Synthesis of ␣-Chemokines IP-10, I-TAC, and MIG Are
Differentially Regulated in Human Corneal Keratocytes
Karla A. McInnis, Andrea Britain, Robert N. Lausch, and John E. Oakes
PURPOSE. Chemokines responsible for recruiting lymphocytes
such as activated T cells into the cornea have not been clearly
defined. IP-10, I-TAC, and MIG are chemoattractants for these
lymphocytes. The goal of this study was to determine whether
human corneal keratocyte (HCKs) in culture synthesize these
chemokines in response to proinflammatory mediators.
METHODS. HCKs grown in vitro were stimulated with IL-1␣,
TNF-␣, or IFN-␥. Induction of ␣-chemokine gene expression
was quantitated by real-time PCR and ELISA. Activation of the
transcriptional activator STAT1 by IFN-␥ receptors expressed
on HCKs was determined by Western blot analysis.
RESULTS. HCKs incubated with TNF-␣, IL-1␣, or IFN-␥ resulted
in a ⬎2000-fold increase in IP-10 protein secretion by 36 hours
after stimulation. In contrast, stimulation with TNF-␣, IL-1␣, or
IFN-␥ induced levels of MIG and I-TAC that were not significantly greater than constitutive levels. Treatment of HCKs with
IFN-␥ activated STAT1 and, in combination with either TNF-␣
or IL-1␣, enhanced MIG and I-TAC synthesis ⬎20-fold.
CONCLUSIONS. IP-10 synthesis is induced in HCKs by IL-1␣,
TNF-␣, and IFN-␥. In contrast, induction of I-TAC and MIG
synthesis in HCKs requires costimulation with IFN-␥ and either
IL-1␣ or TNF-␣. The results suggest therefore, that the upregulation of I-TAC and MIG gene expression at sites of corneal
inflammation are more tightly regulated than that of IP-10. A
role for differential induction of the three ␣-chemokine genes
in corneal inflammatory processes at the eye surface is
discussed. (Invest Ophthalmol Vis Sci. 2005;46:1668 –1674)
DOI:10.1167/iovs.04-1010
T
he cornea is a specialized epidermal surface composed
primarily of an outer layer of epithelial cells overlaying a
layer of transparent connective tissue containing fibroblasts
called keratocytes. In addition to the transmission of light, the
cornea also protects the interior of the eye by acting as a
barrier to infectious agents. Inflammation is an important
mechanism of host resistance to infectious agents that are able
to penetrate the epidermal surface of the cornea and invade its
stromal layer. The major effector cells that participate in these
inflammatory responses include neutrophils and activated T
cells.1–10 The effector functions mediated by these cells in
response to infection can damage corneal tissue and lead to
corneal opacity, neovascularization, and vision loss.1–15
Chemokines are low-molecular-weight secreted peptides
possessing chemotactic properties for leukocytes. Most cheFrom the Department of Microbiology and Immunology, College
of Medicine, University of South Alabama, Mobile, Alabama.
Supported by National Eye Institute Grant EY12713.
Submitted for publication August 23, 2004; revised January 11,
2005; accepted January 13, 2005.
Disclosure: K.A. McInnis, None; A. Britain, None; R.N. Lausch,
None; J.E. Oakes, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: John E. Oakes, 307 University Boulevard,
MSB 2100, Mobile, AL 36688; joakes@jaguar1.usouthal.edu.
1668
mokines fall into two groups on the basis of structure.16,17 The
␣-chemokines are peptides in which the first two cysteines are
separated by an intervening amino acid (CXC), and ␤-chemokines are peptides in which the first two cysteines are adjacent
to each other (CC). In addition, members of the ␣-chemokine
family can be segregated into two groups on the basis of
protein structure.16,17 One group of ␣-chemokines possesses
an ELR tripeptide motif at the N-terminal positions 4, 5, and 6.
This motif is essential for high-affinity binding to CXCR1 and -2
receptors found on neutrophils. Thus, ELR-positive (ELR⫹)
␣-chemokines specifically chemoattract neutrophils to sites of
acute inflammation and therefore play an major role in acute
inflammatory responses. The second group of ␣-chemokines is
characterized by the lack of an ELR motif at the N-terminal
position. These ␣-chemokines possess affinity to the CXCR3
receptors found on cells such as activated T cells and NK
cells.17–19 Therefore, unlike ELR⫹ ␣-chemokines, ELR-negative
(ELRⴚ) ␣-chemokines are potent chemoattractants for lymphocytes involved in cell-mediated immunity.
