821

Anti–Interleukin-6 Antibodies Inhibit Herpes Simplex Virus Reactivation

John D. Kriesel, Bryan M. Gebhardt, James M. Hill, Departments of Medicine and Pathology, University of Utah School of
Sarah A. Maulden, Ivan P. Hwang, Thomas E. Clinch, Medicine, Moran Eye Center, University of Utah, and Paradigm
Xia Cao, Spotswood L. Spruance, and Barbara A. Araneo Biosciences, Inc., Salt Lake City, Utah; LSU Eye Center, Louisiana
State University, New Orleans, Louisiana

Herpes simplex viruses (HSVs) infect epithelial cells, become localized in neurons, and can reacti-
vate in response to a variety of stimuli, including ultraviolet light and hyperthermia. The sequence
of gene activation during viral replication is known, but the molecular linkage between exogenous
stimuli and HSV reactivation has not been determined. It was hypothesized that interleukin (IL)-
6 acts as a signal between exogenous stimuli and neurons, stimulating HSV reactivation from
latency. Mouse corneas were infected with HSV-1, and ocular reactivation was induced 5 – 7 weeks
later by thermal stress or corneal exposure to ultraviolet light. Anti – IL-6 monoclonal antibodies

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were administered to the latently infected mice 8 – 12 h before the reactivation stimulus. Treatment
with anti – IL-6 antibodies resulted in significantly lower frequencies of ocular reactivation compared
with those in mice treated with a control immunoglobulin. These results support the hypothesis
that IL-6 plays a role in HSV reactivation from latency.

At least 20% of the US population experiences periodic
vesicular herpes simplex virus (HSV) lesions on the lip, face,
or mouth [1]. There are Ç50,000 cases of new or recurrent
ocular HSV disease per year in the United States, with stromal
scarring leading to reduced visual acuity in Ç12% of the pa-
tients [2]. Herpes simplex keratitis is a leading indication for
corneal transplantation in developed countries. Ultraviolet
(UV) light exposure, fever, hyperthermia, hypothermia, dental
trauma, and surgical manipulation of the trigeminal ganglion
are stimuli associated with reactivation of HSV type 1 (HSV-1)
[3 – 9]. The sequence of HSV gene activation during productive
infection is well known, but the mechanism by which reactiva-
tion stimuli, including hyperthermia and UV light, stimulate
latent virus to replicate is unknown [10, 11].
Ciliary neurotrophic factor (CNTF) is produced and stored
primarily in myelinating Schwann cells [12, 13]. CNTF is not
actively secreted but is released following damage to peripheral
nerves, possibly to promote repair. CNTF promotes the survival
or differentiation of a wide range of cell types in the vertebrate
Received 29 July 1996; revised 1 November 1996.
Presented in part: National meeting of the Association for Research in Vision
and Ophthalmology, Ft. Lauderdale, Florida, 25 April 1996 (abstract 4361);
Interscience Conference on Antimicrobial Agents and Chemotherapy, New
Orleans, 18 September 1996 (paper H082).
Studies were conducted according to US Department of Agriculture, Univer-
sity of Utah, and Louisiana State University guidelines for the handling of
research animals.
Financial support: Paradigm Biosciences; Utah Lions Eye Bank; Foundation
for Research to Prevent Blindness; NIH (EY-08701, EY-02672, EY-06311,
and EY-02377); and National Eye Institute, Bethesda, Maryland.
Reprints or correspondence: Dr. John D. Kriesel, University of Utah School
of Medicine, Division of Infectious Diseases, Room 4B-322 SOM, 50 N.
Medical Dr., Salt Lake City, Utah 84132.
The Journal of Infectious Diseases 1997;175:821–7
q 1997 by The University of Chicago. All rights reserved.
0022–1899/97/7504–0012$01.00
nervous system and has been investigated for the treatment of
neurodegenerative diseases in humans [14, 15]. During a clini-
cal trial for the treatment of amyotrophic lateral sclerosis, hu-
man patients who received high doses of recombinant human
CNTF (Synergen, Boulder, CO) frequently developed herpes
labialis [16]. The relationship between CNTF administration
and HSV reactivation was unexpected, not associated with high
fever, and appeared to be dose-dependent. We suspected that
CNTF was interacting with its receptor at the neuronal cell
surface to facilitate HSV reactivation.
To our knowledge, murine CNTF has not been definitively
isolated and cloned and, therefore, cannot be augmented or
blocked. The observation that CNTF is stored in Schwann cells
and not actively secreted suggests that the reactivation of HSV
following administration of CNTF to humans is a purely iatro-
genic event, in which CNTF is probably mimicking the activity
of other substances in vivo.
The related cytokine interleukin (IL)-6 is a good candidate
for an alternative inflammatory mediator of HSV reactivation
because it is secreted by a variety of somatic cells and is closely
associated with inflammation. Increased levels of IL-6 and its
message are observed during fever and after skin exposure to
UV light [17 – 20]. Production of IL-6 in the skin appears to
be important in the subsequent cellular responses to UV light –
induced cellular damage [21]. The IL-6 ‘‘family’’ of cytokines
(IL-6, IL-11, oncostatin M, leukemia inhibitory factor, and
CNTF) act at the plasma membrane by binding to their specific
receptors (e.g., IL-6R, CNTFR) and by activating the trans-
membrane signal transducing protein gp130 [22 – 25]. Cyto-
kine-induced dimerization of gp130 leads to activation of the
transcription factors STAT3/APRF and nuclear factor – IL-6
[26]. These transcription factors stimulate synthesis of mRNAs
encoding several cytokines and the acute-phase proteins.
On the basis of our observations in patients who received
CNTF and the relationship of CNTF to IL-6, we hypothesized

