Department of Microbiology and Immunology, School of Medicine, University of South Alabama, Mobile, Alabama
Correspondence: Dr. Robert N. Lausch, Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile, AL 36688. E-mail: rlausch{at}jaguer1.usouthal.edu
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-induced
IL-6 and MIP-2 production by both corneal cell types. Endogenous IL-10
synthesis, which also occurred in vivo, was not modulated
by Herpes virus infection or by depletion of neutrophils or natural
killer cells. Antibody to IL-10 given locally at the time of HSV-1
intracorneal infection was associated with significantly
(p<0.05) enhanced production of IL-6, MIP-2, and MIP-1,
increased neutrophil infiltration, and more extensive corneal disease.
Similarly, mice with a disrupted IL-10 gene developed more severe
corneal disease than wild-type controls. Collectively, these
observations suggest that locally produced IL-10 can act in an
autocrine/paracrine fashion to down-regulate the production of
proinflammatory mediators and thus limit corneal inflammation.
Key Words: cytokines • chemokines • IL-10 knockout mice • IL-10 receptor • corneal fibroblasts • corneal epithelial cells
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of endogenous IL-10 mRNA and protein in the HSV-1-infected cornea has been noted in several studies [13 14 15 ]. However, which cells are producing this cytokine and whether IL-10 helps to regulate the host response in this ocular tissue have not been investigated. The present study was undertaken to test experimentally our hypothesis that endogenous IL-10 is produced by resident corneal cells and participates in an autocrine/paracrine regulatory network to down-regulate corneal inflammation. For these studies, the intracorneal route of infection was chosen because this route elicited a more robust acute-inflammatory response than topical infection.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and reagents
Hamster immunoglobulin G (IgG) monoclonal antibodies (mAbs) to
mouse IL-1 and IL-1ß were obtained from Genzyme (Cambridge, MA).
Rat antimurine IL-4 and IL-13 antibodies, murine recombinant
interferon-
(rIFN-), and biotinylated recombinant IL-10 were
purchased from R&D Systems (Minneapolis, MN). Rabbit antiasialo
antibody was obtained from Wako Bio-Products (Richmond, VA). Dibutyryl
cyclic AMP (Bt2 cAMP) and recombinant murine tumor
necrosis factor (TNF-) were purchased from Sigma Chemical Co.
(St. Louis, MO) and Endogen Inc. (Woburn, MA), respectively. Rat
IgG1 antimurine IL-10 and rat IgG2bRB6-8C5 mAb were prepared as previously described
[1
, 10
].
Intracorneal and subconjunctival inoculations
Intracorneal infection was accomplished by first puncturing the
corneal epithelium wall with a 30-gauge disposable needle. A 30 cm,
32-gauge, stainless-steel needle attached to a Hamilton dispenser
(Hamilton, Reno, NV) was then threaded into the corneal stroma, and 1
µl solution containing the desired inoculum of HSV-1 strain
RE was injected. In some experiments, the 1 µl inoculum
contained a 50:50 mixture of virus and anticytokine antibody.
Subconjunctival antibody injections (5 µl) were performed by using a
32-gauge needle to penetrate the perivascular region of the
conjunctiva. The amount of the specific anticytokine antibody
administered on the basis of in vitro assay was sufficient
to neutralize 40 ng IL-10, 75 ng IL-4, or 25 ng IL-13.
Preparation of corneal fibroblast cultures
Corneal fibroblast cultures were prepared as described
previously [16
]. Briefly, normal corneas removed from
Balb/c mice were minced into small pieces and digested 30–45 min with
300 U/ml of collagenase C-1 889 (Sigma) in a 37°C water bath.
Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand
Island, NY), supplemented with 20% fetal bovine serum (FBS), 2.5%
sodium bicarbonate, 10 mM HEPES buffer solution, and antibiotics, was
added after digestion, and the digested tissue was centrifuged for 5
min at 1000 rpm (250 g). The supernatant was discarded, and
the pellet was placed into a 25 cm2 flask with very little
(
100 µl) medium. At 24 h and again at 48 h, additional
medium (80 µl) was added. At 72 h, 5 ml DMEM with 20% FBS was
added, and the tissue cultures were incubated for 4 days in 5%
CO2 at 37°C. The cultures were then trypsinized and
passed to 75 cm2 flasks.
