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* Department of Microbiology, University of Tennessee, Knoxville; and
Department of Immunology, Cleveland Clinic Foundation, Ohio
Correspondence: Dr. Barry T. Rouse, Department of Microbiology, M409 Walters Life Sciences Building, University of Tennessee, Knoxville, TN 37996-0845. E-mail: btr{at}utk.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and one or
both of two chemokine molecules, IP-10 and MIG. Thus IL-12 DNA
administration lacked modulatory effects on HSK in GKO mice, indicating
the necessary involvement of IFN- induction for antiangiogenesis. In
contrast, exposure of GKO mice to IP-10 DNA did suppress the severity
of HSK. Furthermore, treatment with specific antisera to IP-10 and MIG
in HSV-infected mice abrogated the IL-12-induced inhibitory effect on
lesion severity. Taken together, our data indicate that the HSV-induced
ocular immunoinflammatory lesions can be modulated by IL-12 and that
this effect results from chemokine inhibition of angiogenesis. The use
of antiangiogenesis therapy might represent a useful control measure
against HSK.
Key Words: herpetic stromal keratitis • chemokine • cytokine • immunopathology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence in data implicates CD4+ T cells of the type-1
phenotype as the mediator of HSK [3
, 4
].
Moreover pretreatment with type-2 cytokines can suppress HSK severity
[5
, 6
]. In addition, treatment with the
cytokine interleukin (IL)-10, before the full clinical phase develops,
can result in lesion suppression or even resolution [5
].
HSV infection itself results in the production of a notable IL-12
cytokine response [7
], which presumably helps set the
stage for the type-1 T-cell-mediated CD4+ inflammatory
reaction. We anticipated that if animals were exposed to IL-12 prior to
infection, this would potentiate type-1 CD4+ T-cell
responses further and result in more severe HSK lesions. Unexpectedly,
however, ocular exposure to plasmid DNA encoding IL-12 resulted in
diminished rather than exacerbated stromal lesions. Conceivably, the
lesion-modulating effects of IL-12 could be explained by the induction
of antiviral cytokines such as interferon (IFN)- or the induction of
cellular defenses such as natural killer (NK) cells known to exert
protective effects against HSV infection [8
]. In
addition, IL-12 has been shown to express immunosuppressive effects on
CD4+ T-cell priming by inducing IFN- and inducible
nitric oxide synthase (iNOS) activation, which in turn causes T-cell
apoptosis [9
]. Furthermore, IL-12 may inhibit a tumor,
seemingly mediated by an antiangiogenic effect
[10
].
As we have documented elsewhere, angiogenic sprouting into the normally
avascular cornea appears to be an essential event in the pathogenesis
of HSK [11
, 12
]. This process appears as a
necessary prelude to invasion by the CD4+ T cells, which
drive the inflammatory reaction [11
]. The present study
adds support to the notion that IL-12 is antiangiogenic and that the
inhibitory effect of IL-12 on HSK lesion severity proceeds by an effect
on angiogenesis rather than by antiviral or suppressive effects in
T-cell function. As observed with some tumor systems, the
antiangiogenic effect of IL-12 appeared to act indirectly by inducing
IFN-, which in turn caused the expression of two antiangiogenic
factors, IFN-inducible protein-10 (IP-10) and monokine induced by
IFN- (MIG) [13
]. Accordingly, our results demonstrate
that administration of IL-12 DNA to normal but not IFN- -/- mice
caused up-regulation of two cytokines, IP-10 and MIG, in the cornea.
These inhibited HSV-induced angiogenesis and consequently the severity
of HSK. Our results are discussed in terms of novel approaches that
merit testing to control HSK lesions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-/-) mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). GKO mice contain a nonfunctional IFN-
gene and are on a BALB/c background. BALB/c and B6 mice were housed
conventionally, and KO mice were housed in sterile microisolator cages
in the animal facility. All manipulations were performed in a laminar
flow hood. To prevent bacterial infection, all mice received treatment
with sulfamethoxazole/trimethoprim (Biocraft, Elmond Park, NY) at the
rate of 5 ml/200 ml drinking water. All investigations followed
guidelines of the Committee on the Care of Laboratory Animals
Resources, Commission of Life Sciences, National Research Council. The
animal facilities of the University of Tennessee (Knoxville) are fully
accredited by the American Association of Laboratory Animal Care.
