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Institute of Virology, Erasmus Medical Center, Rotterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN-
-producing Th1 cells play a crucial role,
results in the local secretion of immune-modulatory factors and a major
influx of neutrophils causing corneal lesions and blindness. The
Th1-derived cytokine IL-17 has been shown to play an important role in
several inflammatory diseases characterized by a massive infiltration
of neutrophils into inflamed tissue. Here we show that IL-17 is
expressed in corneas from patients with HSK and that the IL-17R is
constitutively expressed by human corneal fibroblasts (HCF). IL-17
exhibited a strong synergistic effect with TNF- on the induction of
IL-6 and IL-8 secretion by cultured HCF. Secreted IL-8 in these
cultures had a strong chemotactic effect on neutrophils. IL-17 also
enhanced TNF-- and IFN--induced secretion of
macrophage-inflammatory proteins 1 and 3, while inhibiting the
induced secretion of RANTES. Furthermore, considerable levels of
IFN--inducible protein 10 and matrix metalloproteinase 1 were
measured in stimulated HCF cultures, while the constitutive secretion
of monocyte chemotactic protein 1 remained unaffected. The data
presented suggest that IL-17 may play an important role in the
induction and/or perpetuation of the immunopathologic processes in
human HSK by modulating the secretion of proinflammatory and neutrophil
chemotactic factors by corneal resident
fibroblasts. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting CD4+ T cells, orchestrating the
extravasation and activation of neutrophils. These neutrophils are
considered to be directly involved in corneal destruction
(9, 10, 11). Additionally, Langerhans cells (LC) and
macrophages, secreting the proinflammatory cytokines IL-1 and TNF-,
are essential mediators (12, 13, 14, 15).
Extensive studies in murine models of HSK have provided insight into
the roles multiple chemokines play in the development of the disease
(5, 8, 16, 17). These studies demonstrated the temporal
expression of chemokines like IL-8, RANTES, macrophage-inflammatory
protein (MIP)-1, monocyte chemotactic protein (MCP)-1, and
IFN--inducible protein (IP)-10 in affected murine corneas. These
chemokines are likely to contribute to the recruitment and activation
of lymphocytes, dendritic cells, and neutrophils, initiating the
clinical phase of HSK. Although the cellular source of the chemokines
involved in HSK remains ill defined, they may be produced by
infiltrating inflammatory cells and/or resident corneal cells. Several
studies have implied that the interaction with inflammatory cells
mainly involves fibroblasts within the corneal stromal layer. The
stromal cell layer of the cornea is made up of fibroblasts providing
mechanical strength to the cornea by supporting a framework of
extracellular matrix. Large numbers of IFN-- and TNF--producing
cells have been observed in the stromal layer of HSV-infected murine
corneas but not in the epithelial or endothelial layers (18, 19). This corresponds to the location of neutrophils that mainly
infiltrate the stromal fibroblast cell layer of the cornea. Similarly,
the induction of IL-8, which exhibits neutrophil chemotactic
properties, has been shown to be associated with HSV replication in
corneal fibroblasts but not in epithelial cells (20).
Evidence is accumulating that activated tissue resident cells,
including fibroblasts, are involved in modulating local immune
responses by expressing adhesion molecules and secreting regulatory
factors like cytokines and chemokines. Macrophage-derived cytokines
like IL-1 and TNF-, and Th1 cytokines like IFN-, have been shown
to activate mesenchymal cells (21, 22, 23, 24, 25, 26). Interestingly,
another recently described Th1 cytokine, IL-17, can exert strong
synergistic and/or antagonistic effects with the aforementioned
proinflammatory cytokines. These effects include the modulation of MHC
and adhesion molecules and the induction of a variety of cytokines and
chemokines by mesenchymal cells (21, 22, 23, 24, 27, 28, 29, 30). This
modulatory effect of IL-17 could therefore have an indirect but
profound effect on the recruitment and activation of different
inflammatory cells into the corneal stroma during human HSK. To address
this issue, we analyzed the induction of the cytokine IL-6, various
chemokines, and matrix metalloproteinase (MMP)-1 by human corneal
fibroblasts (HCF) upon stimulation with IL-1
, TNF-, and IFN-.