Corneal epithelial cell preparations
Corneal epithelial cells were prepared as previously described
[16
]. Corneal buttons trimmed to 2 mm were incubated at
37°C in phosphate-buffered saline (PBS) containing 5 mM tetrasodium
ethylenediaminetetraacetate dihydrate (EDTA). After 20 min incubation,
the epithelial cell layer was separated from the adjacent corneal
tissue by gentle teasing with forceps. The epithelial sheets were used
intact or incubated with 0.25% trypsin-EDTA (Life Technologies) at
37°C for 10 min. The trypsin-treated epithelial sheets were passed
gently through a 25-gauge needle three times. The cell suspension was
washed, counted, and resuspended with serum-free RPMI 1640. Viability
of the cells was greater than 95%, as determined by staining with
trypan blue.
Stimulation of corneal epithelial cells and fibroblasts in
vitro
Corneal epithelial sheets (three/tube) and corneal fibroblasts
(1x105 cells/well) were incubated with 500 µl DMEM
[American Type Culture Collection (ATCC), Rockville, MD],
supplemented with 20% FBS and antibiotic-antimycotic (Gibco BRL, Grand
Island, NY). In selected experiments, the two corneal cell types were
incubated with antibody to IL-10, control IgG (10 µg/ml), or the
desired inducer [TNF-, Bt2 cAMP, lipopolysaccharide
(LPS), or IFN-] in 500 µl DMEM with 5% FBS and
antibiotic-antimycotic solution at 37°C under 5% CO2.
After incubation for 24–72 h, the culture medium was removed and
assayed for the desired chemokines and cytokines by enzyme-linked
immunosorbent assay (ELISA).
FACS analysis for IL-10R on murine corneal epithelial cells and
fibroblasts
FlurokineTM kits were purchased from R&D Systems and used
according to the manufacturer’s instructions. Test cells
(1x105/ml) prepared as described above were resuspended in
25 µl PBS. Biotinylated recombinant human (rh)IL-10 (10 µl)
was added, gently mixed, and incubated on ice for 60 min. Cells were
washed twice and resuspended in 25 µl FlurokineTM buffer.
Avidin-fluorescein isothiocyanate (FITC) reagent (10 µl) was added to
each tube, followed by incubation in the dark at 4°C for 30 min. Then
the cells were washed twice and resuspended in 400 µl
FlurokineTM buffer. Receptor expression was determined by flow
cytometric analysis using 488 nm wavelength laser excitation (Becton
Dickinson, Mountain View, CA).
Analysis of mRNA for IL-10 and IL-10R by reverse
transcription-polymerase chain reaction (RT-PCR)
To detect mRNA for IL-10 and IL-10R in corneal cells, RT-PCR was
carried out as described previously [17
]. Briefly, total
cellular RNA was obtained by the acid guanidinium thiocyanate-
phenol-chloroform extraction method. The RNA was fractioned on a 1.0%
agarose gel that contained 2.2 M formaldehyde and then was stained with
1 µg/ml ethidium bromide to test that RNA spectrophotometric
measurements were accurate and that the RNA had not been degraded. The
PCR primers used were as follows: mIL-10 primers: (forward) 5'CCC TGG
GTG AGA AGC TGA AG; (reverse) 5'GGA AGA ACC CCT CCC ATC AT; and mIL-10
R primers: (forward) 5'AGG CAG AGG CAG CAG GCC CAG CAG ATT GCT;
(reverse) 5'TGG AGC CTG GCT AGC TGG TCA CAG TAG GTC. RNA (1 µg) was
reverse-transcribed to cDNA by using a GeneAmp RNA PCR kit
(Perkin-Elmer, Norwalk, CT). The cDNA products were amplified by PCR
(40 or 45 cycles for IL-10R or IL-10; 95°C for 30 sec, 65°C or
58°C for 30 sec, and 72°C for 2 min). Preliminary experiments
established that the cycles run were within the exponential
amplification phase for each product. The identity of the PCR products
was verified by sequencing. RT-PCR of the glyceradehyde-3-phosphate
dehydrogenase gene (GAPD) was performed under the same conditions
described above to confirm equal loading of RNA. Ethidium
bromide-stained PCR products were photographed and analyzed using image
analysis software (Adobe Systems, San Jose, CA).
Chemokine and cytokine assays
MIP-2, MIP-1, and IL-6 protein levels were determined using
ELISA kits purchased from R&D Systems, and IL-10 and IL-1 proteins
were determined using ELISA kits from Endogen Inc. To test for
chemokines and cytokines in HSV-1-infected tissue, corneas were removed
from infected mice at the designated times after infection. The corneas
were placed individually in 500 µl, serum-free RPMI 1640 medium and
stored at -70°C until assayed. Samples were thawed, homogenized
using a Wheaton Overhead Stirrer, sonicated for 30 s, and
clarified by centrifugation at 150 g for 10 min. The
clarified corneal lysates were then assayed via ELISA.
