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*
National Eye Institute, National Institutes of Health, Bethesda, MD 20892; and
DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the eye, both of these destructive mechanisms contribute to vision
loss. Misdirected autoimmune responses against ocular Ags, with ensuing
inflammation, can occur spontaneously or after ocular injury. Also,
defensive responses to foreign Ags in the eye can cause inflammation.
Whatever the inciting event, even transient inflammation threatens
vision, as it can destroy the transparency of the lens and cornea and
the integrity of retinal components. Currently, ocular inflammation is
responsible for 
10–15% of vision loss in the United States
(1, 2).
To control deleterious immune responses, it is essential to identify the molecules that promote these responses. Proinflammatory cytokines are key players in deleterious autoimmune responses (3). Other important candidates are the chemokines, a family of 8- to 14-kDa proteins that mediate leukocyte emigration from blood vessels into tissues. Over 40 human and mouse chemokines have been identified. In the last several years, chemokines have been shown to play critical roles in the development of inflammation in diverse disease models (4, 5, 6). Chemokines are produced by ocular cells cultured in vitro, and several chemokines have been detected in ocular fluids and tissues (7, 8, 9, 10, 11, 12, 13, 14). However, a complete analysis of chemokine involvement in the development of ocular inflammation has not been conducted.
Quantitative identification of multiple mRNA transcripts in a small tissue sample has recently become possible with the advent of kinetic (real time) RT-PCR (15). In this study, we utilized this technique to characterize the expression of 74 mRNA transcripts of cytokines, chemokines, and chemokine receptors during T cell-induced ocular inflammation. We examined a murine model in which transgenic (Tg)2 mice expressing hen egg lysozyme (HEL) in the lens of the eye are injected with T cells specific for HEL (16). In this model, both Th1- and Th2-polarized cells induce eye inflammation when introduced into irradiated mice. The inflammatory infiltrates induced by Th1 and Th2 cells differ, containing primarily mononuclear (MNL) or polymorphonuclear (PMN) cells, respectively. By analyzing these mice, we were able to compare the complement of cytokine, chemokine, and chemokine receptor mRNA transcripts expressed in the eye during Th1- and Th2-polarized responses attracting distinct leukocyte subsets. We compared these patterns to mediator expression patterns in experimental autoimmune uveitis (EAU), a well-characterized animal model of human autoimmune eye disease. To our knowledge, the findings detailed in this work represent the most comprehensive analysis to date of 1) inflammatory mediator transcript expression during the development of ocular inflammation, and 2) chemokine transcript expression in Th1- vs Th2-induced inflammation. The results of this large-scale study offer a new perspective on the role of individual mediators in the development of inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HEL-Tg mice, expressing membrane-bound HEL, on the FVB/N background, were generated as detailed elsewhere (17). HEL-specific TCR Tg mice, on the B10.BR background, designated 3A9 (18), were a generous gift from M. Davis (Stanford University, Stanford, CA). Tg mice from each of the two lines were mated to produce (FVB/N x B10.BR)F1 hybrids, expressing either one of the two transgenes. Only such F1 hybrid mice were used in all experiments of the present study. The mice were housed in a pathogen-free facility, and all manipulations were conducted in compliance with the National Institutes of Health Resolution on the Use of Animals in Research.
Preparation of HEL-specific Th1 and Th2 cells
Th1 and Th2 cells expressing a HEL-specific TCR were prepared as
follows: spleen and lymph node cells of 3A9 mice were pooled and the T
cell fraction was partially purified on enrichment columns (R&D
Systems, Minneapolis, MN). CD4 cells were then purified by magnetic
sorting with SuperMACS, using beads directly coupled to anti-mouse
CD4 (Miltenyi Biotec, Sunnyvale, CA). Purified (>97%) CD4 cells were
then cultured at 2.5 x 105/ml in RPMI 1640
medium supplemented with 50 µM 2-ME, antibiotics, and 10% FBS
(complete medium) with 2.5 x 106/ml
syngeneic wild-type splenocytes, irradiated with 30 Gy, as APC, in the
presence of either 2 µg/ml HEL (Sigma-Aldrich, St. Louis, MO), 10
ng/ml IL-12 (Sigma-Aldrich), and 10 µg/ml anti-IL-4 Ab (BD
PharMingen, San Diego, CA) for Th1, or 0.2 µg/ml HEL, 10 ng/ml IL-4
(BD PharMingen), 10 µg/ml anti-IFN-
Ab (BD PharMingen), and 10
µg/ml anti-IL-12 Ab (BD PharMingen) for Th2. After 3 days,
cultured cells were expanded with 40 IU/ml IL-2 (Chiron, Emeryville,
CA) for 3–5 days and then restimulated at 2.5 x
105/ml with 2.5 x 106
irradiated syngeneic APC in the presence of either 2 µg/ml HEL, 40
IU/ml IL-2, and 10 ng/ml IL-12 for Th1, or 0.2 µg/ml HEL, 40 IU/ml
IL-2, and 10 ng/ml IL-4 for Th2. Three days later, cells were
harvested, washed, resuspended in RPMI 1640, and injected i.v. into
recipient mice, as indicated.