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822 Kriesel et al.
that IL-6 acts as a signal between inflamed tissues and neurons,
stimulating a cascade of events culminating in HSV reactiva-
tion from latency (figure 1).
Primary HSV-1 infection, ganglionic latency, and virus reac-
tivation can occur in mice under appropriate conditions, making
the murine keratitis model of HSV-1 infection a good analogue
to human ocular and perioral HSV infections. Induction of HSV
ocular reactivation can be achieved in animals by systemic
epinephrine injection, corneal epinephrine iontophoresis, cor-
neal exposure to UV light, whole-body hyperthermia, and cold
stress [27, 28]. Spontaneous ocular HSV shedding in animals
can be partially suppressed by frequent administration of sys-
temic but not topical b-blockers, suggesting that systemic ad-
ministration of specific blocking agents is necessary to prevent
reactivation of ocular HSV [29 – 31].
The hyperthermia- and UV light – induced mouse models of
ocular HSV-1 reactivation were chosen for our studies because
spontaneous reactivation seldom occurs, and murine immuno-
logic reagents are more readily available than those for other
species. To determine whether IL-6 could be involved in HSV
reactivation, a potent anti-murine IL-6 monoclonal antibody
was administered to latently infected mice, and its effect on
the efficiency of subsequent HSV reactivation was assessed
[32, 33].
Materials and Methods
Mice and corneal infection procedures. Pathogen-free, 4- to
6-week-old, female BALB/c mice were used in all experiments.
In animals subjected to hyperthermic stress, both corneas of anes-
thetized mice were scarified in a cross-hatch pattern and inoculated
with 5 1 103 pfu of HSV-1 McKrae strain. HSV-1 infection was
documented by slit-lamp examination and by tissue culture of
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JID 1997;175 (April)
Figure 1. Hypothesized role of IL-6 in HSV reacti-
vation.
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corneal swabs taken 3 and 7 days after inoculation. Animals whose
corneas failed to show dendritic or geographic ulcers or whose
ocular surface failed to yield infectious virus were excluded from
study. Animals were housed for 40 days to recover from acute
HSV-1 keratitis and for viral latency to be established.
Twenty anesthetized mice subsequently subjected to corneal ex-
posure to UV light were infected by applying 106 pfu of HSV-1
McKrae strain directly to the scarified left corneal surface [4].
These animals were pretreated intraperitoneally (ip) with 0.5 mL
of human gamma globulin (Gammar; Armour Pharmaceuticals,
Collegeville, PA) diluted 7.5 fold in PBS. Corneal swabs taken 3
days after the initial infection and placed in tissue culture con-
firmed that HSV infection was established in all animals. The
animals were allowed 35 days to recover from acute infection,
after which viral latency was established. At that time, the animals’
left corneas were swabbed and examined by polymerase chain
reaction (PCR) for HSV DNA [34]. Eighteen mice with HSV-
negative corneal specimens were used in the subsequent UV light–
induced ocular reactivation study.
Antibody preparation and mouse injections. The MP5-20F3
hybridoma cell line produces rat IgG1, which neutralizes murine–
IL-6 [32]. Five liters of supernatant from MP5-20F3 hybridoma
cells was concentrated 10 times using a 30-kDa filter (Amicon,
Beverly, MA). Contaminating proteins, but not IgG1, were re-
