen intermediate NO.

Materials and MethodsAnimals

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 hy-drochloride (Vetalar; Parke-Davis, Morris Plains, NJ) and 0.04 mg ofacepromazine maleate (Aveco, Fort Dodge, IA) in 0.1 ml of HBSS into theleft hind leg.

Virus

The RE strain of HSV-1 was grown in Vero cells, and intact virions werepurified on Percoll (Pharmacia, Piscataway, NJ) as previously described (17).

Corneal infection

Topical corneal infection of anesthetized mice was achieved by superfi-cially scratching the central cornea 15 times with a 30-gauge needle in acrisscross pattern. A 3-ml HSV-1 suspension (105 plaque-forming units(PFU)) was applied topically to the scarified cornea and rubbed in with theeyelids. All experimental procedures conformed to the Association for Re-search in Vision and Ophthalmology resolution on the use of animals inresearch.

In vivo macrophage depletion

Dichloromethylene diphosphonate (clodronic-acid disodium salt tetrahy-drate; Cl2MDP) was a gift from Boehringer Mannheim (Mannheim, Ger-many). Preparation of liposomes containing Cl2MDP or PBS as a controlwas prepared as described previously (18). For in vivo macrophage deple-tion, mice were injected i.v. with 0.2 ml of Cl2MDP liposomes or PBSliposomes as a control (mock depletion) on days 1, 3, and 5 after HSV-1corneal infection.

In vitro enrichment of TG macrophages

TG were excised from 15 mice, 5 or 7 days after infection and incubatedwith collagenase (3 mg/ml) for 1 h at 37°C. The TG tissue was then trit-urated and passed through a 40-mm filter. The resulting single cell suspen-sion was incubated for 2 h at37°C on the plastic surface of a petri dish(Falcon 3001; Becton Dickinson, Franklin Lakes, NJ). The nonadherentcells were removed by vigorous washing of the petri dish, and the adherentand nonadherent populations were dissolved in lysis buffer before totalRNA extraction (RNeasy kit; Qiagen, Santa Clarita, CA) and analysis ofmRNA in an RNase protection assay (RPA).

Aminoguanidine treatment

Mice received three daily i.p. injections of aminoguanidine in PBS (totaldose 400 mg/kg/day) or three injections of PBS as a control starting 1 dayafter 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 Amer-ican Type Culture Collection (Manassas, VA). For cytokine neutralization,mice received i.p. injections of 0.5 mg of each mAb alone or a combinationof 0.5 mg of both mAb on alternate days starting 1 day before HSV-1corneal infection. Similar injections of a control rat mAb (anti-HLA-BW6;American Type Culture Collection) were given to control for possible non-specific mAb effects.

In vivo depletion ofgd TCR1 T lymphocytes

Groups of mice received i.p. injections of 0.5 mg of the GL3 mAb that isspecific for thegd TCR. Injections were initiated 1 day before HSV-1corneal infection and were repeated 1 and 3 days after infection. Controlsreceived similar injections of rat anti-HLA-BW6 mAb. On days 3 and 5after infection, the TG were removed and total RNA was extracted andanalyzed in an RPA assay.

Virus titration

On days 3, 4, 5, and 7 after HSV-1 corneal infection, mice were sacrificedand the ipsilateral TG was excised and frozen in 0.5 ml of RPMI 1640. TheTG were homogenized, subjected to three freeze-thaw cycles, and the sus-pension was sonicated and centrifuged at 6000 rpm for 10 min. The titer ofinfectious HSV-1 in the supernatant fluids was determined in a standardviral plaque assay on Vero cell monolayers. The results are expressed asthe number PFU per TG.

Immunohistochemical and immunofluorescent staining

Frozen sections of TG were prepared and stained using immunohistochem-ical and immunofluorescent staining procedures that were previously de-scribed (3). Briefly, TG were excised, embedded in OCT (optimal cryo-genic temperature; Tissue Tek; Miles, Naperville, IL), snap frozen, and6-mm frozen sections were cut. The sections were fixed and stained asfollows: for HSV-1 Ags using peroxidase-conjugated rabbit anti-HSV type1 (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 rabbitanti-iNOS (Accurate Chemical & Scientific, Westbury, NY) followed bybiotin-conjugated goat anti-rabbit Ig (Zymed Laboratories, San Fransisco,CA) and then FITC-Avidin (PharMingen, San Diego, CA); for TNF-a bysequential treatment with rat anti-TNF-a (MP6-T22.11; American TypeCulture Collection), biotinylated goat anti-rat Ig (Jackson ImmunoResearch Laboratories, West Grove, PA), ABC reagent (Vectastain ABCkit; Vector Laboratories), and DAB substrate; and for IFN-g using FITC-conjugated rat mAb to mouse IFN-g (R4-6A2; American Type CultureCollection).