Adoptive transfer of Th1 and Th2 cells
Upon injection, the T cell preparations were >90% alive and blastic, and >97% CD4 positive. Recipient HEL-Tg mice were irradiated 4–5 h before cell injection with 4.5 Gy. Recipient mice were sacrificed immediately before cell injection, or 2, 4, or 7 days thereafter. At the time of sacrifice, eyes were removed. One eye was frozen for later RNA preparation or microdissection, and the other eye was used for histological analysis. Eye sections were stained by conventional H&E or by an eosinophil stain that highlights cyanide-resistant eosinophil peroxidase (19, 20). Briefly, eye frozen sections were fixed in 1% Formalin in acetone for 30 s, then stained for 10 min in PBS containing 0.4 mg/ml NaCN, 3 µl/ml 3% hydrogen peroxide, and 0.75 mg/ml diaminobenzidine (Sigma-Aldrich). Slides were then counterstained in Gills hematoxylin for 1 min. For comparison, serial sections from the same eyes were stained with H&E.
Assessment of histological changes
The severity of ocular inflammation in recipient mice was scored by two investigators, who separately evaluated the level of inflammation in the anterior segment, vitreous, and retina, on a scale of 0–3. The final score consisted of the sum of the three subscores, with a maximum value of 9. Severity of changes was assessed according to tissue structural changes, intensity of cellular infiltration, and levels of proteinaceous exudates.
EAU induction
EAU was induced as follows. B10.A mice (The Jackson Laboratory, Bar Harbor, ME) were immunized with 50 µg bovine interphotoreceptor retinoid-binding protein (IRBP, a generous gift from B. Wiggert, National Eye Institute, National Institutes of Health), emulsified in CFA containing 2.5 mg/ml Mycobacterium tuberculosis H37RA. The emulsion was injected into the base of the tail and two thighs in a total volume of 0.2 ml, and the mice were concurrently injected i.p. with 0.5 µg pertussis toxin (Sigma). Mice were sacrificed on day 21 postimmunization, and their eyes were processed for RNA analysis and histological examination, as described above.
Quantitative RT-PCR
Real time RT-PCR assays were performed to specifically quantify levels of RNA transcripts. RNA was prepared from frozen eyes as follows. First, each eye was solubilized using TRIzol reagent (Life Technologies, Gaithersburg, MD) in a tissue homogenizer (Kontes Glass Company, Vineland, NJ). RNA was extracted with chloroform/isoamyl alcohol, precipitated with ethanol, and resuspended in diethyl pyrocarbonate water using standard procedures. Isolated RNA was incubated with 10 U DNase I (Boehringer Mannheim, Indianapolis, IN) in the presence of RNasin (Promega, Madison, WI) for 30 min at 37°C. The samples were then heat inactivated at 95°C for 10 min, chilled, and reverse transcribed with Superscript II reverse transcriptase (Life Technologies) with random hexamers, according to the manufacturer’s protocol. Primers were either obtained from Perkin-Elmer (Foster City, CA), or generated with Primer Express software (Perkin-Elmer) and were synthesized in the DNAX core facility, as reported previously (21, 22). Whenever possible, primer pairs were designed to span intron/exon borders. Samples were then subjected to 40 cycles of amplification at 95°C for 15 s, followed by 60°C for 1 min using an ABI Geneamp 5700 sequence detection system and SYBR green buffer, according to the manufacturer (Perkin-Elmer). PCR amplification of the housekeeping gene ubiquitin was performed for each sample to control for sample loading and to allow normalization between samples, according to the manufacturer’s instructions (Perkin-Elmer). Both water and genomic DNA controls were included to ensure specificity. Each data point was examined for integrity by analysis of the amplification plot and dissociation curves.