Human and murine corneal keratocytes (HCKs) can produce several ELR⫹ ␣-chemokines in response to proinflammatory mediators.20 –30 Thus, it is believed that these cells play an
important role in determining the numbers of neutrophils that
accumulate within inflamed corneal tissue. Interferon (IFN)-␥inducible protein 10 (IP-10/CXCL10), IFN-inducible T-cell ␣
chemoattractant (I-TAC/CXCL11), and monokine induced by
IFN-␥ (MIG/CXCL9) belong to the ELRⴚ family of ␣-chemokines.31–33 It is not known whether HCKs produce ELR⫺
␣-chemokines during inflammatory responses. This is an important question, because synthesis of any of the three ELR⫺
␣-chemokines by keratocytes in response to disease or injury
could lead to the recruitment of the effector lymphocytes
responsible for many of the physiological events involved in
the generation of stromal keratitis. The goal of this study
therefore, was to determine whether IP-10, I-TAC, and MIG are
synthesized by HCKs in response to proinflammatory mediators known to be upregulated in inflamed corneal tissue.34 –38
MATERIALS
AND
METHODS
Preparation of HCKs
Human corneas were received from the National Disease Research
Institute Interchange (Philadelphia, PA) within 4 days of enucleation
and were obtained in accordance with the guidelines of the Declaration of Helsinki for research involving human tissue. After the endothelial cell layers were removed from the cornea with forceps, the
corneas were washed repeatedly in keratinocyte serum-free medium
(K-SFM; Invitrogen-Gibco, Grand Island, NY) with supplements (25 mg
bovine pituitary extract and 2.5 ␮g human recombinant EGF; Invitrogen-Gibco) and incubated concave side down overnight in 0.5 mL
grade II Dispase (25 U/mL; Roche, Indianapolis, IN) at 4° C. After
incubation, the epithelial layer was removed with forceps, and the
stromal layer was cut into small pieces and incubated in 3 mL K-SFM
plus supplements and 1000 U/mL type II collagenase for a minimum of
20 minutes and a maximum of 2 hours. At this time, the tissue was
seeded into a 75-cm2 tissue culture flask in 3 mL Dulbecco’s modified
Eagle’s medium (DMEM; Invitrogen-Gibco) containing 20% fetal bovine
Investigative Ophthalmology & Visual Science, May 2005, Vol. 46, No. 5
Copyright © Association for Research in Vision and Ophthalmology
IOVS, May 2005, Vol. 46, No. 5
Synthesis of ␣-Chemokines in Human Corneal Keratocytes
serum, 1⫻ antibiotic-antimycotic (Invitrogen-Gibco), 10 mM HEPES,
1.5% sodium bicarbonate (Invitrogen-Gibco), 0.004 N sodium hydroxide, and 0.2 ␮g/mL kanamycin (Sigma-Aldrich, St. Louis, MO). Once the
cells reached confluence, they were harvested and seeded into 25-cm2
flasks in DMEM containing 10% fetal bovine serum. At 80% confluence,
the medium was then replaced daily with SFM (Opti-MEM I; InvitrogenGibco) supplemented with 5 ␮g/mL gentamicin (Invitrogen-Gibco).
After 3 days, the medium was replaced with 2.0 mL SFM containing
selected concentrations of either human recombinant interleukin
(IL)-1␣ (R&D Systems, Inc., Minneapolis, MN), human recombinant
tumor necrosis factor (TNF)-␣ (Genzyme, Cambridge, MA), or human
recombinant IFN-␥ (PBL Biomedical Laboratories, New Brunswick, NJ).