moved by caprylic acid precipitation, and the supernatant was
concentrated again using a 30-kDa filter. Retained proteins and
immunoglobulin were dialyzed against 10,000 vol of PBS, using
tubing with a 50-kDa cutoff (Spectrum, Houston), reconcentrated
with a 30-kDa filter, and dialyzed again in an identical fashion. A
Bradford protein assay showed that the final anti–IL-6 antibody
concentration was 10 mg/mL. Experimental mice were injected ip
with 5.0 mg/0.5 mL of this preparation. An equivalent volume and
quantity of rat immunoglobulin (Jackson Immunoresearch, West
Grove, PA) was concurrently injected into control mice.
Reactivation stimuli and detection of HSV. Hyperthermic
stress was induced by submerging latently infected mice up to
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JID 1997;175 (April) Anti–IL-6 Antibodies Inhibit HSV Reactivation
their necks in 427C water for 10 min [3]. Eye swabs were obtained
daily from these mice for 4 days after hyperthermia, and the sur-
rounding medium was inoculated onto Vero cell monolayers. The
monolayers were observed for 21 days, and HSV was detected
by characteristic cytopathic effect. Tissue culture readings were
blinded, and positive cultures were confirmed by subculturing.
The left corneas of anesthetized, latently infected mice were
exposed to 600 mJ/cm2 UV B (UVB) light emitted from a gel box
[4]. A Dacron swab dipped in virus isolation medium was gently
touched to the left corneal surface daily for 4 days after UV light
exposure. The specimens were digested and extracted, and the
DNA was precipitated according to the method of Cone et al. [34].
Thirty-two amplification cycles were performed using a single set
of HSV type common gB primers in a thermal cycler (GeneAmp
9600; Perkin-Elmer Cetus, Norwalk, CT) under the following con-
ditions: 1.5 mM MgCl2 , denaturation at 947C for 15 s, annealing
at 637C for 15 s, and extension at 727C for 30 s. The 488-bp
amplification product was isolated using a 2% agarose electropho-
resis gel containing 1 mg/mL ethidium bromide.
Ocular cytokine and cytokine mRNA determinations. Groups
of 3 uninfected animals were sacrificed before, 2 h after, or 4 h
after exposure to 600 mJ/cm2 UVB light. Half the animals were
pretreated with 5.0 mg of ip anti–IL-6 antibodies, and the other
half received 5.0 mg ip control rat immunoglobulin 8 h before UV
light exposure. The eyes were removed, rinsed in saline, and stored
at 0707C. One eye was used for cytokine ELISA, and the other
for reverse transcriptase PCR. Immunologic reagents were ob-
tained from Pharmingen (San Diego) unless specified otherwise.
The ocular specimens were thawed, and cytokine ELISAs were
done as previously described [35, 36]. Coating antibodies were
anti–IL-6 MP5-20F3 (10 mg/mL; prepared in-house as described
above) and anti–tumor necrosis factor (TNF)-a MP6-XT3 (2 mg/
mL). Biotinylated monoclonal anti–IL-6 MP5-32C11 (0.5 mg/mL)
and biotinylated polyclonal rabbit anti–TNF-a (0.5 mg/mL) were
used as detecting antibodies. Standard curves were established
using 78–10,000 pg/mL recombinant murine TNF-a and 4–500
pg/mL of recombinant murine IL-6. Sensitivities of the assays
were Ç300 pg/mL for TNF-a and 4 pg/mL for IL-6.
The frozen ocular specimens were homogenized with a mor-
tar and pestle before RNA extraction and reverse transcription
[37]. Primers specific for b-actin (5*-ATGGTGGGAATGGGT-
CAGAAG-3*; 5*CACGCAGCTCATTGTAGAAGG-3*), IL-6
(5*-GAAATGAGAAAAGAGTTGTGC-3*; 5*-CACTAGGTT-