The combinative effect of IL-17 with these cytokines on HCF activation
was emphasized.
Our data show that IL-17 is expressed in corneas of HSK patients and has a regulatory effect on the induction of immune-modulatory factors by cytokine-stimulated HCF. These results indicate that the interaction of IL-17 with HCF may play a significant role in the initiation and perpetuation of the inflammatory processes in human HSK.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant IL-1, IL-17, TNF-, and IFN- were
obtained from PeproTech (London, U.K.). For blocking experiments,
neutralizing mAbs directed to human IL-17 (R&D Systems, Abingdon,
U.K.), IL-8, and isotype-matched control mAbs were used
(PeproTech).
Human intracorneal T cell lines (TCL) and HCF cultures
The generation of HSV-specific TCL used in this study, obtained from affected corneas of two HSK patients, has been described previously (31). To determine IL-17 mRNA expression, TCL were either left unstimulated or stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml). Primary HCF cultures were generated from three individual donor corneas that had been rejected for transplantation use due to low endothelial cell counts, and from one transplanted HSK-diseased cornea. The central part of each cornea was finely minced and digested with collagenase essentially as described elsewhere (31). Adherent cells were cultured in six-well plates in medium consisting of a 1:1 ratio of DMEM and Ham F12 nutrient mixture (Life Technologies, Breda, The Netherlands) supplemented with 10% FCS and antibiotics. HCF cultures, with a fibroblast-like morphology, were grown in bulk in 162-cm2 flasks and cryopreserved. All cells stained positive for vimentin by immunofluorescence staining, whereas almost no reactivity was found for both the acidic and basic subfamilies of cytokeratin (data not shown), indicating that HCF cultures were not contaminated with corneal epithelial or endothelial cells. Third- or fourth-passage corneal fibroblasts were used in all experiments. For cytokine stimulation experiments, HCF from donor corneas were grown in medium of DMEM and Ham F12 with 10% FCS and antibiotics. At confluence, medium was replaced with a serum-free medium consisting of DMEM supplemented with 2 mM L-glutamine, 20 µg/ml cholesterol, and 0.3% BSA (all obtained from Sigma-Aldrich, St. Louis, MO). HCF were left for 5 days on serum-free medium before stimulation with cytokines. Serum-free medium was used to maintain a more native biosynthetic phenotype and appearance (32) and to reduce background levels of cytokine and chemokine production (data not shown). Stimulatory cytokines were added to the HCF cultures and supernatants were collected after 48 h. All tested stimulatory conditions were repeated in at least eight individual experiments unless stated otherwise. For blocking experiments, the stimulating mixture of cytokines was preincubated with a neutralizing anti-IL-17 mAb (10 µg/ml) at room temperature 30 min before addition to the cell culture. Inhibition of cytokine and chemokine expression was induced by pretreatment of HCF cultures with 10 ng/ml of the anti-inflammatory drug dexamethasone (Sigma-Aldrich) for 2 h.
RNA isolation and RT-PCR analyses
Total cellular RNA was extracted from human HSK corneas, cultured HCF, or intracorneal TCL with TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. The cornea buttons analyzed were obtained from three patients with a fulminant HSV-induced necrotizing stromal keratitis after therapeutic penetrating keratoplasty. HSK classification and disease status were defined on the basis of clinical criteria (33). None of the patients were treated with steroids. For RT-PCR analyses, total RNA was converted into cDNA using oligo(dT) and reverse transcriptase. The following synthetic oligonucleotides were used for PCR amplification: for IL-17 amplification, primers 5'-ATCTCCACCGCAATGAGGAC-3' and 5'-GTGGACAATCGGGGTGACAC-3' (232-bp amplicon); for IL-17R, primers 5'-CTAAACTGCACGGTCAAGAAT-3' and 5'-CTGAGCTCATGCATGGCGTGG-3' (456-bp amplicon). As an internal control for the amount of cDNA, the GAPDH gene was amplified with primers 5-GGTGAAGGTCGGAGTCAACG-3' and 5'-CAAAGTTGTCATGGATGACC-3' (496-bp amplicon). Amplification was performed in standard PCR buffer with 1 U AmpliTaq Gold DNA polymerase (PerkinElmer, Groningen, The Netherlands), 200 µM dNTPs, and 25 pmol of each primer in a total volume of 50 µl. DNA amplification was started with a 5-min incubation at 95°C, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. The final extension was at 72°C for 7 min. PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Southern blotting and labeling with a specific 32P-labeled oligonucleotide probe was performed to confirm the specificity of the amplicons.