Virus
HSV-l strain RE (kindly provided by Dr. Robert Lausch,
University of Alabama, Mobile) was used in all procedures. Virus was
grown in Vero cell monolayers (American Type Culture Collection,
Manassas, VA; Cat. no. CCL81 ), titrated, and stored in aliquots at
-80°C until used.
Plasmid DNA preparation
Plasmid DNA encoding murine IL-12 was kindly provided by Dr.
Kenji Okuda (Yokohama City University School of Medicine, Japan).
Plasmid DNA encoding murine IP-10 was constructed by polymerase chain
reaction (PCR) amplification of the full-coding region. All plasmid
DNAs used in this work were inserted into the pCDNA3 expression vector
(Invitrogen, San Diego, CA). The plasmid DNAs were purified by
polyethylene glycol precipitation by the method of Sambrook et al.
[14
] with some modifications. The quality of DNA was
measured by electrophoresis on 1% agarose gel. The protein expression
of the different plasmids was determined by reverse transcriptase
(RT)-PCR and dot blot after in vitro transfection into Chinese hamster
ovary (CHO) cells.
Corneal HSV infection
Corneal infections of all mouse groups were conducted under deep
anesthesia induced by the inhalant anesthetic methoxyfurane (Methofane;
Pittman Moore, Mondelein). The mice were scarified lightly on their
corneas with a 27-gauge needle, and a 2.5 µl drop containing 1 x 106 plaque-forming units (PFU) of HSV-1 RE for BALB/c,
1 x 107 PFU of HSV-1 RE for B6 and iNOS KO mice, and
1 x 104 PFU of HSV-1 RE for GKO mice was applied to
the eye and gently massaged with the eyelids.
Clinical observations
The eyes were examined on different days after infection for the
development of clinical lesions by slit-lamp biomicroscopy (Kawa Co.,
Nagoya, Japan), and the clinical severity of keratitis of individually
scored mice was recorded. The scoring system was as follows: 0, normal
cornea; +1, mild corneal haze; +2, moderate corneal opacity or
scarring; +3, severe corneal opacity but iris visible; +4, opaque
cornea and corneal ulcer; +5, corneal rupture and necrotizing stromal
keratitis. The severity of angiogenesis was recorded as described
previously [12
]. According to this system, a grade of 4
for a given quadrant of the circle represents a centripetal growth of
1.5 mm toward the corneal center. The score of the 4 quadrant of the
eye was then summed to derive the neovessel index (range 0–16) for
each eye at a given time point [12
].
Virus recovery and titration
Swabs of the corneal surface were collected at various time
points post-infection. The swabs were put into sterile tubes containing
500 µl Dulbecco’s modified Eagle’s medium with 10 international
units (IU) penicillin/ml and 100 µg streptomycin (Life Technologies,
Grand Island, NY)/ml and were stored at -80°C. For detection and
quantification of virus in the swabs, the samples were thawed and
vortexed. Individual subsamples (200 µg each sample) were
diluted further, and viral titers were determined by a plaque assay
performed on Vero cells as described elsewhere [15
].
Plasmid DNA administration
Plasmid DNA (100 µg) was suspended in 4 µl sterile
phosphate-buffered saline (PBS). Corneas were scarified using a
27-gauge needle in a criss-cross pattern, and the plasmid was
administered intraocularly on 6 and 3 days before virus infection.