Supernatants were then collected from cultures at selected times after
stimulation and analyzed for human IP-10, MIG, and I-TAC with ELISA
kits (R&D Systems, Inc.). Synergy was determined by treating the HCKs
with select combinations of TNF-␣ and IFN-␥ or IL-1␣, and IFN-␥ and
comparing the results with single cytokine treatment. Minimum levels
of detection were as follows: hIP-10, 1.67 pg/mL; hMIG, 3.84 pg/mL;
and hI-TAC, 13.9 pg/mL). Results were read on a microplate reader
(MRX; Dynatech Laboratories, Chantilly, VA) at 450 and 550 nm. A
small paired t-test was used to calculate significant differences between
chemokine levels.
Analysis of mRNA Levels by Real-Time PCR
Total cellular RNA was extracted from cell cultures by the acid guanidium thiocyanate-phenol-chloroform method, as described previously.23 RNA was then converted into cDNA with an RNA PCR core kit
(GeneAmp; Applied Biosystems/Roche, Branchburg, NJ) according to
the manufacturer’s instructions. The DNA was then amplified by realtime PCR performed on a PCR thermocycler (iCycler iQ Multi-Color
Real Time PCR Detection System; Bio-Rad, Hercules, CA). Primers were
selected by computer (Beacon Designer v.2.13 software; Premier Biosoft, Palo Alto, CA) and then analyzed by BLAST (www.ncbi.nlm.nih.
gov/blast/ provided in the public domain by the National Center for
Biotechnology Information, Bethesda, MD) to ensure that the primers
were specific and thus amplified only the gene of interest during PCR.
The primer sequences chosen were human IP-10 primers: mRNA
forward oligonucleotide 5⬘-CTTCCAAGGATGGACCACACA-3⬘ and
mRNA reverse oligonucleotide 5⬘-CCTTCCTACAGGAGTAGTAGCAG3⬘; and human GAPD primers: mRNA forward oligonucleotide 5⬘GAAGGTGAAGGTCGGAGTC-3⬘; and mRNA reverse oligonucleotide
5⬘-GAAGATGGTGATGGGATTTC-3⬘.
1669
IFN-␥ receptor mRNA. RT-PCR products were analyzed by adding equal
volumes (15 ␮L) of the PCR-amplified product to individual lanes of a
1.5% agarose gel containing 0.5 ␮g/mL ethidium bromide. Electrophoresis was then performed at 100 V in 1⫻ Tris-acetate-EDTA (TAE)
electrophoresis buffer containing 0.1 ␮g/mL ethidium bromide. The
gel was then viewed with a transilluminator and the image captured
with a zoom digital camera (Digital Science DC120; Eastman Kodak
Co., Rochester, NY). The RT-PCR band intensities were analyzed with
the accompanying software (ID Image Analysis Software for Windows
ver. 2.0.3; Eastman Kodak Co.). The constitutively expressed gene
GAPD served as the positive RT-PCR control to ensure that each
experimental sample contained equal amounts of purified mRNA.
Western Blot Analysis
After incubation for 3 days in KSFM, total protein extracts were
prepared by aspirating the medium and washing cells with ice-cold
PBS. The cells were then scraped and pelleted in a 1.5 mL microcentrifuge tube. They were then resuspended in 100 ␮L SDS lysis buffer
(200 mM Tris-HCl [pH 6.8], 15% glycerol, 4% SDS, and 6% ␤-mercaptoethanol) and pipetted vigorously to lyse the cells. Cell lysates were
stored at ⫺70°C until use.
For Western blot analysis experiments, 20 ␮L of total protein
extracts was boiled for 3 minutes in the SDS lysis buffer and separated
on a 10% Tris-glycine gel (Novex, San Diego, CA) with SDS running
buffer (25 mM Tris-base, 190 mM glycine, and 0.1% SDS). The contents
of the gel were then transferred to a polyvinylidene difluoride (PVDF)
membrane (Invitrogen) with a transfer apparatus (Western Transfer
Apparatus; Novex). Membranes were then blocked with 6% nonfat dry
milk in TBS-T (Tris-buffered saline ⫹ 0.1% Tween) for 1 hour. The
membrane was then washed with TBS-T and incubated overnight with
anti-STAT1 (M-22), anti-p-STAT1 (Tyr 701), or anti-␤-actin (C-11; Santa
Cruz Biotechnology, Santa Cruz, CA) at room temperature in TBS-T
with 0.5% BSA. Blots were washed three times with TBS-T and TBS and
then incubated with the corresponding horseradish-peroxidase (HRP)–
conjugated secondary antibody (␣-rabbit-HRP; Pierce, Rockford, IL;
␣-goat-HRP, Sigma-Aldrich) for 2 hours in TBS-T with 0.5% BSA. The
blot was then washed as just described and immunoblot signals detected with chemiluminescent substrate (Super Signal West Dura Extended Duration Substrate; Pierce).