RNase protection assay

Total RNA was extracted from pools of 4 TG using an RNeasy kit ac-cording to the manufacturer’s instructions. A 2-mg aliquot of the RNA wasstored for a semiquantitative RT-PCR analysis. The remaining RNA wasanalyzed in a multiprobe RPA assay (PharMingen). The template set in-cluded probes specific for the following mRNA:gd TCR, IL-12 (IL-12p-40), TNF-a, CD4, CD8, IL-2, F4/80, IFN-g, and the housekeeping genesL32 and glyceraldehyde phosphate dehydrogenase (GAPDH). The RPAwas performed according to the manufacturer’s instructions, the resultingbands were visualized on a PhosphorImager (PSI-PC; Molecular Dynam-ics, Sunnyvale, CA), and the results were analyzed using ImageQuaNTsoftware.

Semiquantitative RT-PCR

A total of 2 mg of RNA from TG of each treatment group was used tosynthesize first strand cDNA using the Promega Reverse Transcription kit(Stratagene, La Jolla, CA), and PCR was performed using 1% of the cDNAobtained as template. The following primer sequences were used: iNOSsense, 59-TTT GCT TCC ATG CTA ATG CGA AAG-39; iNOS anti-sense,59-GCT CTG TTG AGG TCT AAA GGC TCC G-39; hypoxanthine-guanine phosphoribosyl transferase (HPRT) sense, 59CTC GAA GTGTTG GAT ACA GGC-39; and HPRT anti-sense, 59-GAT AAG CGA CAATCT 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) andHPRT was amplified for 20 cycles. Preliminary studies determined thatthese cycle numbers were in the linear range of amplification for eachprimer set. PCR products were separated by agarose gel electrophoresisand blotted onto nylon membrane (Zeta Probe; Bio-Rad Laboratories,Richmond CA). The iNOS and HPRT cDNA were detected by hybridiza-tion with a 32P-labeled 4.1-kbNotI andSfiI fragments of plasmid pPQRSthat contains cloned fragments of mouse iNOS and HPRT gene sequences(gifts from Dr. Steve Reiner, University of Chicago, Chicago, IL). Thehybridized bands were visualized on a PhosphorImager and the resultsanalyzed using ImageQuaNT software.

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ResultsMacrophages regulate leukocytic accumulation in the infectedTG

Groups of HSV-1-infected mice were treated with Cl2MDP lipo-somes to deplete macrophages. HSV-1-infected control mice weremock depleted with PBS liposomes. At 3, 5, and 7 days afterinfection, the infected TG were removed and total RNA was ex-tracted from pools of four ganglia from each treatment group. Leu-kocytic infiltration of the TG was monitored using a multiprobeRPA assay to analyze the expression of mRNA for various leuko-cyte subpopulation markers. The experiment was repeated fourtimes, and an identical pattern emerged from each experiment. Theresults of two representative experiments are shown in Fig. 1.

The TG of noninfected mice contained a low level of mRNA forF4/80, suggesting that small numbers of macrophages are presentin the normal TG (data not shown). No mRNA for T cell sub-population markers or cytokines was detectable in the normal TG.The level of expression of the housekeeping gene transcripts L32and GAPDH in normal TG was roughly equivalent to that in theTG of the macrophage-depleted group (Fig. 1A, lane 2) on days 3and 5 after infection. Our previous immunohistochemical studiesestablished that the TG is infiltrated with leukocytes within 3 daysafter HSV-1 corneal infection (3). Our current findings are in goodagreement with our previous results (Fig. 1). Three days afterHSV-1 corneal infection, mRNA for the macrophage marker F4/80was prominent in the TG and continued to increase through day 7after infection. The mRNA for T lymphocyte subpopulation mark-ers including thegd TCR, CD4, and CD8 were also present butexpressed at a much lower level.

Macrophage depletion markedly reduced the level of F4/80mRNA during the period of virus replication in the ganglion (Fig.1). The level of expression of the T cell subpopulation markers waslow at days 3 and 5 after infection and was not influenced bymacrophage depletion. However, by day 7 after infection, macro-phage depletion did cause a marked reduction in mRNA forgdTCR, CD4, and CD8. Thus, macrophages regulate the infiltrationand/or retention ofgd TCR1 and TCR-ab1 T lymphocytes in theinfected TG.

Cytokine production in the infected TG

RNA transcripts specific for the cytokines IL-12, TNF-a, andIFN-g were readily detectable in the TG by 3 days after HSV-1corneal infection (Fig. 1). Macrophage depletion dramatically re-duced 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-gwere also reduced, although the reduction was not consistentlyseen until day 7 after infection. The reduction of cytokine messagewas associated with a significant reduction of cells expressingIFN-g and TNF-a protein in the HSV-1-infected TG of macrophage-depleted mice (Table I).