ELISA
Culture supernatants from corneal fibroblasts stimulated for
48 h, referred to as conditioned medium, were harvested and
cleared of cellular debris by centrifugation. Secretion levels of IL-6,
IL-8, RANTES, MIP-3, IP-10, and MMP-1 were measured with
commercially available ELISA kits (R&D Systems). MIP-1 content was
measured in an ELISA using a coating mAb, recombinant human MIP-1 as
a standard, and an HRP-conjugated detection mAb (R&D Systems). The
amount of MCP-1 in culture supernatants was determined using a
commercially available ELISA kit obtained from BioSource (Nivelles,
Belgium). The detection limits of the ELISA were as follows: IL-6,
<0.7 pg/ml; IL-8, <10 pg/ml; RANTES, <8 pg/ml; MIP-3, <0.9
pg/ml; IP-10, <4.5 pg/ml; MMP-1, 21 pg/ml; MIP-1, <10 pg/ml; and
MCP-1, <20 pg/ml.
Neutrophil chemotaxis
Polymorph mononuclear cells (PMN) were isolated from fresh peripheral blood of healthy, adult volunteers using Polymorph Prep (Nycomed, Oslo, Norway) and residual erythrocytes were lysed. To analyze the chemotactic activity of conditioned medium of HCF, PMN were brought to a final concentration of 2 x 106 cells/ml in serum-free medium. The chemotaxis assay was performed in a 24-well Transwell system (Costar, Badhoevedorp, The Netherlands). The bottom wells of the chamber were filled with 100 µl of recombinant human IL-8 (10 ng/ml) as a positive control for neutrophil chemotaxis, the control solution, or conditioned medium from stimulated HCF cultures, diluted 1/10 in serum-free medium. The upper wells, holding a polycarbonate filter with a pore size of 3 µm, were placed on top and filled with 150 µl of neutrophil suspension. The Transwell system was incubated in humidified air with 5% CO2 at 37°C for 45 min, and the number of cells that had migrated through the filter into the bottom well were counted. Inhibition of IL-8-mediated neutrophil recruitment involved a preincubation of the conditioned medium with 10 µg/ml anti-IL-8 mAb 30 min before the experiment at room temperature. As a control, conditioned medium was incubated under similar conditions with an irrelevant isotype-matched control mAb.
Statistical analysis
Results are expressed as the mean ± SD. The statistical significance of the modulatory effect of IL-17 on cytokine-treated was determined by the Mann-Whitney U test. Differences resulting in p values <0.05 were considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whereas the normal healthy cornea is essentially devoid of T
cells, HSK is considered an immunopathologic disease orchestrated by
corneal-infiltrating CD4+ Th1 cells secreting
IL-2 and IFN- (9, 10, 11). To assess the possible role of
the Th1 cytokine IL-17 in human HSK, the expression of IL-17 mRNA was
determined in corneas from three patients with fulminant HSK who
underwent corneal transplantation to restore sight. IL-17 transcripts
could be detected in all HSK corneas, while no IL-17 was detected in
control corneas (Fig. 1
A).
Mitogenic stimulation of two intracorneal TCL generated from corneas of
two other HSK patients induced IL-17 mRNA expression. This demonstrates
that corneal-infiltrating T cells, at least in part, are able to
express IL-17 mRNA upon activation (Fig. 1B).