RESULTS
Reverse Transcription–Polymerase
Chain Reaction
IP-10, I-TAC, and MIG Synthesis in HCKs after
Stimulation with IL-1␣ and TNF-␣
Total RNA was extracted and analyzed for the presence of IFN-␥
receptor mRNA by RT-PCR as previously reported.23 Primers were
selected with the program Primer Quest (purchased from IDT, Coralville, IA, at http://www.idtdna.com) and analyzed with BLAST. The
primer sequences selected and the expected RT-PCR product size are
as follows: human IFN-␥ receptor primers (mRNA 321-bp product):
mRNA forward oligonucleotide 5⬘-CCAAGTCCTTGATCTCTGTGG-3⬘;
and mRNA reverse oligonucleotide 5⬘-CTGCCAGGTTCAGACTGGTT3⬘; and human GAPD primers (mRNA 417-bp product): mRNA forward
oligonucleotide 5⬘-CCAAAGGGTCATCATCTCTGC-3⬘; mRNA reverse
oligonucleotide 5⬘-ATTTGGCAGGTTTTTCTAGACGG-3⬘. For the PCR
reaction, a master mix containing DNA polymerase (AmpliTaq; Applied Biosystems/Roche) and appropriate primers was prepared according to the manufacturer’s instructions. Twelve-microliter aliquots
were then dispersed into PCR tubes along with 3 ␮L cDNA, for a total
volume of 15 ␮L. RT-PCR products were amplified using an initial
thermocycle of 2 minutes at 95°C followed by thermocycles of 30
seconds at 95°C, 30 seconds at 65°C, and 2 minutes at 72°C. Preliminary experiments established that 20 to 22 cycles of amplification were
within the exponential amplification phase of GAPD, whereas 28
cycles of amplification were needed to amplify detectable levels of
Previous studies in our laboratory have shown that HCKs
produce maximum levels of ELR⫹ ␣-chemokines in response to
1000 U/mL of IL-1␣ or 500 U/mL of TNF-␣.23–25,30 Therefore,
preliminary experiments were performed to determine
whether IP-10, I-TAC, and MIG expression could be induced in
HCKs in response to these doses of proinflammatory mediators. Over the course of 36 hours, supernatants were collected
from HCKs stimulated with either IL-1␣ (1000 U/mL) or TNF-␣
(500 U/mL), and protein levels of IP-10, MIG, and I-TAC were
analyzed by ELISA. It was found that TNF-␣ stimulation increased IP-10 expression ⬎15,000-fold, whereas IL-1␣ stimulation enhanced IP-10 expression ⬎3000-fold (Fig. 1A).
IP-10 mRNA levels were also increased in IL-1␣- and TNF␣-stimulated cells by ⬎1000-fold, as determined by real-time
PCR (Fig. 1B). Dose–response experiments demonstrated that
IP-10 gene expression is responsive to concentrations of IL-1␣
and TNF-␣ as low as 5 U/mL (Fig. 1C). In contrast, IL-1␣ did not
stimulate detectable synthesis of either MIG or I-TAC, whereas
TNF-␣ stimulated low-picogram levels of MIG synthesis that
were only detectable at 36 hours after stimulation (data not
shown).
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IOVS, May 2005, Vol. 46, No. 5
FIGURE 2. IFN-␥ receptor mRNA was constitutively expressed in
HCKs. Total RNA was extracted from cells from two different donors
and stimulated with 100 U/mL IFN-␥ or media alone for 18 hours. IFN-␥
receptor and GAPD mRNA levels were analyzed by RT-PCR.
To determine whether HCKs express message for IFN-␥
receptors, RNA was extracted from HCKs and analyzed for the
presence of receptor mRNA by RT-PCR. It was found that IFN-␥
receptor mRNA was constitutively expressed by HCKs (Fig. 2).