Macrophages are an important source of IL-12 and TNF-a butnot of IFN-g. Therefore, we proposed that macrophage depletiondirectly removed the source of IL-12 and TNF-a but indirectlyreduced IFN-g production by another cell, such as agd TCR1 Tcell. Support for this hypothesis came from two types of experi-ments. First, single cell suspensions of TG obtained 7 days aftercorneal infection were divided into plastic adherent and nonadher-ent populations. Total RNA from these populations was subjectedto RPA. The plastic adherent population was enriched for mRNAspecific for the macrophage marker F4/80 and for the cytokinesIL-12 and TNF-a (Fig. 2). There was a very weak band forgdTCR mRNA and for IFN-g mRNA in the nonadherent population

and no discernible bands for these mRNA species in the adherentpopulation (Fig. 2).

To determine the contribution ofgd TCR1 T cells to the IFN-gmessage in the infected TG, mice were depleted ofgd TCR1 Tcells by injection of GL3 mAb 1 day before and on alternate daysafter HSV-1 corneal infection. On days 3 and 5 after infection, theTG were excised and IFN-g mRNA was quantified by RPA. De-pletion of gd TCR1 T cells caused an 89 and 60% reduction inIFN-g mRNA in the TG on days 3 and 5 after infection, respec-tively (Fig. 3). These findings provide strong support for the notionthat macrophages are the main source of IL-12 and TNF-a andgdTCR1 T cells are the main source of IFN-g during the peak ofHSV-1 replication in the TG.

Activated macrophages also produce NO through the activity ofthe enzyme iNOS. NO can strongly inhibit HSV-1 replication invitro. To determine whether iNOS mRNA is present in the infectedTG, and if it is produced by macrophages, the level of iNOSmRNA expression in the TG of macrophage-depleted and mock-depleted mice was determined by a semiquantitative RT-PCR as-say. As shown in Fig. 4, iNOS mRNA was readily detectable ininfected TG of control mice by 3 days after infection and began todecline by day 7 after infection. The level of expression of iNOSmRNA was markedly reduced in the ganglia of macrophage-de-pleted mice. In separate experiments, frozen sections of infectedTG that were obtained 7 days after HSV-1 corneal infection werestained for iNOS. Numerous iNOS1 cells were found in directapposition to neuron cell bodies within the ophthalmic branch ofthe TG (Fig. 5). Thus, HSV-1 corneal infection leads to a rapidinfiltration of iNOS1 macrophages into the ophthalmic branch ofthe TG.

Macrophages control HSV-1 replication in the infected TG

Because activated macrophages are present in the infected TG andproduce cytokines that inhibit HSV-1 replication in vitro, it wasreasonable to propose that macrophages might contribute to thecontrol of HSV-1 replication in the TG after corneal infection. Totest this possibility, groups of five to six mice received cornealinfections with HSV-1 followed by macrophage depletion withCl2MDP liposomes or mock depletion with PBS liposomes. Atvarious times after infection the corneas and TG were removed,homogenized, and infectious HSV-1 titers were determined in astandard virus plaque assay.

Macrophage depletion did not significantly enhance or prolongHSV-1 replication in the infected corneas as determined either bycorneal examination or by viral plaque assay of corneal extracts(data not shown). In the TG of control mice, replicating virus wasdetectable by day 3, reached peak titers by day 4, and was nolonger detectable by day 7 after corneal infection (Fig. 6). Thekinetics of HSV-1 replication in the TG was not markedly alteredby macrophage depletion, although very low levels of replicatingvirus were routinely detected in the TG of macrophage-depletedmice on day 7, when replicating virus was no longer detectable inthe TG of control mice. However, macrophage depletion did sig-nificantly increase HSV-1 titers throughout the course of virus rep-lication in the TG (Fig. 6). The increased virus load in the TG ofmacrophage-depleted mice was associated with increased HSV-1dissemination within the ganglion. Thus, the number of HSV-1Ag-positive neurons in the TG of macrophage-depleted mice(59.1 6 8.73 and 13.76 2.65 on days 5 and 7 after infection,respectively) was significantly higher (p , 0.01) than the numberin mock-depleted mice (28.26 3.65 and 2.26 0.13 on days 5 and7, respectively) as illustrated in Fig. 7. Our findings establish thatmacrophages play an important role in controlling HSV-1 replica-tion and dissemination within the TG after corneal infection.