Myeloperoxidase (MPO) assay
MPO, a marker for neutrophils, was detected according to the
method of Bradley et al. [18
], as previously
described.
Histological examination
Immunohistochemical staining was performed using a slight
modification of the procedure of Hendricks et al.
[19
]. Six micron sections were cut at -20°C with a
microtome cryostat (Carl Zeiss, Inc., Microscope Division, Thornwood,
NY) and placed on polylysine hydrobromide-precoated slides
(Polysciences Inc., Warrington, PA). After blocking with normal goat
serum, 50 µl appropriately diluted, primarily antibody was added and
incubated overnight at 4°C in a moist chamber. The sections were then
stained using the streptavidin-biotin complex (S-ABC)
immunoperoxidase-staining procedure (Zymed Laboratories, South San
Francisco, CA) [1
]. Finally, the slides were washed in
distilled water, counterstained with Mayer’s hematoxylin, covered with
a glass coverslip, and examined under light microscopy. Pictures were
taken via camera-enhanced Olympus BX50 light microscopy (Olympus
Optical Co., Tokyo, Japan).
Neutrophil and natural killer (NK) cell depletion in
vivo
Neutrophil and NK cell depletions were performed as previously
described [1
, 20
]. Briefly, mice were given
0.5 mg purified RB6-8C5 mAb intraperitoneally (i.p.) 5 h before
HSV-1 infection. To deplete NK cells, 0.8 mg rabbit asialo GM1
antiserum was given i.p. 1 day before virus infection.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(5 ng/ml; unpublished results). When studies
were conducted with excised mouse corneal buttons instead of isolated
cells, 30–80 pg IL-10 were detected per button. Exposure of this
ocular tissue to varying doses of TNF- (0.1–5 ng), LPS (0.1–100
ng/ml), IFN- (5–500 U/ml), or Bt2 cAMP (0.1–1.0 mM)
did not enhance IL-10 synthesis significantly. We conclude that IL-10
is made constitutively by corneal epithelial cells and fibroblasts and
that synthesis is not up-regulated by well-established biological
inducers.
 View larger version (13K): [in a new window] |
Figure 1. Constitutive secretion of IL-10 protein by resident corneal cells.
Epithelial cells and fibroblasts (lx105 cells/well) were
incubated in DMEM supplemented with 20% FBS. At the indicated times,
aliquots of culture supernatants were collected and assayed for IL-10
by ELISA. The bars show the mean ± SE of
three cultures at each time point.
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 View larger version (47K): [in a new window] |
Figure 2. Constitutive expression of mRNA for IL-10 and IL-10R in murine corneal
epithelial cells and fibroblasts. Murine corneal epithelial cells and
fibroblasts were incubated in medium with 20% FBS. At desired
incubation times, total RNA was extracted. mRNA levels for IL-10,
IL-10R, and GAPD were determined as described in Materials and Methods.
The numbers in the brackets indicate the PCR cycles run.
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-induced synthesis of
MIP-2 and IL-6 markedly [10
, 11
]. This
suggested that at least certain resident corneal cells expressed
functional IL-10 receptors (IL-10Rs). To investigate this hypothesis,
first we tested whether isolated epithelial cells or cultured corneal
fibroblasts expressed IL-10R mRNA. Figure 2
shows that message was
detected in both cell types. Expression of IL-10Rs on the cell surface
was evaluated via fluorescein-activated cell sorter (FACS) analysis. It
was found that epithelial cells and fibroblasts stained positively when
incubated with biotinylated IL-10 (Fig. 3
). Addition of unlabeled IL-10 but not an irrelevant protein
reduced the fluorescence intensity substantially (unpublished results).
Collectively, these results indicated that both cell types expressed
IL-10Rs on their surface. The relatively narrow pattern of fluorescence
intensity seen with fibroblasts contrasted with the broader staining
pattern displayed by epithelial cells and suggests that receptor
expression on fibroblasts is more uniform than that on epithelial
cells. Alternatively, the fibroblast phenotype may have been selected
during in vitro culturing.
 View larger version (28K): [in a new window] |
Figure 3. Expression of IL-10 receptors on normal murine cornea epithelial cells
and fibroblasts. Epithelial sheets were mechanically peeled off from
normal Balb/c mouse corneas, and a single cell suspension was prepared.