Functional IFN-␥ receptors transduce a signal that leads to
activation of STAT1 through tyrosine phosphorylation, inducing formation of STAT1:STAT1 homodimers.43,44 To determine
whether the IFN-␥ receptors synthesized by HCKs are membrane bound and functional, cell cultures were treated with
IFN-␥ at 100 U/mL for a period of 0.5 to 6 hours. Total protein
extracts were then collected and analyzed by Western blot for
the presence of phosphorylated STAT1 and total STAT1. ␤-Actin served as the internal control (Fig. 3). Phosphorylation of
STAT1 was detected as early as 30 minutes after stimulation
(Fig. 3, lane 2). After reaching its peak at 1 hour after stimulation, levels of activated STAT1 began to decline until they were
no longer detectable at 6 hours (Fig. 3, lanes 3– 6). These
results suggest that the IFN-␥ receptors are expressed on the
HCK membranes and are capable of responding to IFN-␥.
To determine whether IFN-␥ activated receptors induce
IP-10, I-TAC, and MIG synthesis, production of the three ␣-chemokines was analyzed in HCKs after stimulation with increas-
FIGURE 1. IP-10 protein synthesis and intracellular IP-10 mRNA levels
in HCKs stimulated with IL-1␣ or TNF-␣. (A) Cells were stimulated with
either 1000 U/mL IL-1␣ or 500 U/mL TNF-␣ for 36 hours. Culture
supernatants were harvested at the time indicated, and IP-10 levels
were quantitated by ELISA. (B) Total RNA was isolated from the
cultures, and intracellular mRNA levels were analyzed by real-time PCR
and calculated as the increase (x-fold) over media treated samples. (C)
Supernatants were collected from cells treated with increasing concentrations (5, 10, 20, 30, 50, and 100 U/mL) of TNF-␣ or IL-1␣ for 18
hours and assayed for IP-10 by ELISA. Quantitative results for four
donors are shown as mean ⫾ SEM (*P ⬍ 0.05).
IP-10, I-TAC, and MIG Synthesis in HCKs after
Stimulation with IFN-␥
In many cell types, the genes encoding IP-10, I-TAC, and MIG
are responsive to IFN-␥ stimulation.39 – 42 Because the presence
of the IFN-␥ receptor on HCKs has not been firmly established,
it was of interest to determine whether these cells possess
IFN-␥ receptors and, if so, whether IFN-␥ has the capacity to
induce synthesis of the three ␣-chemokines.
FIGURE 3. IFN-␥ receptor was functionally capable of signal transduction in HCKs. Total protein was extracted from cells (two donors) that
were stimulated with IFN-␥ (100 U/mL) for up to 6 hours or media
alone (6 hours) and separated by gel electrophoresis. The gel was then
blotted onto a PVDF membrane, which was probed for p-STAT1, total
STAT1, or ␤-actin. The proteins were visualized on a luminescent
image analyzer (LAS-1000 Plus; Fuji, Tokyo, Japan).
IOVS, May 2005, Vol. 46, No. 5
Synthesis of ␣-Chemokines in Human Corneal Keratocytes
1671
18 hours. This time point was selected because HCKs stimulated with IL-1␣, TNF-␣, or IFN-␥ for 18 hours failed to produce
MIG or I-TAC. Therefore, if synthesis of either chemokine is
induced in HCKs stimulated with combinations of inducers for
18 hours, the results would suggest that synergy was involved
in the induction. Figure 5 shows that HCKs stimulated with
combinations of either IFN-␥ and IL-1␣ or IFN-␥ and TNF-␣
produced ⬍1500 pg of MIG (Fig. 5A) and ⬎500 pg of I-TAC
(Fig. 5B). These levels were ⬎24 times higher that those
obtained when HCKs were individually induced with IL-1␣,
TNF-␣, or IFN-␥ alone or with combinations of IL-1␣ and
TNF-␣.
We also tested whether IP-10 synthesis could be synergistically enhanced after exposure to a combination of cytokines. It
was found that when HCKs were exposed to IL-1␣ (10 and 20
U/mL) and TNF-␣ (10 and 20 U/mL) in combination with IFN-␥
(10 and 20 U/mL), the levels of IP-10 produced were enhanced
more than fourfold above that obtained with a single stimulant
(Fig. 6). In contrast, treatment of HCKs with a combination of
IL-1␣ and TNF-␣ did not synergistically enhance IP-10 synthesis. These results demonstrate that cytokine combinations containing IFN-␥ elevate IP-10 gene expression.