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The keratogenic properties of Th1 cytokines in HSK may in part be
due to their modulatory effect on corneal resident cells.
Corneal-infiltrating T cells are mainly observed in the corneal stroma,
suggesting an interaction between T cells and corneal fibroblasts by
means of direct cell-cell contact or soluble factors like IL-17. To
evaluate whether corneal fibroblasts express the IL-17R, RT-PCR was
performed on RNA extracted from three primary HCF cultures. Two primary
HCF cultures had been generated from two healthy donor corneas and one
from a transplanted HSK-diseased cornea. RT-PCR analysis showed that
HCF expressed IL-17R mRNA (Fig. 2).
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-induced IL-6 and IL-8
secretion by HCF
Studies in HSK mouse models have shown a strong induction of IL-6
and IL-8 after HSV-1 infection of the cornea (5, 20). By
mimicking the inflammatory situation in the corneal stroma during HSK
development in vitro, we determined the modulatory effect of IL-17 (100
ng/ml) on IL-1 (100 ng/ml), TNF- (50 ng/ml), and IFN- (100
U/ml) to induce secretion of IL-6 and IL-8 by HCF. Optimal
concentrations of stimulatory cytokines had been determined in
preliminary experiments (data not shown).
HCF showed a low level of background secretion of both IL-6 and IL-8,
while stimulation with IL-17 or IFN- alone or in combination did not
show any considerable increase in either IL-6 or IL-8 levels (Fig. 3). However, stimulation of HCF with
IL-1 or TNF- did have a clear stimulatory effect on the secretion
of both factors. Although IL-17 itself had no effect, incubation of HCF
with combinations of stimulating cytokines showed that it had a
synergistic effect on the TNF-- and TNF-/IFN--induced
secretion of both IL-6 and IL-8. This synergistic effect was almost
completely neutralized by preincubating the stimulating cytokine
mixture with an anti-IL-17 mAb, but not with an isotype-matched
control mAb (Fig. 3). Furthermore, a 2-h pretreatment of HCF with
dexamethasone, an immunosuppressive drug commonly used in treating
human HSK, almost completely abolished the induced secretion of IL-6
and IL-8 (Fig. 3).
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In addition to IL-6 and IL-8, several other chemokines are
expressed in affected corneas and are assumed to play a critical role
in the development of HSK (5, 8). Because HCF are a
probable cellular source for these chemokines, we also analyzed the
effects of stimulation with IL-1, TNF-, IL-17, and IFN- on
chemokine production (including MCP-1, RANTES, MIP-1, MIP-3, and
IP-10) and MMP-1 by HCF.
The observation by Tumpey et al. (34) that MIP-1
knockout mice fail to develop HSK suggests that this chemokine is a key
factor in the development of the disease. MIP-1 secretion by HCF,
although in low amounts (up to 30 pg/ml), was observed solely upon
costimulation of IL-17 with TNF-, IFN-, or both cytokines
combined (Fig. 4). Whereas MIP-1 is
mainly chemotactic for neutrophils, MIP-3 is a powerful
chemoattractant for LC precursors (35). Both IL-1 and
TNF- induced the secretion of MIP-3 by HCF, whereas neither IL-17
nor IFN- stimulated MIP-3 secretion (Fig. 4). IL-17 showed a
clear synergistic effect with TNF- and TNF-/IFN- on MIP-3
secretion by HCF, but not with IL-1 or IFN-.
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, whereas IP-10 production was augmented by TNF-
and IFN- (Fig. 4
). Stimulation of HCF with TNF- and IFN-
combined had a synergistic effect on the secretion of both RANTES and
IP-10. IL-17 showed a remarkable inhibition on the secretion of RANTES
by HCF upon stimulation with TNF- and TNF-/IFN-, while IP-10
production was only slightly inhibited by IL-17 (Fig. 4).
Although the role of MCP-1 is not clear at this point, it has been
reported to play a role in CD4+ T cell
recruitment and appears to be rapidly up-regulated and persistently
expressed in HSV-1-infected corneas (5). In our
experiments, HCF constitutively secreted MCP-1, and no evident changes
were observed by incubation with stimulatory cytokines (Fig. 4).