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FIGURE 1. Macrophages regulate leukocyticinfiltration and cytokine production in the TG. Ondays 1, 3, and 5 after HSV-1 corneal infection,groups of four mice were depleted of macro-phages by i.v. injection of 0.2 ml of Cl2MDP li-posomes or were mock depleted with PBS lipo-somes. At the indicated times, TG from eachtreatment group were pooled, total RNA was ex-tracted, and mRNA for leukocyte subpopulationmarkers, cytokines, and the housekeeping genes,L32 and GAPDH, were detected in a multiprobeRPA. The resulting bands were visualized with aPhosphorImager (A). Thelane designations are:1, mock depleted and2, macrophage depleted.The relative amounts of leukocyte and cytokinemessage were analyzed using ImageQuaNT soft-ware. The quantitative analysis of the bands inAis shown graphically inB. Quantitative data froma similar experiment are shown inC. The cross-hatched bars represent TG from mock-depletedmice, and the solid bars represent TG from mac-rophage-depleted mice.

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Table I. Cytokine-producing cells in the infected TG

Day 5a Day 7a

Control Mac. Depl. p valueb Control Mac. Depl. p valueb

TNF-a1 cellsc 128.26 15.1 95.56 6.8 0.0952 139.46 11.6 92.86 8.5 0.0159IFN-g1 cells 45.26 7.2 8.26 2.5 0.0079 156.06 21.7 73.86 10.8 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 controlmice that were mock depleted with PBS-liposomes. Frozen sections of TG were prepared and stained for TNF-a or IFN-g.

b Mann-WhitneyU 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 (n5 5).

FIGURE 1. (continued)

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IFN-g, TNF-a, and NO contribute to the control of HSV-1replication in the TG

Because mRNA for the cytokines, TNFa and IFN-g, and for iNOSwas readily detectable in the infected TG, we determined that thecorresponding proteins contributed to the control of virus replica-tion. Groups of mice received in vivo treatment with neutralizingAbs to IFN-g, TNF-a, or a combination of both Abs. At varioustimes after HSV-1 corneal infection, the TG were excised andHSV-1 titers in individual ganglia were determined in a virusplaque assay. Individual neutralization of IFN-g or TNF-a causeda significant increase in HSV-1 titers on days 3–7 after infection(Fig. 8). Simultaneous neutralization of both cytokines had a syn-ergistic effect on virus replication in the ganglion.

In separate experiments, mice were treated with the iNOS in-hibitor, aminoguanidine alone, or in combination with the neutral-izing Ab to TNF-a. Inhibition of iNOS significantly increased vi-rus replication, particularly when combined with TNF-aneutralization (Fig. 9). Thus, TNF-a, IFN-g, and NO all contributedirectly or indirectly to the control of HSV-1 replication in the TG.

IFN-g and TNF-a control leukocyte accumulation in the TG

Groups of four mice received in vivo treatment with neutralizingAbs to IFN-g, TNF-a, or a combination of both mAbs. At varioustimes after HSV-1 corneal infection, the TG were excised andpooled, and total RNA was extracted and subjected to RPA anal-ysis. The results of two representative experiments are shown inFig. 10. Individual neutralization of TNF-a or IFN-g reduced leu-kocytic infiltration of the ganglia as illustrated by a marked reduc-tion in the levels of mRNA forgd TCR, CD4, CD8, and F4/80.Simultaneous neutralization of both TNF-a and IFN-g had a syn-ergistic inhibitory effect on leukocytic infiltration of the TG. Thereduction in the accumulation of leukocytes in the TG was asso-ciated with a corresponding decrease in cytokine mRNA. Thus,neutralization of TNF-a and IFN-g individually and synergisti-cally decreased the levels of mRNA for IL-12, TNF-a, and IFN-g.The experiment was repeated four times with an identical patternemerging from each experiment.

DiscussionAfter primary HSV-1 infection at a peripheral site, the virus istransmitted to the neuron cell bodies of the sensory ganglia (1).The virus then replicates in the ganglion for 5–6 days. A role foran adaptive immune response in controlling HSV-1 replication inthe sensory ganglia is now well established (6, 7, 19). This studydemonstrates an important role for innate immunity in controllingearly virus replication in the ganglion as well.

Our studies used a multiprobe RPA assay to screen for cytokineand leukocyte subpopulation marker gene expression in the in-fected TG. This assay permits screening for a large number of

FIGURE 2. Cytokine profile of plastic adherent and nonadherent cellsobtained from TG that were excised 7 days after HSV-1 corneal infection.Single cell suspensions were prepared from 30 infected TG and allowed toadhere to the plastic surface of a tissue culture flask for 2 h. The nonad-herent cells were removed and total RNA was extracted from the plasticadherent and nonadherent cells. The mRNA for leukocyte subpopulationmarkers and cytokines was analyzed in a multiprobe RPA. The relativeamount of message for F4/80, TNF-a, gd TCR, IFN-g, and IL-12 in ad-herent cells (solid bars) and nonadherent cells (crosshatched bars) is shownas PhosphorImager units.