FIGURE 4. Dose–response curve and kinetics for IP-10 synthesis in
HCKs stimulated with IFN-␥. (A) Supernatants were collected from
cells stimulated with increasing concentrations (5, 10, 20, 30, 50, and
100 U/mL) of IFN-␥ for 18 hours. Supernatants were then assayed for
IP-10, MIG, and I-TAC by ELISA. (B) Supernatants were collected from
HCKs stimulated with IFN-␥ (100 U/mL) over a 36-hour period and
analyzed for IP-10 by ELISA. Quantitative results for at least two donors
are shown as the mean ⫾ SEM (*P ⬍ 0.05).
ing doses of IFN-␥ (Fig. 4). In cell cultures receiving media
only, IP-10, I-TAC, and MIG synthesis was below the level of
detection. When treated with IFN-␥ (100 U/mL), HCKs produced ⬎400 pg of IP-10 (Fig. 4A). In contrast, neither the MIG
nor I-TAC level was increased significantly after IFN-␥ stimulation when compared with HCKs treated with media alone. We
also performed kinetic studies to determine the levels of IP-10
produced by HCKs, in response to IFN-␥ over a 36-hour period.
IP-10 was secreted at levels ⬎2000-fold higher than untreated
cells (Fig. 4B). These results indicate that HCKs exposed to
IFN-␥ produce an increased amount of IP-10 but little to no
MIG or I-TAC.
I-TAC and MIG Synthesis in HCKs Stimulated with
IFN-␥ and IL-1␣ or IFN-␥ and TNF-␣
Both IL-1␣ and TNF-␣ can synergize with IFN-␥ to enhance
I-TAC and MIG gene expression in human astrocytes and neutrophils.40,41 Therefore, we tested whether costimulation
would provide a stimulus strong enough to induce synthesis of
these ELRⴚ ␣-chemokines. MIG and I-TAC synthesis was determined in cells stimulated with combinations of cytokines for
FIGURE 5. Combinations of IFN-␥ and IL-1␣ or IFN-␥ and TNF-␣ increase MIG and I-TAC production by HCKs. Cells were stimulated with
combinations of IFN-␥, TNF-␣, and IL-1␣ at 10 or 20 U/mL for 18 hours.
Supernatants were then collected and assayed for MIG (A) and I-TAC
(B) by ELISA. Quantitative results for three donors are shown as the
mean ⫾ SEM (*P ⬍ 0.05).
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McInnis et al.
FIGURE 6. Combinations of IFN-␥ and TNF-␣ or IFN-␥ and IL-1␣ increase IP-10 production in HCKs. Cells were stimulated with combinations of IFN-␥, TNF-␣, and IL-1␣ at 10 or 20 U/mL for 18 hours.
Supernatants were then collected and assayed for IP-10 by ELISA.
Quantitative results for three donors are shown as the mean ⫾ SEM
(*P ⬍ 0.05).
DISCUSSION
IL-1␣ and TNF-␣ induce gene expression by activating the
cytoplasmic transcriptional activator NF-␬B,45,46 whereas IFN-␥
induces gene expression by activating the cytoplasmic transcriptional factor STAT1.43,44 Because the promoters upstream
of the genes for IP-10, MIG, and I-TAC all possess binding sites
for NF-␬B and STAT1, one would expect that expression of all
three genes would be inducible by IL-1␣, TNF-␣, and IFN-␥
stimulation. This hypothesis is supported by the fact that the
three inducers can individually upregulate IP-10, I-TAC, and
MIG gene expression in selected cell types and strains, such as
astrocytes, neutrophils, and NIH 3T3 cells.39 – 41 Therefore, the
unexpected finding in this study was that IL-1␣, TNF-␣, and
IFN-␥ stimulated HCKs to synthesize nanogram levels of IP-10
but little I-TAC or MIG. It is not known why IP-10 is differentially upregulated in HCKs. One possible explanation is that the
number and arrangements of NF-␬B and STAT1 binding motifs
on the three promoters are not identical.33,47,48 For DNA
bound transcriptional activators to upregulate gene expression, they must interact with several cotranscriptional factors
that act as a bridge between the activator and the transcriptional apparatus bound to the transcriptional start site.49 –51
Therefore, it is possible that the mix of cotranscriptional factors produced by HCKs are more efficient at interacting with
NF-␬B and STAT1 complexes bound to IP-10 promoters than to
the promoters of the I-TAC and MIG genes.