TGCCGAGTAGAT-3*), and TNF-a (5*-CCTCTTCTCATTCCT-
GCTTGT-3*; 5*-CACTTGGTGGTTTGCTACGAC-3*) were
used. Twenty (b-actin), 30 (TNF-a), or 35 (IL-6) amplification
cycles were performed in a thermal cycler (GeneAmp 9600; Per-
kin-Elmer Cetus) under the following conditions: 1.5 mM MgCl2 ,
denaturation at 947C for 15 s, annealing at 507C (TNF-a) or 557C
(b-actin, IL-6) for 15 s, and extension at 727C for 30 s. Amplifica-
tion products were detected using a 2% agarose electrophoresis
gel containing 1 mg/mL ethidium bromide.
Statistical analysis. In experiments in which both eyes were
infected with HSV-1 (hyperthermic stress model), isolation of vi-
rus from individual eyes could not be treated as independent
events. Logistic regression analysis for correlated data was used
to obtain a P value corrected for nonindependence in these experi-
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823
ments [38]. Fisher’s exact and the x2 trend tests were used to
compare groups when the events were independent.
Results
Hyperthermia has been demonstrated to rapidly induce virus
reactivation within neurons of the trigeminal ganglion in mice
latently infected with HSV-1. A marked reduction in the rate of
HSV isolation from the eyes was seen among latently infected
BALB/c mice pretreated with 2.0 mg of anti – IL-6 antibodies
compared with those that received 0.2 mg of anti – IL-6 antibod-
ies (P Å. 04) or 2.0 mg of control rat immunoglobulin (P õ
.001) (figure 2A). To obtain a fully independent measure of
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HSV reactivation for each animal, the data were reanalyzed by
scoring whether HSV was isolated from either eye on a given
day. The association between the administration of anti – IL-6
antibody and a reduced rate of HSV isolation was confirmed
in this reanalysis (P Å .01, .03, and .20 for days 1, 2, and 3,
respectively; x2 test for trend) (figure 2B).
Another experiment was done using the UV light – induced
model of HSV reactivation, with HSV detection at the ocular
surface by PCR. Ten latently infected mice were treated with
5.0 mg of anti – IL-6 antibodies, and 8 control mice received 5.0
mg of control rat immunoglobulin 8 h before ocular exposure to
UV light. The animals were anesthetized, and their left corneas
were exposed to 600 mJ/cm2 of UVB light. The left cornea of
each animal was gently swabbed 1, 2, and 4 days after UV
light exposure. Two (20%) of 10 animals that received anti –
IL-6 antibodies, compared with 6 (75%) of 8 controls, had
HSV detected by PCR at the corneal surface after the UV light
stimulus (P Å .05) (figure 3).
The possibility that these antibody preparations contained
other substances that inhibit in vitro HSV replication was as-
sessed. Vero cell monolayers were incubated overnight with
three different concentrations of anti – IL-6 or control rat anti-
bodies. The monolayers were then infected with identical ali-
quots containing Ç70 pfu of HSV-1 and overlaid with agarose
containing identical concentrations of these antibodies (table
1). Five days after tissue culture inoculation, the number of
HSV-1 in IL-6 – treated groups was not significantly reduced
compared with numbers in controls maintained in medium
alone. We conclude that neither of the antibody preparations
used in our studies contained significant nonspecific antiviral
activity.
IL-6 is part of a well-described cascade of inflammatory
cytokines, which includes IL-1 and TNF-a. To determine
whether the UV light exposure procedure used in the preceding