FIGURE 3. In vivo depletion of gd TCR1 T cells reduces IFN-gmRNA in the infected TG. Groups of four mice were depleted ofgd TCR1

T cells by injection of the GL3 mAb on days21, 12, and14 after HSV-1corneal infection or they were mock depleted by similar treatment with acontrol mAb. The TG from each treatment group were excised on thedesignated day after infection, pooled, and total RNA was extracted andanalyzed with the multiprobe RPA. The relative amount of IFN-g mRNAin TG from mock-depleted control mice (solid bars) and fromgd TCR1 Tcell-depleted mice (cross-hatched bars) is shown.

FIGURE 4. Macrophage depletion dramatically reduces the mRNA foriNOS in the HSV-1-infected TG. The TG were excised on days 3 (lanes 1and2), 5 (lanes 3and4), and 7 (lanes 5and6) from groups of macrophage-depleted (lanes 2,4, and6) and mock-depleted (lanes 1,3, and5) mice;total RNA was extracted. A 2-mg aliquot of each RNA preparation wasanalyzed for iNOS mRNA and housekeeping gene (HPRT) mRNA by asemiquantitative RT-PCR assay. The bands were identified by Southernblot analysis.

FIGURE 5. Cells expressing iNOS in the HSV-1-infected TG. InfectedTG were excised 7 days after HSV-1 corneal infection. Frozen sectionswere prepared and stained for iNOS using an indirect immunofluorescentstaining technique. The primary anti-iNOS Ab was omitted as a control(A). Numerous cells exhibiting cytoplasmic staining for iNOS (arrows) areseen surrounding neuron cell bodies in the ophthalmic branch of the TG (B).

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transcripts in a single sample. However, there are unique problemsassociated with the use of this or other molecular biologicalscreening assays at sites of inflammation, especially those inducedby HSV-1. Most RPA assays use housekeeping genes to standard-

ize for RNA quantity and hybridization efficiency. However, at aninflammatory site, particularly in nervous tissue, a significant pro-portion of the housekeeping gene transcripts is contributed by in-filtrating inflammatory cells. Moreover, an HSV-1 virion protein,referred to as virus host shutoff, has been shown to destabilize anddegrade host mRNA.

Our studies clearly demonstrate an inverse correlation betweenthe degree of leukocytic infiltration into the TG and the amount ofHSV-1 replication. Thus, any treatment that reduces inflammationwould not only reduce the leukocytic contribution to the pool ofhousekeeping gene mRNA, but would also result in increasedvirus-induced destabilization of this mRNA pool. For this reason,

FIGURE 6. Macrophage depletion augments HSV-1 replication in theTG. On days 1, 3, and 5 after HSV-1 corneal infection, groups of six micewere depleted of macrophages by i.v. injection of 0.2 ml of Cl2MDP li-posomes or were mock depleted with PBS liposomes. The HSV-1 titers inextracts of individual TG obtained at the indicated time after corneal in-fection of mock-depleted (solid bar) and macrophage-depleted (cross-hatched bar) mice were determined by a viral plaque assay and recorded asthe number of PFU/TG.pp, Difference in HSV-1 titers in TG from mock-depleted and macrophage-depleted mice was significant (p , 0.01) byStudent’st test.

FIGURE 7. Macrophage depletion results in increased HSV-1 dissem-ination in the TG. On days 1, and 3, after HSV-1 corneal infection, groupsof six mice were depleted of macrophages by i.v. injection of 0.2 ml ofCl2MDP liposomes or were mock depleted with PBS-liposomes. On day 5and day 7 after infection, the TG were excised, frozen sections were pre-pared, and HSV Ag-positive neurons were identified by immunohisto-chemical staining. On day 5 (A andB) and day 7 (C andD), there weremore HSV Ag-positive neurons in TG from macrophage-depleted mice (BandD) than in TG from mock-depleted mice (A andC).

FIGURE 8. In vivo neutralization of TNF-a and IFN-g augmentsHSV-1 replication in the TG. Groups of six mice received i.p. injections ofneutralizing rat mAb to TNF-a, IFN-g, or a combination of both mAbsbeginning 1 day before HSV-1 corneal infection and continuing on alter-nate days after infection. Controls were treated with a control rat mAb ofirrelevant specificity. The HSV-1 titers in extracts of individual TG ob-tained at the indicated time after corneal infection were determined by aviral plaque assay and recorded as the mean6 SE number of PFU/TG. Thesignificance of differences in HSV-1 titers in TG from mice that receivedcontrol mAb or cytokine-neutralizing mAb was assessed by a Student’sttest and indicated as (p, p , 0.05; pp, p , 0.01).