We also observed that IP-10 protein levels increased in a
time-dependent manner even though expression of IP-10
mRNA remained almost constant after 12 hours. One possible
explanation is that sustained stimulation of HCKs with chemokine inducers enhances the efficiency of IP-10 mRNA translation.52 A second observation made in the kinetic studies of
IP-10 synthesis was that while all three cytokines induced IP-10
synthesis, the secreted levels of IP-10 were higher in cells
treated with TNF-␣ (⬎11-fold) and IL-1␣ (⬎3.5-fold) than those
in IFN-␥–treated cells. It is quite possible that TNF-␣- and
IOVS, May 2005, Vol. 46, No. 5
IL-1␣-activated NF-␬B enhances transcription more efficiently
than IFN-␥-activated STAT1.
We further observed that, whereas HCKs exposed to a
single cytokine did not produce significant levels of I-TAC and
MIG, both were produced at levels ranging from 250 picograms to ⬎1 ng when HCKs were costimulated with IFN-␥
preparations containing either IL-1␣ or TNF-␣. Because both
the I-TAC and MIG promoters possess a STAT1 binding site in
addition to a NF-␬B binding site, these results suggest that
I-TAC and MIG gene expression can only be activated in HCKs
after the promoters of the two genes have interacted with both
NF-␬B and STAT1.33,53,54 In addition to I-TAC and MIG, the
levels of IP-10 produced by IL-1␣- and TNF-␣-stimulated HCKs
were also significantly enhanced when IFN-␥ was added into
the IL-1␣ or TNF-␣ preparations used to stimulate the cells.
These results are probably attributable to the fact that cobinding of NF-␬B and STAT1 transcriptional activators to the IP-10
promoter also significantly enhances expression of this ELRⴚ
␣-chemokine gene.
IL-1␣ and TNF-␣ are the primary proinflammatory cytokines
released into corneal tissue at sites of injury or disease.34 –38
These proinflammatory mediators activate synthesis of a number of ELR⫹ ␣-chemokines that play an important role in
chemoattracting neutrophils into sites of inflammation.20 –25,30
It is interesting that the only ELR⫺ ␣-chemokine produced by
IL-1␣- and TNF-␣-stimulated HCKs is IP-10. This finding has
potential physiological relevance. Corneal epithelial cells store
physiologically active IL-1␣.36,55,56 After damage to the epithelial cell membrane, IL-1␣ can be immediately released into
corneal extracellular spaces. Based on the results of this study,
one can speculate that IP-10 is produced in diseased or injured
corneal tissue before I-TAC and MIG. One activity that IP-10
possesses is the capacity to enhance binding of activated T
cells to endothelial cells lining the walls of blood vessels.57
Thus, one in vivo function of the IP-10 produced by HCKs in
response to IL-1␣ may be to enhance extravasation of activated
T cells from blood vessels located in limbal areas of the cornea.
In contrast to IP-10, the synthesis of I-TAC and MIG by HCKs
require costimulation with IFN-␥. Because IFN-␥-producing
cells are predominantly lymphocytes,58 synthesis of I-TAC and
MIG may not occur within inflamed corneal tissue until IFN-␥producing lymphocytes have infiltrated the site of inflammation.
In conclusion, it has been found that HCKs can synthesize
the three ELRⴚ ␣-chemokines essential in chemoattracting activated T-cell subsets to sites of inflammation. It has been
reported that HCKs influence the intensity of innate immune
responses generated in stromal layers of the cornea by producing multiple ELR⫹ ␣-chemokines.23–25,30 The results of this
study suggest that the ELRⴚ ␣-chemokines produced by HCKs
may also play an important role in regulating acquired immune
responses.
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