experiments induced measurable levels of IL-6 or TNF-a
mRNA in the eye, reverse transcriptase PCR was done on
ocular explants from mice 2 and 4 h after UV light exposure.
Ocular IL-6 message was induced by UV light exposure, re-
gardless of whether animals were treated with anti – IL-6 or
control antibodies (table 2). TNF-a mRNA was detected in
UC: J Infect
824 Kriesel et al.
all ocular specimens, regardless of antibody pretreatment and
whether the eyes were exposed to UV light.
TNF-a can inhibit the replication of viruses, including HSV
[39 – 41]. To detect possible alterations in TNF-a or IL-6 (pro-
tein) levels following anti – IL-6 antibody treatment, the effects
of these antibodies on serum and ocular cytokine expression
were investigated in mice. Serum was collected from mice 2
and 4 h after hyperthermic stress, and ocular tissue was col-
lected 2 and 4 h after corneal UV light exposure. IL-6 and
TNF-a were not detected in the serum of untreated animals
after hyperthermic stress, nor were these cytokines detected in
mouse ocular specimens after UV light exposure (table 2).
Animals pretreated with anti – IL-6 or control rat antibodies,
followed by ocular UV light exposure, still had undetectable
levels of ocular IL-6 and TNF-a 2 and 4 h later (table 2). These
studies showed that UV light exposure specifically induced IL-
6 mRNA in ocular tissue, expression of TNF-a and IL-6
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JID 1997;175 (April)
Figure 2. Ocular HSV detection among latently in-
fected mice following hyperthermic stress. A, eyes.
Groups of 10 latently infected BALB/c mice were
pretreated intraperitoneally with 0.2 mg of anti – IL-
6, 2.0 mg of anti – IL-6, or 2.0 mg of control rat immu-
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noglobulin. Results are % of HSV culture – positive
eyes (n Å 20/group) on each of days 1 – 4 after hyper-
thermia. Isolation of HSV was significantly less fre-
quent among animals that received 2.0 mg of anti –
IL-6 antibodies compared with controls (P õ .001)
or those treated with 0.2 mg of anti – IL-6 antibodies
(P Å .04). B, mice. Analysis of same experiment,
shown as % of culture-positive mice (n Å 10/group)
on each of days 1 – 4 after hyperthermic stimulus. Mice
were determined to be culture positive if HSV was
detected from either eye on a given day. Difference
between the groups was observed 1, 2, and 3 days
after hyperthermic stress (P Å .01, .03, and .20, for
days 1 – 3, respectively; x2 test for trend).
mRNAs were not eliminated by treatment with anti – IL-6 anti-
bodies, IL-6 and TNF-a were undetectable in the sera of mice
after hyperthermic stress and undetectable in explanted mouse
eyes after UV light exposure, and anti – IL-6 antibody pretreat-
ment did not induce detectable levels of TNF-a in ocular speci-
mens after UV light exposure.
Discussion
We conclude that systemic administration of anti – IL-6 anti-
bodies significantly reduced the frequency of virus detection
at the corneal surface in 2 different animal models of HSV-1
ocular reactivation. Our data support the hypothesis that IL-6
plays an important role in the interaction between environmen-
tal stimuli and HSV reactivation. We propose that tissue dam-
age creates an environment favorable for the replication of
HSV and that IL-6 ‘‘signals’’ the neuron latently infected with
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JID 1997;175 (April) Anti–IL-6 Antibodies Inhibit HSV Reactivation 825