FIGURE 9. In vivo inhibition of iNOS augments HSV-1 replication inthe TG. Groups of six mice received i.p. injections of control mAb ofirrelevant specificity or of neutralizing mAb to TNF-a on alternate daysbeginning 1 day before infection; or received daily i.p. injections of theiNOS inhibitor aminoguanidine (AG); or received a combination of AGand mAb to TNF-a. The HSV-1 titers in extracts of individual TG obtainedat the indicated time after corneal infection were determined by a viralplaque assay and were recorded as the mean6 SE number of PFU/TG.p,Significant (p, 0.05) difference in HSV-1 titers when compared with micetreated with control mAb as assessed by Student’st test.

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FIGURE 10. In vivo neutralization ofTNF-a and IFN-g reduces leukocytic ac-cumulation and cytokine production in theTG. Groups of four mice received i.p. in-jections of neutralizing mAb to TNF-a,IFN-g or a combination of both mAbs be-ginning 1 day before HSV-1 corneal in-fection and continuing on alternate daysafter infection. Controls were treated witha rat mAb of irrelevant specificity. Ondays 3, 5, and 7 after infection the four TGof each treatment group were removed,pooled, and total RNA was extracted. ThemRNA for leukocyte subpopulation mark-ers and cytokines was analyzed in a mul-tiprobe RPA. The resulting bands areshown (A) for RNA from TG of micetreated with: control mAb (lane 1), anti-TNF-a (lane 2), anti-IFN-g (lane 3), oranti-TNF-a and anti-IFN-g (lane 4). Thequantitative analysis of the bands inA isshown graphically inB. Quantitative datafrom a similar experiment are shown inC.

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standardization of these assays is virtually impossible. In our as-says, no attempt was made to adjust the quantity of RNA or house-keeping gene message in each sample. Thus, the results reflect thetotal amount of mRNA for a particular protein in a constantamount of tissue (pool of four TG) for the various treatments. Infour separate TG pools from each treatment group, the pattern ofmRNA expression was identical, suggesting that the differencesobserved were not artifactual. Therefore, we believe that the use ofa multiprobe RPA assay as a screening device is very useful evenin situations that defy normal approaches to standardization. More-over, the levels of mRNA for leukocyte Ags and cytokines de-tected by RPA are in close agreement with levels of the corre-

sponding proteins as determined immunohistochemically in thisand previous studies (3).

Our results establish that IFN-g, TNF-a, IL-12, and iNOS areexpressed in the TG within 3 days after HSV-1 corneal infection.At this time, gd TCR1 T cells and macrophages are readily de-tectable and surround neurons in the ophthalmic branch of the TG(3). In contrast, TCR-ab1 T cells are barely detectable in theganglion and are not localized to the neuron cell bodies at thistime. At sites of infection, macrophages are often a major sourceof IL-12, TNF-a, and iNOS. Thegd TCR1 subpopulation of Tcells is an important source of IFN-g in certain infections andregulates macrophage function with this molecule (10). Several

FIGURE 10. (continued)

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observations in the current study point to macrophages as the pri-mary source of TNF-a, IL-12, and iNOS in the HSV-1-infectedTG. First, the temporal pattern of expression of mRNA for TNF-aand IL-12 was very similar to the pattern of expression of mRNAfor the macrophage marker F4/80. Second, in vivo depletion ofmacrophages resulted in a dramatic decrease in mRNA for IL-12,TNF-a, and iNOS. Third, plastic adherent cells derived from theHSV-1-infected TG were highly enriched for mRNA for the mac-rophage marker F4/80 and for IL-12 and TNF-a. Our findings alsosuggest thatgd TCR1 T cells are the main source of IFN-g in theinfected TG. There was a very similar pattern of expression ofmRNA for gd TCR and for IFN-g in the infected TG. The plasticnonadherent TG cells were enriched for mRNA forgd TCR andfor IFN-g. In addition, in vivo depletion ofgd TCR1 T cells withGL3 mAb dramatically reduced mRNA for IFN-g 3 and 5 daysafter HSV-1 corneal infection.

Our findings also suggest thatgd TCR1 T cells and macro-phages cross-regulate their accumulation and activation in the in-fected TG. Depletion of macrophages markedly diminishedgdTCR1 T cell accumulation and IFN-g mRNA and protein produc-tion in the infected TG. Although macrophage depletion signifi-cantly reduced the number of IFN-g1 cells in the TG on days 5 and7 after infection, the effect was proportionately greater on day 5(Table I). This may simply reflect variation in the efficiency ofmacrophage depletion. As can be seen in Fig. 1,B and C, moreefficient macrophage depletion tends to be associated with agreater reduction in mRNA forgd TCR and IFN-g. Alternatively,TCR-ab1 T cells may contribute to the IFN-g production by day7 and be less dependent on macrophage regulation. Macrophagedepletion also markedly reduced the mRNA for IL-12, a potentregulator of IFN-g production bygd TCR1 T cells (20).