Although MMP-1 is not a chemokine, it was included in this study
because MMP-1 is known to degrade fibrilar collagens (37),
an essential component of the corneal stroma. HCF showed a constitutive
secretion of MMP-1, which was not affected by IFN-. Compared with
the mild induction by IL-17, IL-1 and TNF- induced a strong
enhancement of MMP-1 secretion. Interestingly, IL-17 had a synergistic
effect with IFN- on MMP-1 secretion while showing a slight
inhibitory effect in combination with IL-1 or TNF- (Fig. 4).
HCF induce neutrophil recruitment through IL-8 release
Chemokines expressed within HSV-1-infected corneas are believed to
induce the corneal infiltration of neutrophils involved in the
development of corneal lesions (3, 5, 8, 16, 17). To test
the PMN chemotactic properties of chemokines secreted by HCF,
conditioned medium from control and cytokine-stimulated HCF cultures
were tested in a neutrophil chemotaxis assay. Conditioned medium from
HCF stimulated with the combination of IL-17, TNF-, and IFN- was
used because it contained the highest levels of IL-8 and MIP-1 (see
Figs. 3 and 4), both potent PMN chemoattractants. rIL-8 (10 ng/ml) was
used as a positive control, while fresh serum-free medium served as
background control. Compared with the medium control, conditioned
medium from stimulated HCF induced a strong migration of PMN. This
effect could be blocked by pretreatment of the conditioned medium with
a neutralizing anti-IL-8 mAb 30 min before the experiment (Fig. 5). Conditioned medium from nonstimulated
HCF caused a mild migration of PMN, probably due to a low level of
background production of IL-8 in these cultures (see Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-) or Th1 cell (IFN-) origin,
has previously been shown to activate and modulate the secretion of
chemokines (21, 22, 23, 24, 25, 26), suggesting a role of resident cells
such as fibroblasts in the recruitment and activation of inflammatory
cells to sites of inflammation. Furthermore, recent observations have
shown that activation of resident cells can be heavily modulated by the
Th1-derived cytokine IL-17 (21, 22, 23, 24, 27, 28, 29, 30). Accordingly,
the potential interactive role of IL-17 and corneal fibroblasts in the
induction and perpetuation of inflammatory processes in human HSK was
hypothesized.
The expression and pathogenic properties of IL-1, TNF-, and
IFN- in HSK have been extensively studied in the experimental HSK
mouse model. These proinflammatory cytokines probably do not have a
direct immunopathologic effect by recruiting and stimulating
inflammatory cells, but rather exert their effects through interaction
with corneal resident cells in an autocrine or paracrine fashion. IL-17
is secreted by activated CD4+ T cells, mainly Th0
and Th1 (27), and exhibits pleiotropic biological
activities on various human tissue resident cells including
fibroblasts. IL-17 shares mainly properties with IL-1 and TNF- in
that these three cytokines activate the transcription factor NF-
B in
a variety of cell types and all stimulate secretion of proinflammatory
factors in mesenchymal cells (27). The present study shows
IL-17 expression in affected corneas of patients with fulminant
HSK, whereas no IL-17 expression was detectable in control corneas.
Mitogenic stimulation of intracorneal TCL obtained from corneas of HSK
patients induced IL-17 mRNA expression. The HCF constitutively
expressed the IL-17R, consistent with its broad tissue distribution
(38).
IL-17 indirectly stimulates granulopoiesis both in vitro
(27) and in vivo (39), and has also
previously been marked in playing an important role in the
pathogenesis of other immune-mediated diseases associated with a
massive infiltration of neutrophils into inflamed tissues
(40, 41, 42, 43, 44, 45). The functional relevance of IL-17 signaling in
host defense has recently been demonstrated in an experimental model of
Klebsiella pneumoniae lung infection in IL-17R knockout mice
(46). IL-17R signaling appeared to be critical for the
local induction of G-CSF and MIP-2 and subsequent PMN recruitment
into the alveolar space. The murine C-X-C chemokines MIP-2 and
KC, homologs of human IL-8, have also been associated with the
development of experimental murine HSK (5). Neutralization
of MIP-2 decreased corneal PMN infiltration and significantly reduced
corneal pathology, demonstrating that MIP-2 is the major chemokine that
attracts PMN into the HSV-1-infected cornea in mice (17).