HSV-specific lymphoproliferation
This assay has been described in detail elsewhere
[16
]. Briefly, at day 15 following HSV ocular infection,
the splenocytes of vector, IL-10, or IL-12 DNA-treated mice were
enriched for T cells by a nylon wool column and used as responder
populations. These T cells were restimulated in vitro with irradiated
syngeneic-enriched, naïve dendritic cell (DC) or with DC
infected with UV-inactivated HSV [multiplicity of infection (MOI) of
1.5 before UV inactivation] and were incubated for 5 days at 37°C.
Concanavalin A (Con A; 5 µg/ml) was used as a polyclonal-positive
control and incubated for 3 days. Eighteen hours before harvesting,
[3H] thymidine was added to the cultures.
Cytokine assay
For cytokine (IFN-) assay, splenocytes from mice were
suspended in 10% RPMI-1640, and 106 cells in 1 ml were
stimulated in vitro with irradiated syngeneic-enriched DC pulsed with
UV-inactivated HSV (MOI, 5.0 before UV inactivation). A similar number
of cells were Con A-stimulated (5 µg/106 cells/ml) in
96-well plates. Plates were incubated at 37°C for 72 h. The
supernatant fluid was collected and stored at -80°C until use. These
supernatants were screened for the presence of IFN- by enzyme-linked
immunosorbent assay (ELISA) as described previously
[16
].
Isolation of RNA
At day 3 post-infection, corneas were dissected carefully, freed
of scleral tissues, minced, and homogenized in TRI reagent (Molecular
Research Center, Cincinnati, OH). Total RNA was isolated by the
manufacturer’s protocol. All procedures including RT-PCR were
performed in a laminar flow hood.
RT-PCR
Total cellular RNA (10 µg/ml) was reversed-transcribed using
oligo(dT) primers and RT (Promega, Madison, WI), according to protocols
described previously [17
]. The cDNA was made by the
reverse-transcription reaction incubated at 42°C for 90 min. The cDNA
(2 µl) was subjected to 35 cycles of amplification as described
[5
] using primers. The primers used follow: ß-actin-1,
5'-GTGGGGCGCCCCAGGCACCA-3'; ß-actin-2, 5'-CTCCTTAATGTCACGCACGAT-3';
IFN--1, 5'-ATGAACGCTACACACTGCAT C-3'; IFN--2,
5'-GCAGCGACTCCTTTTCCGCTT-3'; IP-10-1,
5'-ACCATGAACCCAAGTGCTGCCGTC-3'; IP-10-2, 5'-
GCTTCACTCCAGTTAAGGAGCCCT-3'; MIG-1, 5'-ACTCAGCTCTGCCATGAACTCCGC-3';
MIG-2, 5'-AAAGGCTGCTCTGCCAGGGAAGGC-3'; IL-10-1,
5'-ATGAAATATACAAGTTATATC-3'; IL-10-2, 5'-TTAGCTTTTCATTTTGATCAT-3'. The
PCR products were separated by agarose gel electrophoresis.
Preparation and administration of antibodies (Abs)
Rabbit polyclonal Abs to IP-10 and MIG were produced by
Biosynthesis (Lewisville, TX) using synthetic peptides selected from
the IP-10 and MIG protein sequences (CIHIDDGPVRMRAIGK and
CISTSRGTIHYKSLKDLKQFAPS) coupled to carrier-protein keyhole limpet
hemocyanin. Mice were given 250 µl antibodies intraperitoneally
(i.p.) to IP-10 and/or MIG 1 day before infection and 3 days after
infection.
Corneal micropocket assay
In vivo angiogenic activity was assayed in the avascular cornea
of BALB/c mouse eyes, as described previously [18
].