In adoptive transfer experiments, replicate samples were treated as follows. For experimental samples, mRNA from eyes of three replicate mice was extracted and analyzed separately. For control mice, mRNA from three replicate eyes was extracted and reverse transcribed, then equivalent amounts of individual cDNA reactions were combined to create pooled samples for real time RT-PCR.
Data analysis
For quantitative RT-PCR experiments, mRNA levels for each primer pair were normalized to the housekeeping gene ubiquitin, according to the manufacturer’s instructions (Perkin-Elmer). Ubiquitin-normalized values from experiments performed with identical reaction conditions can be compared with each other, although variations in primer pair efficiency make these comparisons approximate. Quantitative RT-PCR for all adoptive transfer experiment samples and controls were performed at the same time to enable comparisons of ubiquitin-normalized values. Similarly, all PCR reactions for EAU experiments and relevant controls were performed at the same time to facilitate comparison. Note, however, that the ubiquitin-normalized values between these two data sets should not be directly compared.
Calculations of the fold increase in mRNA level from baseline (shown in
Table I
) were performed as follows. For
adoptive transfer experiments, the maximal mean mRNA level detected at
any time point (usually at day 4 for Th1-induced disease and day 7 for
Th2-induced disease) was used as the peak mRNA level. The mRNA level
present in pooled samples at time zero (after irradiation, but before T
cell injection) was used as the baseline level. Presented in Table I is
the following ratio: peak mRNA level/baseline mRNA level. For EAU
experiments, peak level was the mean mRNA level at day 21, and
the baseline was the mean mRNA level in unmanipulated B10A mouse eyes.
All values were rounded to two significant digits. Ratios of the fold
increase in Th1-induced disease to the fold increase in Th2-induced
disease (which also represents the ratio of the peak value in
Th1-induced disease to the peak value in Th2 disease) are also shown in
Table I. In compiling the data, molecules that were undetectable at
time zero were assigned a baseline value of 1 for the purpose of
calculating a ratio. Most values represent the average of three mRNA
levels determined from three replicate mouse eyes; some represent the
average of two levels if one reaction failed.
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Retinal pigment epithelium (RPE) cells or infiltrating leukocytes were isolated from mouse eye frozen sections using a PixCell II laser microdissection microscope (Arcturus, Mountain View, CA). Infiltrating leukocytes or RPE cells were identified by light microscopy and removed to a membrane using laser settings of power 35 mW, duration 3 ms, spot size 7.5 µm, or power 40 mW, duration 3.5 ms, spot size 7.5 µm, respectively. The captured RPE samples also contained small numbers of adjacent resident cells, mostly choroidal capillary endothelial cells and fibroblasts.
RT-PCR of microdissected samples
RNA was extracted from RPE cells or infiltrating leukocytes
captured by laser microdissection as follows. A total of 200 µl
TRIzol (Life Technologies) was used to lyse the cells. RNA was isolated
by chloroform extraction and isopropanol precipitation, according to
the manufacturer’s instructions. After digestion with DNase, total RNA
was used for cDNA synthesis. A Superscript II RNase
H- reverse transcriptase system (Life
Technologies) and random primers (Promega) were employed. PCR was
performed with 2 µl cDNA, 3 pmol of each
32P-labeled primer, 4 nmol of each dNTP, 1x
GeneAmp buffer, 1 U AmpliTag Gold Polymerase (Perkin-Elmer), and a
final concentration of 1.5 mM MgCl2. The
following primer sequences were used for PCR amplification: monokine
induced by IFN- (MIG)/CXCL9, 5'-GATCAAACCTGCCTACATCC-3'
and 5'-GGCTCTGTAGAACACAGAGT-3'; macrophage-inflammatory protein
(MIP)-1/CCL9, 5'-GCCCACTAAGAAGATGAAGCCT-3' and
5'-CCTTCTCTAAAGCAAATGTAA-3'; eotaxin/CC chemokine ligand
(CCL)11, 5'-TAGGT AAGCAGTAACTTCCATCTGTCTC-3' and
5'-TGACTAAATCAAGCAGTTCTTAGGCTCTG-3'; RANTES/CCL5,
5'-CCTCACCATCATCCTCACTGC-3' and 5'-TCTTCTCTGGGTTGGCACACA-3';
C-10/CCL6, 5'-ATAACGCGTATGCAGGCCTCATACAAGAAATGG-3' and 5'-TAC
TGCAGTCAAGCAATGACCTTGTTC-3'; 18S, 5'- AGGAATTGACGGA AGGGCAC-3' and
5'-GGACATCTAAGGGCATCACA-3'.