Interestingly, in our experiments, conditioned medium from HCF
stimulated with the combination of TNF-, IFN-, and IL-17 induced
chemotaxis of PMN in vitro (Fig. 5). Despite the presence of multiple
other chemokines in these conditioned media, this chemotactic activity
on PMN was mainly induced by IL-8, because preincubation of conditioned
medium with a neutralizing IL-8 mAb almost completely abrogated PMN
migration.
The data presented here demonstrate that cytokine-stimulated HCF
secrete chemokines associated with HSK. Chemokine secretion was
differentially regulated by Th1 cell- and macrophage/monocyte-derived
cytokines. Compared with IFN- and IL-17, IL-1 and TNF- were
more effective in stimulating HCF to secrete IL-6, IL-8, MIP-3, and
MMP-1. Whereas simultaneous treatment with TNF- and IFN- induced
significant secretion of RANTES and IP-10 by HCF, no detectable amounts
of MIP-1 were observed upon stimulation with these cytokines.
However, neither Th1 cell-derived nor macrophage/monocyte-derived
cytokine seemed to significantly alter the constitutive expression of
MCP-1.
The induced secretion of IL-6 and IL-8 by HCF stimulated with either
IL-1 or TNF- was consistent with observations previously
described (47, 48). Similarly, induction of IL-6 secretion
upon stimulation with IL-1 or TNF- has also recently been
observed in myofibroblast cells (49). In addition, such
cells do not secrete any MIP-1 upon stimulation with IL-1 or
TNF- (26), comparable with our results. However, in
comparison with another report on chemokine production by HCF
(50), we observed some contradiction in the induction of
RANTES and MCP-1 secretion by treatment of HCF with IL-1 or TNF-. In
this report stimulation of HCF with IL-1 or TNF- resulted in
increased levels of RANTES and MCP-1 production. In contrast,
stimulation of HCF in our experiments with IL-1 did not induce
secretion of detectable amounts of RANTES, nor did IL-1 or TNF-
significantly alter MCP-1 secretion. Although the different effects of
IL-1 and IL-1 on HCF are understandable, the differential effect
of TNF- on MCP-1 production might be related to the usage of serum
in cell cultures that might contain costimulatory factors that were
absent in our experiments. In a recent report about rheumatoid
arthritis in man, RANTES and MCP-1 have been shown to induce IL-6 and
IL-8 (51). These chemokines thus appear not to solely play
a role in inflammatory cell migration, but also to be involved in the
activation of synoviocytes. Consequently, the secretion of the
chemokines detected in conditioned medium of cytokine-stimulated HCF
may only in part be directly induced by the cytokines added.
As in human HSK corneas, IL-17 expression has been observed in other
inflamed tissues, such as the lungs in asthma (52) and the
synovium in arthritis (53). IL-17 exerts a modulatory
effect on IL-1-, TNF--, and IFN--induced chemokine secretion by
human mesenchymal cells (21, 22, 23, 24, 27, 28, 29, 30). Furthermore,
IL-17 has been described to stimulate the secretion of IL-1 and
TNF- by macrophages (54). In our experiments, IL-17 had
a synergistic effect on TNF--induced HCF secretion of IL-6, IL-8,
and MIP-3, which was similar to observations by others (23, 25, 27, 52, 55). Additionally, a synergistic effect was observed
for IL-17 with TNF- and IFN- on the induction of MIP-1,
whereas, similar to findings in keratinocytes (21), IL-17
antagonized TNF-- and/or IFN--induced secretion of RANTES.