Table 2. Results of ocular cytokine and cytokine mRNA determina-

tions among groups of 3 mice each before and 2 or 4 h after exposure
to 600 mJ/cm2 of ultraviolet B light (UV).

TNF-a:mRNA/
IL-6:mRNA/protein protein

Pretreatment group Pre-UV Post-UV Pre-UV Post-UV

Anti – IL-6 (5.0 mg) 0/0 //0 //0 //0

Control antibody (5.0 mg) 0/0 //0 //0 //0

Figure 3. Representative gel showing corneal swab amplification NOTE. Data represent detectable levels of mRNA/cytokine. Ocular IL-6
products. Lanes 1 – 10: corneal swab specimens from animals pre- mRNA was induced by UV exposure, while tumor necrosis factor (TNF)-a
treated with 5.0 mg of anti – IL-6 antibodies. Lanes 12 – 19: animals message was produced constitutively. Ocular IL-6 and TNF-a protein levels
pretreated with 5.0 mg of control rat immunoglobulin. Lanes 11 and were undetectable by ELISA before and after UV exposure, regardless of

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20: medium control specimens. Positive control specimen (/) was whether mice were pretreated with anti – IL-6 or control antibodies. /, present;
0, absent.
derived by extracting and precipitating aliquots of HSV-1 McKrae
strain cultured in vitro. Negative control specimen (0) excludes con-
tamination of the polymerase chain reaction mix. MW, molecular
weight.
failed to induce high levels of reactivation, suggesting that
depletion of NGF alone may be insufficient to induce virus
reactivation in vivo [45]. Of note, subconjunctival saline injec-
HSV that such a situation has occurred. Studies directly exam- tion alone, presumably a local inflammatory stimulus, induced
ining the roles of gp130, the IL-6 receptor, and IL-6 itself HSV reactivation in this study. Latently infected trigeminal
in HSV reactivation will be necessary to fully explore this ganglion explants treated in vitro with TNF-a reactivated more
hypothesis. frequently than did untreated controls [46], raising the possibil-
Reagents that block or directly augment the activity of mu- ity that this cytokine may also play a role in HSV reactivation
rine gp130 are not currently available. Targeted disruption of or that TNF-a induced IL-6 production in these cultures, lead-
the murine gp130 gene is lethal [42]. The murine IL-6 receptor ing to virus reactivation. Once virus reactivation begins, syn-
has been blocked by raising polyclonal antibodies in guinea thesis of IL-6 by productively infected cells could amplify
pigs [43], but monoclonal anti-murine IL-6 receptor antibodies reactivation [47].
are not available. In vitro HSV latency model studies have Recombinant human IL-6 (rhIL-6; Sandoz Pharmaceuticals,
shown that removal of nerve growth factor (NGF) from the East Hanover, NJ) has been tested for antitumor activity in
culture medium facilitates reactivation [44]. However, multiple humans with advanced malignancies. Subcutaneous doses (1 –
injections of latently infected mice with anti-NGF antibodies 30 mg/kg) of rhIL-6 raised serum IL-6 levels up to 3 ng/mL
and reliably induced fever [48, 49]. These serum IL-6 levels
are similar to those seen in human subjects exposed to doses
Table 1. Results of plaque inhibition assay for nonspecific antiviral of whole-body UV irradiation sufficient to induce sunburn.
activity of anti – IL-6 and control rat antibody preparations. Induction of HSV reactivation was not specifically investigated
in these reports, but would be of interest in light of our findings.
Treatment, concentration (mg/mL) No. of plaques P*
The procedure used to prepare the anti – IL-6 monoclonal
Anti – IL-6 antibodies for use in our studies virtually precludes the presence
2.0 54 { 1 .66 of interferons. Nonspecific antiviral activity of the anti – IL-6
0.2 46 { 3 .18 or control immunoglobulin preparations was not observed in
0.02 65 { 8 .49 a plaque inhibition assay. Treatment of animals with anti – IL-
Rat immunoglobulin
6 antibodies or control rat immunoglobulin did not differen-
1.0 63 { 2 .65
0.1 60 { 3 .87 tially alter the expression of IL-6 or TNF-a ocular message
0.01 61 { 5 .74 after UV light exposure. Measurable levels of TNF-a, a cyto-
Medium control 58 { 5 — kine with direct antiviral activity, were not induced by anti –
IL-6 antibody treatment. However, a more sensitive technique
NOTE. Data are mean { SE. Vero cells were incubated overnight with
0.01 – 2.0 mg/mL anti – IL-6 or rat control antibody preparations, inoculated for the detection of murine TNF-a would be helpful in future
with HSV-1, and overlaid with medium containing identical antibody concen- studies to exclude a reciprocal increase in levels of this cyto-
trations. Plaques were counted 5 days later by blinded observer. No significant kine. A specific effect of anti – IL-6 antibodies on the expression
antiviral activity was observed in any group, compared with medium controls.
* Compared with medium control values, determined by unpaired t test for of other cytokines cannot be excluded by these experiments.
continuous variables. Cross-reactivity of the anti – IL-6 antibodies with other cyto-