Corneal fibroblasts were grown in vitro, and a single cell
suspension was prepared. The expression of IL-10 receptors was detected
by FACS using biotinylated IL-10 (–-). Biotinylated soybean trypsin
inhibitor served as the negative control (—).
|
induction of MIP-2 and IL-6. Figure 4
shows that antibody treatment resulted in 2.4- and 3.2-fold
increases in MIP-2 production by corneal epithelial cells and
fibroblasts, respectively, and IL-6 levels were enhanced by 2.9- and
2.3-fold. In contrast, addition of neutralizing antibody to IL-4 (10
µg/ml) was without effect (unpublished results). These results
suggest that endogenous IL-10 acts in an autocrine/paracrine manner to
down-regulate MIP-2 and IL-6 production by resident corneal cells.
 View larger version (15K): [in a new window] |
Figure 4. Effect of antibody neutralization of endogenous IL-10 on the expression
of IL-6 and MIP-2 by cultured corneal epithelial cells and fibroblasts.
Neutralizing antibody to IL-10 (10 µg/ml) or control IgG was added to
washed cultures. Two hours later, cell cultures were exposed to
rTNF- (50 pg/ml). Culture supernatants were collected 24 h
later and assayed for IL-6 and MIP-2 via ELISA. The bars show the
mean ± SE of three cultures in each group.
*Significantly (p<0.05) different from the IgG-treated
controls.
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production was
elevated in a dose-response way. Additional studies performed 1, 2, 3,
or 5 days after a 104 PFU/cornea challenge dose revealed
that IL-10 corneal levels were not elevated significantly above
preinfection levels (unpublished results).
 View larger version (16K): [in a new window] |
Figure 5. IL-10 and IL-1 expression in the normal and HSV-1-infected cornea.
Balb/c mice were infected intracorneally with the indicated dose of
HSV-1. Six hours later, corneas were removed, and individually prepared
lysates were assayed for IL-10 and IL-1 via ELISA. The bars show the
mean ± SE of four corneas. *Significantly
(p<0.001) different from uninfected corneas.
|
expression by
80% (p<0.05), demonstrating that NK cells were reduced
significantly [4
]. We found that a reduction in either
cell type failed to affect IL-10 corneal levels significantly when
animals were examined 2 days after 104 PFU intracorneal
challenge. Collectively, these results indicate that IL-10 is produced
constitutively in the normal cornea and is not or is only marginally
elevated after virus infection. Furthermore, resident epithelial cells
and fibroblasts appear to be the principal cells producing this
cytokine, at least during the first 2 days after infection.
Antibody neutralization of endogenous IL-10 enhances ocular
inflammation
To determine whether endogenous IL-10 influenced corneal
inflammation, neutralizing antibody was admixed with the virus inoculum
(103 PFU/cornea). Simultaneously, additional antibody was
given subconjunctivally. After infection (24 h), the mean corneal
opacity score for the six anti-IL-10-treated mice (1.3±0.2) was
significantly higher (p<0.05) than that observed in the
IgG-treated control hosts (0.5±0.1). Histologically, the architecture
of the control IgG-treated corneas was intact (Fig. 6A
). Modest numbers of leukocytes were present in the central
corneal, most of which stained positively with RB6-8C5 mAb. In
contrast, corneas from mice given anti-IL-10 antibody were noticeably
swollen and displayed extensive neutrophil infiltration (Fig. 6B)
. MPO
activity measured in the antibody-treated corneas was some 2.7-fold
higher than that detected in the IgG-treated control corneas
(Fig. 7
).
 View larger version (96K): [in a new window] |
Figure 6. Enhancement of corneal inflammation following treatment with
neutralizing antibody to IL-10. Animals were inoculated with IL-10
antibody or control IgG admixed with HSV-1 (1x103
PFU/cornea). An additional 5 µl IgG was given subconjunctivally at
the time of infection. Day 1 after infection, eyes were removed and
embedded in tissue-freezing medium. Frozen sections were prepared and
stained for neutrophils using specific antibody (RB6-8C5). (A)
Representative example of a control IgG-treated cornea. The
architecture of the cornea is normal, and there is a modest influx of
neutrophils (arrows). (B) Cornea from a mouse treated with neutralizing
IL-10 antibody. Note the extensive neutrophil infiltrate in the swollen
cornea. Original magnification, x200.