Neutralization of IFN-g, which appears to be produced pri-marily by gd TCR1 T cells, reduces the accumulation of mac-rophages and their expression of TNF-a in the infected TGduring the period of HSV-1 replication. This finding is consis-tent with the established capacity ofgd TCR1 T cells to induceTNF-a and iNOS production by macrophages (21) throughIFN-g. Moreover, neutralization of TNF-a and IFN-g individ-ually and synergistically reduced macrophage andgd TCR1 Tcell accumulation in the HSV-1-infected TG. This result is con-sistent with the observation that TNF-a and IFN-g can individ-ually and synergistically induce vascular endothelial cells toproduce the chemokine RANTES (22), a chemoattractant forboth monocytes andgd TCR1 T cells (23). Therefore, it ap-pears thatgd TCR1 T cells and macrophages reciprocally reg-ulate each other’s accumulation and activation in the HSV-1-infected TG. The TG of noninfected mice exhibited a low levelof mRNA for F4/80 and no detectable mRNA for T cell markersor cytokines. Thus, the reciprocal activation and accumulationof macrophages andgd TCR1 T cells may be initiated by res-ident macrophages after early HSV-1 replication in the TG.

Because the sensory neurons cannot be regenerated, it is es-sential that virus replication is controlled without destruction ofthe infected neuron. The cytokines, TNF-a and IFN-g, and thenitrogen radical NO have all been shown to inhibit HSV-1 rep-lication in vitro (11, 14 –16). Our findings clearly establish thatin vivo neutralization of IFN-g or TNF-a or inhibition of NOproduction by iNOS results in a dramatic increase in virus rep-lication in the ganglion. The elaboration of these cytokines bygd TCR1 T cells and macrophages that are in direct appositionto the infected neurons may terminate virus replication withoutneuronal toxicity.

This and our previous study (5) demonstrate that the virusload in the TG after HSV-1 corneal infection is markedly in-

creased in the absence ofgd TCR1 T cells and macrophages.The increased virus titers were associated with increased num-bers of infected neurons. Thus, one function of macrophagesandgd TCR1 cells may be to prevent the lateral disseminationof HSV-1 from one neuron to the next within the ganglion.However, virus replication in the ganglion was ultimately ter-minated with similar kinetics in the presence or absence of ei-ther of these inflammatory cells. It is not yet clear whethertermination of virus replication in neurons requires exogenoushelp or whether it is determined by factors that are endogenousto the neurons. If the latter is true, then one may question thesignificance of controlling the level of HSV-1 replication in theneurons. We propose and are currently testing two alternativehypotheses. The first is that by preventing the lateral spread ofHSV-1 from neuron to neuron and by limiting virus replicationwithin each neuron,gd TCR1 T cells and macrophages reducethe number of latently infected neurons and the number of cop-ies of latent viral genome within each neuron. The second pos-sibility is that a high level of HSV-1 replication leads to viraldestruction of the neuron and to fewer latently infected neurons.Thus, control of virus replication bygd TCR1 T cells and mac-rophages may represent a compromise between the virus andthe host. Immune protection from viral destruction of host neu-rons and the resulting loss of corneal sensation may increase thenumber of latently infected neurons, permitting the virus to beretained in the sensory ganglia for the lifetime of the host. Al-though the factors that influence the likelihood of HSV-1 reac-tivation from latency are poorly defined, there is evidence thatthe frequency of latently infected neurons and the number ofcopies of viral genome in each neuron could be contributingfactors (24). Thus, the effectiveness of the innate immune re-sponse during acute HSV-1 replication in the sensory gangliamight dramatically influence the likelihood and frequency ofrecurrent herpetic disease in later life.

References1. LaVail, J. H., W. E. Johnson, and L. C. Spencer. 1993. Immunohistochemical

identification of trigeminal ganglion neurons that innervate the mouse cornea:relevance to intercellular spread of herpes simplex virus.J. Comp. Neurol. 327:133.

2. Gebhardt, B. M., and J. M. Hill. 1992. Cellular neuroimmunologic responses toocular herpes simplex virus infection.J. Neuroimmunol. 28:227.

3. Liu, T., Q. Tang, and R. L. Hendricks. 1996. Inflammatory infiltration of thetrigeminal ganglion after herpes simplex virus type 1 corneal infection.J. Virol.70:264.

4. Shimeld, C., J. L. Whiteland, N. A. Williams, D. Easty, and T. J. Hill. 1996.Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion:an in vivo study of virus antigen and immune cell infiltration.J. Gen. Virol.77:2583.