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826 Kriesel et al.
kines, including the related cytokines CNTF, IL-11, oncostatin
M, and leukemia inhibitory factor, also cannot be excluded.
Although the expression of TNF-a mRNA may be inducible
in ocular tissues under some circumstances [50], the constitu-
tive production of TNF-a mRNA demonstrated in our analysis
of ocular tissues is well described in the literature, with control
over TNF-a protein production usually exerted at a posttran-
scriptional level [51]. By contrast, the induction of IL-6 mRNA
after ocular UV light exposure suggests that this cytokine is
produced locally within the eye in response to this stimulus
but that IL-6 itself may be present only transiently or below
the threshold of ELISA detection. This interpretation is sup-
ported by the findings of Kilgus et al. [52] in a study in which
a damaging stimulus to the skin resulted in localized IL-6
mRNA synthesis and specific IL-6 bioactivity. Immunohisto-
chemical techniques may be helpful to demonstrate IL-6 and
TNF-a in UV light – exposed eyes. Similarly, the lack of detect-
able IL-6 and TNF-a in the animals’ sera after the hyperthermic
stimulus could be explained by cytokine production below the
threshold of the detection assays, interference with ELISA de-
tection due to cytokine-receptor binding, rapid clearance of the
cytokines, or transient production of these cytokines.
The apparent reduction in the frequency of HSV reactivation
seen after administration of anti – IL-6 antibodies could be spe-
cific or might also occur when other cytokines, such as IL-1,
TNF-a, or IFN-g, are blocked. Given the complex and often
synergistic nature of cytokine activities in biologic responses,
several cytokines and other substances may be involved in
induction or repression of HSV reactivation in vivo. Investiga-
tion of HSV reactivation in cytokine knockout mice might be
revealing, but the establishment of HSV latency in genetically
deficient mice could be problematic, and subsequent HSV reac-
tivation studies would be subject to the compensatory effects
of one cytokine for another often seen in these models [53]. The
studies described here do not distinguish between the effects of
IL-6 itself and those of proteins induced by IL-6, including the
acute-phase proteins. However, the systemic effects of the
acute-phase proteins contrast with the local production and
activity of IL-6, a more logical candidate to facilitate neuronal
HSV reactivation after tissue injury.
Acknowledgments
We thank Motomi Mori for her statistical expertise and assis-
tance and T. A. Dowell for expertise in preparing monoclonal
antibodies.
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