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 View larger version (13K): [in a new window] |
Figure 7. Effect of antibody neutralization of endogenous IL-10 on expression of
cytokines and chemokines. Animals were inoculated intracorneally with
neutralizing antibody to IL-10 or control IgG, admixed with HSV-1
(1x103 PFU/cornea). An additional 5 µl IgG was given
subconjunctivally at the time of infection. Twenty-four hours later,
corneal lysates were prepared and individually assayed for IL-6, MIP-2,
and MIP-l via ELISA. MPO activity was also measured. The bars show
the mean ± SE of four mice in each group.
*Significantly (p<0.05) different from the IgG-treated
controls.
|
, and IL-6
in corneas were measured by ELISA. Figure 7
shows that the levels of
MIP-2, MIP-1, and IL-6 in the antibody-treated hosts were 2.6-,
3.9-, and 3.2-fold higher, respectively, than those seen in the
controls (p<0.05). Treatment with neutralizing antibody to
IL-4 or IL-13 (25 µg/eye) did not increase the level of any of the
three mediators significantly (unpublished results). When the virus
challenge dose was increased tenfold (104 PFU/cornea),
IL-10 neutralizing antibody treatment was associated again with
elevated cytokine/chemokine levels, but the differences from the
controls were not statistically significant (p>0.05).
Disruption of IL-10 gene enhances ocular inflammation
Mice, in which the IL-10 gene has been disrupted, develop
inflammatory bowel disease uniformly [21
]. When we
examined these animals, the ocular tissues were found to be clinically
normal, and neither MPO nor MIP-2 could be detected in uninfected
corneal lysates (Fig. 8
). To investigate whether disruption of the IL-10 gene influenced
corneal inflammation, these mice and their WT counterparts were
infected with 103 PFU HSV-1. Two days postinfection, the
mean corneal score for five IL-10 knockout hosts was 2.8 ± 0.2.
This was significantly higher (p<0.001) than the mean
opacity score (0.8±0.2) observed in the WT controls. In addition,
MIP-2 levels and MPO activity in IL-10 knockouts were each some
threefold higher than those seen in the controls (Fig. 8)
.
 View larger version (16K): [in a new window] |
Figure 8. Effect of IL-10 gene knockout on expression of MIP-2 and
myeloperoxidase. IL-10 knockout or WT mice were inoculated
intracorneally with HSV-1 (1x103 PFU/cornea). Two days
later, corneal lysates were prepared and individually assayed for MIP-2
and myeloperoxidase. The bars show the mean ± SE of
four mice in each group. *Significantly (p<0.01) different
from the WT-infected controls.
|
production in the cornea and
vice versa induces corneal cells
to make MIP-2 and IL-6 [4
, 16
]. Thus, IL-10
could be inhibiting the synthesis of these mediators indirectly by
interfering with IL-1 production. Therefore, we tested whether
intracorneal inoculation of neutralizing antibodies to IL-10 at the
time of HSV-1 infection resulted in elevated IL-1 production in the
cornea. Figure 9A
shows that 24 h postinfection, IL-1 levels were not
significantly different from the controls.
 View larger version (14K): [in a new window] |
Figure 9. Effect of neutralizing antibody to IL-10 or to IL-1/ß on cytokine
expression in the HSV-1-infected cornea. Animals were inoculated
intracorneally with HSV-1 admixed with neutralizing antibody to IL-10
or antibodies to IL-1 plus IL-1ß. Controls received IgG. One day
after infection, corneas were removed, lysates prepared, and
individually assayed for IL-1, IL-10, or IL-6 via ELISA. The bars
show the mean ± SE of four animals in each group.
*Significantly (p<0.01) different from the IgG controls.
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/ß because
the IL-6 levels were reduced by >85% (Fig. 9C)
. |
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced
IL-6 and MIP-2 production, strongly indicating that IL-10 was acting in
an autocrine/paracrine manner to down-regulate expression of these
proinflammatory cytokines. Importantly, local neutralizing antibody
treatment in vivo not only elevated proinflammatory mediator
synthesis but also increased the severity of corneal disease.
Furthermore, mice with a disrupted IL-10 gene exhibited significantly
heightened corneal disease and a threefold increase in the levels of
MIP-2 and MPO relative to that seen in the WT controls. In our ocular
infection model, MIP-2 is the major chemoattractant of neutrophils
[4
]. Antibody neutralization of MIP-2 has been shown to
reduce neutrophil infiltration and corneal inflammation. Also, IL-6 is
produced strongly in the virus-infected cornea [13
], and
IL-6 knockout mice develop less severe corneal disease (unpublished
results). Therefore, blocking a negative regulator of these two
mediators would be predicted to enhance corneal inflammation, and this
is what occurred.