5. Sciammas, R., P. Kodukula, Q. Tang, R. L. Hendricks, and J. A. Bluestone. 1997.T cell receptor-g/d cells protect mice from herpes simplex virus type 1-inducedlethal encephalitis.J. Exp. Med. 185:1969.

6. Goldsmith, K., W. Chen, D. C. Johnson, and R. L. Hendricks. 1998. Infected cellprotein (ICP) 47 enhances herpes simplex virus neurovirulence by blocking theCD81 T cell response.J. Exp. Med. 187:341.

7. Simmons, A., and D. C. Tscharke. 1992. Anti-CD8 impairs clearance of herpessimplex virus from the nervous system: implications for the fate of virally in-fected neurons.J. Exp. Med. 175:1337.

8. Cantin, E. M., D. R. Hinton, J. Chen, and H. Openshaw. 1995.g interferonexpression during acute and latent nervous system infection by herpes simplexvirus type 1.J. Virol. 69:4898.

9. Heise, M. T., and H. W. Virgin. 1995. The T-cell-independent role ofg interferonand tumor necrosis factora in macrophage activation during murine cytomega-lovirus and herpes simplex virus infections.J. Virol. 69:904.

10. Jones-Carson, J., J. V. Torres, H. C. van der Heyde, T. Warner, R. D. Wagner,and E. Balish. 1995. [Gamma]/d cell-induced nitric oxide production enhancesresistance to mucosal candidiasis.Nat. Med. 1:552.

11. Karupiah, G., Q. Xie, R. M. L. Buller, C. Nathan, C. Duarte, andJ. D. MacMicking. 1993. Inhibition of viral replication by interferon-g-inducednitric oxide synthase.Science 261:1445.

12. Baskin, H., S. Ellermann-Eriksen, J. Lovmand, and S. C. Mogensen. 1997. Her-pes simplex virus type 2 synergizes with interferon-g in the induction of nitricoxide production in mouse macrophages through autocrine secretion of tumornecrosis factor-a. J. Gen. Virol. 78:195.

2904 CONTROL OF HSV-1 IN THE PERIPHERAL NERVOUS SYSTEM

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13. Croen, K. 1998. Evidence for an antiviral effect of nitric oxide.J. Clin. Invest.91:2446.

14. Feduchi, E., M. A. Alonso, and L. Carrasco. 1989. Humang interferon and tumornecrosis factor exert a synergistic blockade on the replication of herpes simplexvirus. J. Virol. 63:1354.

15. Feduchi, E., and L. Carrasco. 1991. Mechanism of inhibition of HSV-1 replica-tion by tumor necrosis factor and interferong. Virology 180:822.

16. Karupiah, G., and N. Harris. 1995. Inhibition of viral replication by nitric oxideand its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites.J. Exp.Med. 181:2171.

17. Pertoft, H. 1980. Purification of herpes simplex virus using Percoll.PharmaciaFine Chemicals, Separation News 3:2.

18. van Rooijen, N., and A. Sanders. 1998. Liposome mediated depletion of macro-phages: mechanism of action, preparation of liposomes and applications.J. Im-munol. Methods 174:83.

19. Gesser, R. M., T. Valyi-Nagy, and N. W. Fraser. 1994. Restricted herpes simplexvirus type 1 gene expression within sensory neurons in the absence of functionalB and T lymphocytes.Virology 200:791.

20. Skeen, M. J., and H. K. Ziegler. 1995. Activation ofgd T cells for production ofIFN-g is mediated by bacteria via macrophage-derived cytokines IL-1 and IL- 12.J. Immunol. 154:5832.

21. Nishimura, H., M. Emoto, K. Hiromatsu, S. Yamamoto, K. Matsuura, H. Gomi,T. Ikeda, S. Itohara, and Y. Yoshikai. 1995. The role ofg/d T cells in primingmacrophages to produce tumor necrosis factor-a. Eur. J. Immunol. 25:1465.

22. Marfaing-Koka, A., O. Devergne, G. Gorgone, A. Portier, T. J. Schall,P. Galanaud, and D. Emilie. 1995. Regulation of the production of the RANTESchemokine by endothelial cells: synergistic induction by IFN-g plus TNF-a andinhibition by IL-4 and IL-13.J. Immunol. 154:1870.

23. Roth, S. J., T. G. Diacovo, M. B. Brenner, J.-P. Rosat, J. Buccola, C. T. Morita,and T. A. Springer. 1998. Transendothelial chemotaxis of humana/b andg/d Tlymphocytes to chemokines.Eur. J. Immunol. 28:104.

24. Maggioncalda, J., A. Mehta, Y. H. Su, N. W. Fraser, and T. M. Block. 1996.Correlation between herpes simplex virus type 1 rate of reactivation from latentinfection and the number of infected neurons in trigeminal ganglia.Virology225:72.

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