Briefly, mice were pretreated with plasmid DNA encoding IP-10 twice
intraocularly before implantation. Pellets for insertion into the
cornea were made by combining recombinant human (rh) vascular
endothelial growth factor (VEGF)-165 (40 µg; R&D Systems,
Minneapolis, MN), sulcralfate (10 mg; Bulch Meditec, Vaerlose,
Denmark), and hydron polymer in ethanol (120 mg/1 ml ethanol;
Interferon Sciences, New Brunswick, NJ) and by applying the mixture to
a 15 x 15mm2 piece of synthetic mesh (Tetko, Depew,
NY). The mixture was allowed to air-dry, and fibers of the mesh were
pulled apart, yielding pellets containing 90 ng VEGF. Pellets
containing rhVEGF were implanted into an intracorneal pocket (1 mm from
the limbus), after which the eyes were evaluated for corneal
neovascularization. The extent of the neovessel in-growth was recorded
by direct measurement using calipers (Symbol of Quality, Biomedical
Research Instruments, Rockville, MD) under stereomicroscopy. The number
of vessels originating from the limbus was counted over the entire
orbit, and the area of angiogenesis was calculated according to the
formula for an ellipse. A = [(clock hours)x0.4x(vessel length
in mm)x
]/2. Each clock hour is equal to 30° at the circumstance.
Statistical analysis
Significant differences among groups were evaluated using the
Student’s t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression
in the cornea, which in turn could be antiviral. As documented in a
later section, exposure of the cornea to IL-12 DNA did result in
IFN- mRNA expression. However, measurement of the duration and
concentration of virus in ocular washing at various times after
infection in IL-12 and vector-treated mice revealed no significant
differences (Fig. 2
). A second explanation for the inhibitory effect of IL-12 DNA was
that IL-12 could exert immunosuppressive effects on CD4+
T-cell priming, as demonstrated in some other systems
[9
]. As shown in Figure 3
, evidence for any such immunosuppression was not obtained.
Accordingly, measurement of HSV-specific proliferative (mainly a
function of CD4+ T-cell function; P=0.3) and
cytokine production of splenocytes at 15 days post-infection
(P=0.4) revealed no evidence of immunosuppression. In
contrast, pretreatment with IL-10 DNA did result in diminished
HSV-specific proliferative responses.
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induction
[19
, 20
]. Other activities such as
immunosuppression may be the consequence of iNOS induction
[9
, 21
, 22
]. To test the role
of IFN- and iNOS activity during the IL-12-induced inhibition of
HSK, the effects of IL-12 pretreatment were compared in ocularly
infected normal and KO mice. Figure 5 C
records the inhibitory effects of IL-12 DNA pretreatment in
BALB/c and IFN- -/- mice. Whereas IL-12 pretreatment inhibited the
severity of HSK in BALB/c mice, the effect was not evident in GKO mice
(P=0.7). In the latter mice, animals were infected with
102 less virus than given to BALB/c mice, because GKO mice
die of encephalitis if infected with the virus doses used to produce
HSK in BALB/c mice [23
]. To measure the influence of
iNOS expression, B6 mice and B6 background iNOS -/- mice were used.
Both groups of mice were IL-12 or vector DNA-treated prior to HSV
infection, and the severity of HSK lesions was compared. Figure 5A
and 5B
, shows that IL-12 DNA pretreatment resulted in diminished
HSK-lesion severity in B6 and iNOS -/- mice, and the IL-12 inhibitory
effect of IL-12 was even more apparent in the iNOS -/- mice. These
data indicate that the anti-inflammatory effect of IL-12 does not
likely proceed via NO production, as has been shown to occur in some
other systems [9
, 22
].
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production, which in turn induced IP-10 and MIG antiangiogenic
factors [13
, 24
]. The modulating effect of
IL-12 DNA pretreatment on HSK and corneal angiogenesis would also seem
to involve a similar mechanism. Thus, as shown in Figure 6
, topical administration of IL-12 DNA led to increased expression
of mRNA for IFN- as well as for IP-10 and MIG. There was no
up-regulation of IL-10 mRNA, another possible mediator of HSK
suppression as shown in previous work [5
,
6
]. The up-regulation of IP-10 and MIG mRNA appeared to
be the consequence of IFN- expression, because measurement of IP-10
and MIG mRNA in IL-12 DNA-treated GKO corneas revealed no detectable
IP-10 and MIG mRNA expression.