The molecular events responsible for IL-10-mediated down-regulation of
corneal cell-produced MIP-2 and IL-6 are not known. The genes for both
of these mediators are regulated by activation of nuclear factor
(NF)-
B [22
23
24
25
]. Lentsch et al.
[26
] have shown that the anti- inflammatory activity of
IL-10 in their lung-injury model was associated with increased
stability of I-B, the cytoplasmic inhibitor of NF-B. Studies
to determine whether this mechanism is operative in corneal cells are
in progress.
Evidence of autocrine action of IL-10 in the cornea is compatible with previous studies showing that exogenous IL-10 could suppress corneal cell-derived proinflammatory mediators [10 11 12 ]. Furthermore, it is well-documented that IL-10 acts in an autocrine way to suppress the production of proinflammatory cytokines in macrophages and monocytes [27 28 29 30 ]. More recently, there have been studies showing that IL-10 can serve as an autocrine growth factor in Epstein-Barr virus (EBV)-infected B cells [31 , 32 ], malignant B cells [33 ], and in the Ly-1 B cell subset [34 ]. In addition, this cytokine has been shown to be an autocrine regulator of cytotrophoblast matrix metalloproteinase -9 [35 ]. Thus, apparently IL-10 autocrine action occurs in various cell types and mediates a diverse array of biological effects.
Others have shown that endogenous IL-10 is an important, natural regulator of lung-inflammatory injury elicited by deposition of IgG immune complexes [36 ] or bacterial infection [37 ]. IL-4 and IL-13 were also shown to function as regulatory cytokines in the lungs [38 ]. However, no IL-4 could be detected in the herpes virus-infected corneas (ELISA sensitivity was 5 pg/ml). Moreover, in vivo administration of neutralizing antibody to IL-4 or IL-13 did not enhance corneal disease or up-regulate production of MIP-2 and IL-6. Thus, IL-4 and IL-13 are unlikely to play an important anti-inflammatory role in the HSV-1-infected cornea.
The synthesis of IL-10 has been shown to be up-regulated in macrophages
following exposure to TNF-, LPS, Bt2 cAMP, and IFN-
[39
40
41
42
]. However, when cultured corneal cells were
exposed to these inducers, IL-10 production was not enhanced.
Similarly, increasing the intracorneal virus-challenge dose, which
significantly enhanced IL-1 levels, had little or no effect on IL-10
expression. The failure of various inducers as well as HSV-1 infection
to elevate IL-10 synthesis suggests that IL-10 is highly regulated in
the cornea, and thus, this cytokine’s capacity to down-regulate
proinflammatory cytokine production is limited. Indeed, we found that
endogenous IL-10 impaired development of corneal disease at a
103 PFU virus-challenge dose but in agreement with our
earlier study [13
] could not do so when a
104 PFU-infectious dose was given. The identification of a
reagent that could boost endogenous IL-10 production by corneal cells
might be therapeutically useful in controlling ocular inflammation.
The host response to a pathogen in tissue such as the lungs, joints,
and gut has been demonstrated to be regulated by proinflammatory and
anti-inflammatory mediators that are balanced so as to provide an
adequate defense while minimizing destruction of self tissue
[43
]. Our results suggest that in the virus-infected
cornea, IL-10 serves to counterbalance the effects of IL-1. It is
interesting that the in vivo neutralizing antibody
experiments indicated that IL-10 did not influence IL-1 expression.
This result is in agreement with our previous studies involving
exogenous IL-10 [10
]. The antibody studies also showed
that IL-l /ß did not affect IL-10 expression. Thus, IL-1 and IL-10
appear to act independently of each other as they regulate
proinflammatory mediator expression in the cornea. It should be noted
that IL-10 can down-regulate IL-1 expression in cells of the
macrophage/monocyte lineage [44
, 45
]. These
different outcomes highlight the complex, cell type-specific nature of
cytokine regulation. Constitutive production of IL-10 and IL-10
receptor may reflect nature’s attempt to maintain a clear cornea.
In summary, the present results establish that IL-10 is an important player in limiting inflammation at the ocular surface. Recently, D’Orazio and Niederkorn [46 ] have demonstrated that IL-10 produced by TGF-ß-treated antigen-presenting cells played a critical role in anterior chamber- associated immune deviation. Whether IL-10 produced by resident corneal cells contributes to immune privilege in the eye remains to be tested.
Received April 26, 2000; revised July 13, 2000; accepted July 17, 2000.
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