However, in contrast with some of these reports (21, 27),
no synergistic effect of IL-17 with IL-1 or IFN- was observed,
which might be a cell type-specific phenomenon.
As its name suggests, IP-10 is known to be induced by IFN-, as was
also observed in our experiments. Strikingly, IP-10 secretion by HCF
was strongly increased upon costimulation with both IFN- and
TNF-, which is in accordance with a previous report
(56). Whereas a previous report (57) showed
no significant stimulatory effect of IL-17 on IP-10 induction in
keratinocytes, our results showed a slight inhibitory effect of IL-17
on IP-10 secretion by HCF. Secretion of IL-6 and IL-8, induced by HCF
stimulation with a combination of IL-17, TNF-, and
IFN-—resulting in the highest secretion levels (Fig. 3)—was
inhibited by pretreatment with an anti-IL-17 mAb to levels similar
to those of TNF- plus IFN-, demonstrating the specific modulatory
effect of IL-17. Glucocorticoids such as dexamethasone are widely used
in the treatment of corneal inflammatory diseases. The data presented
here show that secretion of IL-6 and IL-8 by cytokine-stimulated HCF
can be completely blocked by pretreatment with dexamethasone. This is
compatible with the idea that the beneficial effects of glucocorticoids
in clinical practice are due to inhibition of exaggerated cytokine
production. This effect of glucocorticoids is mediated by their
antagonistic action on transcription factors such as NF-B, which are
required for cytokine transcription (58).
The collection of chemokines found to be secreted by HCF upon cytokine
stimulation exhibits a broad range of leukocyte-recruiting and
-activating potentials. These activities include the migration and
activation of T and NK cells (e.g., RANTES, MCP-1, MIP-3, and
IP-10), LC (e.g., MIP-3), macrophages/monocytes (e.g., MCP-1), and
PMN (e.g., IL-8 and MIP-1). Studies on expression profiles of
different chemokines in the HSK mouse models have shown that most
proinflammatory cytokines and chemokines are expressed throughout both
the preclinical and clinical phase of HSK (8). Exceptions
to this are the chemokine MIP-1, which was detected only during the
clinical phase, and the chemokines IP-10 and RANTES, which can only be
detected early after HSV-1 infection of the cornea, but not during the
clinical phase of the disease (8). Remarkably, in the
present study IL-17 exerted a strong stimulatory signal on the
induction of IL-8, MIP-1, and MIP-3, whereas it inhibited the
secretion of RANTES and, to a lesser extent, IP-10. This IL-17-specific
inhibitory effect on RANTES and IP-10 secretion might reflect a
down-regulatory signal by activated T cells to limit the recruitment of
additional T lymphocytes, and corresponds with the transient detection
of these chemokines in the cornea early after infection. In contrast,
increased secretion of IL-8 and MIP-1 would create an inflammatory
environment in the corneal stroma with a high potential for PMN
extravasation and activation, where they directly or indirectly cause
tissue damage. Although MMP-1 is not a chemokine, its secretion in the
corneal stroma might explain some clinical aspects of HSK. As described
previously, IL-1 and TNF- augmented secretion of MMP-1 in human
fibroblasts (59). MMP-1 degrades structural type I and
type II collagen, and its production in the corneal stroma during HSK
might thus contribute to the development of corneal lesions.
Accordingly, IL-17 has previously been implicated in synovium matrix
destruction in rheumatoid arthritis by similar modes of action
(60).
In conclusion, our study shows the ability of cytokine-stimulated HCF to secrete inflammatory mediators that are potentially involved in the immunopathogenesis of HSK. The Th1 cytokine IL-17, expressed in human HSK corneas, modulated these responses in a synergistic or antagonistic fashion. We speculate that during the development of HSK, in addition to corneal-infiltrating cells, corneal fibroblasts are an important cellular source of proinflammatory cytokines and chemokines. Among these is IL-8, which primarily mediates the extravasation and activation of pathogenic PMN into the cornea. Furthermore, our results suggest that IL-17 might be an important player in the immune activation of HCF. Future studies in murine models might elucidate any potential role of IL-17 in the inflammatory processes leading to HSK.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Georges M. G. M. Verjans, Institute of Virology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands. E-mail address: Verjans{at}viro.fgg.eur.nl
3 Abbreviations used in this paper: HSK, herpetic stromal keratitis; MIP, macrophage-inflammatory protein; MCP, monocyte chemotactic protein; MMP, matrix metalloproteinase; IP, IFN--inducible protein; HCF, human corneal fibroblast; LC, Langerhans cell; TCL, T cell line; PMN, polymorph mononuclear cell.