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-induced IP-10
and MIG expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and one or both of two chemokine
molecules IP-10 and MIG. Thus, IL-12 DNA administration had no
modulatory effect on HSK in GKO mice, indicating the necessary
involvement of IFN- induction. In contrast, exposure of GKO mice to
IP-10 DNA did suppress the severity of HSK. Furthermore, suppression
with specific antisera of IP-10 and MIG expression in HSV-infected mice
abrogated the IL-12-induced inhibitory effect on HSK. Taken together,
our results indicate that the ocular immunoinflammatory lesion that
results from HSV infection can be modulated by IL-12 and that this
effect results from chemokine inhibition of angiogenesis.
As first shown by our group [5
] and confirmed by others
[28
], the surface application of plasmids encoding
various proteins is a convenient means of expressing molecules in the
cornea. By such an approach, we showed that pre-exposure to T-helper
cell type 2 (Th2) chemokines, such as IL-4 and IL-10, reduced HSK
lesions caused by HSV infection. Such lesions are immunoinflammatory,
and CD4+ T cells produce type-1 cytokines, such as IFN-
and IL-2, the principal mediators of the inflammation
[4
]. During lesion resolution, Th2 cytokines may
predominate [5
]. Initial experiments with IL-12 DNA
administered to the eye were done in an attempt to exacerbate lesions
and to facilitate disease induced by nonvirulent mutant viruses.
Surprisingly, however, lesions were diminished rather than exaggerated.
Because IL-12 induces IFN-, as shown to occur in the cornea in the
present study, a logical explanation for the modulatory effect on
HSK-lesion severity was the antiviral activity of IFN-. Indeed,
clearance of HSV infection from peripheral sites often appears to be a
correlate of IFN- production [29
, 30
].
In line with this observation, animals unable to produce IFN- (GKO
mice) are markedly more susceptible to HSV infection
[23
]. Nevertheless, such mice can still express HSK, as
long as they are infected with lower, nonlethal doses of virus
[23
]. In our model, although IL-12 DNA resulted in
IFN- expression, this appeared inadequate to curtail viral
replication. Thus, in IL-12 DNA-treated mice, levels and duration of
viral expression were almost identical to those in control vector
DNA-treated animals.
The HSK lesions are mediated principally by CD4+ T cells, although the identity of antigens that drive these cells has yet to be identified. Conceivably, the inhibitory effects of IL-12 administration might result from immunosuppressive effects of IL-12 on CD4+ T-cell priming. In fact, such a mechanism was advocated to explain the inhibitory effects of IL-12 on CD4+ T-cell-mediated uveitis [9 ]. In this instance, suppression was mediated by up-regulation of iNOS, which led to NO production. The latter interfered with Bcl 2-regulated apoptosis in developing CD4+ effector cells [9 ]. Such a mechanism appeared not to be the explanation for our observations. Thus, the HSV-specific CD4+ T-cell responses appeared normal in IL-12-treated mice. In addition, the inhibitory effect of IL-12 on HSK expression was unimpaired in mice unable to express iNOS because of gene-KO. Indeed, in such mice, the inhibitory effects of IL-12 were even more marked, but we have no explanation for this observation.
The hypothesis favored to explain the inhibitory effect of IL-12 on HSK
lesions was an effect on corneal neovascularization. Accordingly,
ocular HSV infection results in angiogenic sprouting into the normally
avascular cornea [12
]. A molecular explanation for such
events is lacking, but the VEGF family of potent angiogenesis factor
appears as involved [12
]. Angiogenesis appears necessary
during HSV pathogenesis to permit appropriate access of
CD4+ T cells and some other inflammatory components to the
corneal stroma [11
]. In support of such ideas, we have
shown elsewhere that inhibition of angiogenesis with a cytokine that
causes vascular endothelial-cell apoptosis results in diminished HSK
lesions [12
]. The present study demonstrates further the
relationship of angiogenesis and HSK-lesion expression. Thus, we show
that IL-12 pretreatment results in diminished angiogenesis, which
correlated with reduced HSK lesions. The effect of IL-12 appeared as
indirect, and IL-12 served to up-regulate IFN-, which in turn caused
expression of two CXC chemokines, IP-10 and MIG. These latter molecules
are the actual angiogenesis inhibitors. At least for IP-10, using a
corneal micropocket assay, we demonstrated that it could inhibit the
angiogenesis effect of VEGF, a factor involved in HSV angiogenesis
[12
]. Others have also shown the inhibitory effect of
IP-10 against angiogenesis caused by fibroblast growth factor
[31
]. Moreover, it was apparent that inhibition with
specific antisera of IP-10 or MIG (but preferably both simultaneously)
reversed an IL-12-induced effect on angiogenesis and HSK expression.
That IFN- was an essential component of the antiangiogenesis was
supported by observations that the IL-12-induced effect did not occur
in GKO mice. Taken together, our data support the mechanism advocated
to explain the antitumor effect of IL-12 observed in some systems,
namely that IL-12 induces IFN-, which then up-regulates
antiangiogenic chemokines [13
, 25
,
32
].
The molecular mechanism by which IP-10 and MIG inhibit angiogenesis has yet to be established. To this end, studies in the cornea may represent a more convenient and accessible model than those in solid tumors or in vitro systems. Evidence supports the fact that at least two types of receptors can be involved in responses to IP-10 and MIG [33 34 35 ]. These are heparan sulfate proteoglycans (HSPG) as well as CXCR3. Curiously, CXCR3 can be expressed on effector T lymphocytes, and engagement of the receptor by IP-10 or MIG can result in chemotaxis. Such an event could recruit inflammatory T cells to the cornea and conceivably serve to increase the severity of HSK. However, because neovascularization may be necessary to permit invasion by CD4+ T cells, the effect on angiogenesis, likely mediated by HSPG-receptor engagement [35 ], will be dominant. Future therapy of HSK could benefit from targeting angiogenesis receptors such as HSPG. Such issues merit further investigation.
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ACKNOWLEDGEMENTS |
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Received September 26, 2001; revised November 14, 2001; accepted November 16, 2001.
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REFERENCES |
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and IL-2 are protective in the skin but pathologic in the corneas of HSV-1 infected mice J. Immunol. 149,3023-3034[Abstract]
, nitric oxide, and apoptosis J. Exp. Med. 189,219-230 production J. Immunol. 153,1697-1706[Abstract]
production by IL-12 and IL-18 Curr. Opin. Immunol. 10,259-264[Medline]
induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect J. Exp. Med. 9,1603-1610
knockout mice J. Immunol. 155,3964-3971[Abstract]
antagonizes Herpes simplex virus type 1 ocular infection through CD4+ and CD8+ T lymphocytes J. Immunol. 164,6435-6443 in immunity to Herpes simplex virus J. Leukoc. Biol. 60,528-532[Abstract]
-inducible protein 10 (IP-10), a member of the CXC chemokine family, is an inhibitor of angiogenesis Biochem. Biophys. Res. Commun. 210,51-57[Medline]
affect tumorigenicity and response to IL-12 therapy and antiangiogenesis Immunity 9,25-34[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or vector DNA
(•). Three days after the second treatment, animals were infected
with 106 PFU of HSV-1 RE on their scarified corneas and
subsequently scored for lesion severity by slit-lamp biomicroscopy. The
data are compiled from seven independent experiments.
), IP-10 (
), or anti-IP-10 plus anti-MIG (
) 1
day before virus infection and 5 days after virus infection. Groups of
mice received 106 PFU of HSV-1 RE on their scarified
corneas. The mice were examined clinically by slit-lamp biomicroscopy,
and the severity of lesions was scored on a 0-to-5 scale. Data are
compiled from two independent experiments. *, A significant reduction
of the effect of IL-12 DNA in anti-IP-10 plus anti-MIG-treated mice
(P<0.01).