Received for publication April 22, 2002. Accepted for publication September 24, 2002.
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  J. R. Nichols, A. L. Aldrich, M. M. Mariani, D. Vidlak, N. Esen, and T. Kielian TLR2 Deficiency Leads to Increased Th17 Infiltrates in Experimental Brain Abscesses J. Immunol., June 1, 2009; 182(11): 7119 - 7130. [Abstract] [Full Text] [PDF]
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 Y. Liu, K. Kimura, R. Yanai, T.-i. Chikama, and T. Nishida Cytokine, Chemokine, and Adhesion Molecule Expression Mediated by MAPKs in Human Corneal Fibroblasts Exposed to Poly(I:C) Invest. Ophthalmol. Vis. Sci., August 1, 2008; 49(8): 3336 - 3344. [Abstract] [Full Text] [PDF]
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S. J. Divito and R. L. Hendricks Activated Inflammatory Infiltrate in HSV-1-Infected Corneas without Herpes Stromal Keratitis Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1488 - 1495. [Abstract] [Full Text] [PDF]
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 S. J. Molesworth-Kenyon, R. Yin, J. E. Oakes, and R. N. Lausch IL-17 receptor signaling influences virus-induced corneal inflammation J. Leukoc. Biol., February 1, 2008; 83(2): 401 - 408. [Abstract] [Full Text] [PDF]
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R. Duan, L. Remeijer, J. M. van Dun, A. D. M. E. Osterhaus, and G. M. G. M. Verjans Granulocyte Macrophage Colony-Stimulating Factor Expression in Human Herpetic Stromal Keratitis: Implications for the Role of Neutrophils in HSK Invest. Ophthalmol. Vis. Sci., January 1, 2007; 48(1): 277 - 284. [Abstract] [Full Text] [PDF]
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 T. Kielian, A. Haney, P. M. Mayes, S. Garg, and N. Esen Toll-Like Receptor 2 Modulates the Proinflammatory Milieu in Staphylococcus aureus-Induced Brain Abscess Infect. Immun., November 1, 2005; 73(11): 7428 - 7435. [Abstract] [Full Text] [PDF]
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M. Numasaki, M. Watanabe, T. Suzuki, H. Takahashi, A. Nakamura, F. McAllister, T. Hishinuma, J. Goto, M. T. Lotze, J. K. Kolls, et al. IL-17 Enhances the Net Angiogenic Activity and In Vivo Growth of Human Non-Small Cell Lung Cancer in SCID Mice through Promoting CXCR-2-Dependent Angiogenesis J. Immunol., November 1, 2005; 175(9): 6177 - 6189. [Abstract] [Full Text] [PDF]
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M. J. Ruddy, F. Shen, J. B. Smith, A. Sharma, and S. L. Gaffen Interleukin-17 regulates expression of the CXC chemokine LIX/CXCL5 in osteoblasts: implications for inflammation and neutrophil recruitment J. Leukoc. Biol., July 1, 2004; 76(1): 135 - 144. [Abstract] [Full Text] [PDF]
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 M. J. Ruddy, G. C. Wong, X. K. Liu, H. Yamamoto, S. Kasayama, K. L. Kirkwood, and S. L. Gaffen Functional Cooperation between Interleukin-17 and Tumor Necrosis Factor-{alpha} Is Mediated by CCAAT/Enhancer-binding Protein Family Members J. Biol. Chem., January 23, 2004; 279(4): 2559 - 2567. [Abstract] [Full Text] [PDF]
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