Primer sequences for RANTES/CCL5 (23), C-10/CCL6
(24) eotaxin/CCL11, MIP-1/CCL9, and MIG/CXCL9
(25) were described in the corresponding cited
publications. MIG/CXCL9, MIP-1/CCL9, and C10/CCL6 primers were
obtained from Life Technologies, RANTES/CCL5 primers were a generous
gift from N. Tuaillon (National Eye Institute, National Institutes of
Health), and eotaxin/CCL11 primers were a generous gift from J. Farber
(National Institute of Allergy and Infectious Diseases, National
Institutes of Health). The 18S primers were purchased from Ambion
(Austin, TX).
Primers were labeled with 32P before PCR. Reactions were conducted in PCR Express Thermal Cycler (Hybaid, Middlesex, U.K.) for 40 cycles with an annealing temperature of 60°C, a denaturing temperature of 94°C, and an extension temperature of 72°C. PCR products were separated on polyacrylamide gel, and radioactive bands were detected on a PhosphorImager Storm 860 (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ocular inflammation was induced by adoptively transferring
HEL-specific polarized Th1 or Th2 cells into Tg recipient mice
expressing HEL in their lens. Fig. 1
summarizes the mean histological severity of ocular inflammation in
groups of recipient mice examined on day 2, 4, or 7 after T cell
injection. Eyes of mice injected with Th1 cells showed no changes on
day 2, mild to moderate inflammation on day 4, and moderate to severe
inflammation on day 7 postinjection. Mice injected with Th2 cells had
no ocular changes on day 2, none to mild inflammation on day 4, and
mild to moderate ocular inflammation on day 7. It is of note that Th1
cells are more potent than Th2 cells in this system, inducing more
severe disease even when injected at a much smaller number than Th2
cells (1 x 106 vs 20 x
106 per recipient, respectively). Fig. 2A demonstrates typical
histological changes in eyes of recipient mice. Th1 cell-induced
disease is characterized by MNL infiltration, proteinaceous exudate,
and retinal folding, whereas Th2 recipient eyes show infiltration with
mostly PMNs, many of which are eosinophils (Fig. 2B). These
ocular changes in Th1 and Th2 recipient mice are consistent with those
observed and described in more detail in our previous study
(16)
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To identify inflammatory mediators involved in eye inflammation,
we measured mRNA levels of 34 cytokines, 26 chemokines, and 14
chemokine receptors by real time RT-PCR in eyes of recipient mice on
days 0, 2, 4, and 7 post cell injection. Fig. 3 summarizes the kinetics of changes in
mRNA levels of molecules that increased significantly in inflamed eyes,
or exhibited high levels of baseline expression. Table I
shows the
complete list of tested molecules and records their baseline levels at
day 0, as well as their highest measured mRNA levels, presented as fold
increase over the baseline. In Fig. 3, molecules are divided into those
that were preferentially expressed in Th1 (a) or Th2
(b) recipients, or those that were similarly expressed in
eyes of both recipient groups (c).
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a, the inflammatory molecules
preferentially expressed in Th1 recipient eyes included mRNA
transcripts of eight cytokines (IL-1
, IL-1
, IL-1R
antagonist, IL-6, IL-18, TNF-, IFN-, and osteopontin),
eleven chemokines (TCA-3/CCL1, MCP-1/CCL2, MIP-1/CCL3,
MIP-1/CCL4, RANTES/CCL5, MCP-3/CCL7, MIP-3/CCL19, MIP-2/CXCL1–3,
MIG/CXCL9, IFN--inducible protein-10 (IP-10)/CXCL10, and
lymphotactin/XCL1), and five chemokine receptors (CCR1, CCR2, CCR5,
CXCR3, and CCR7). In the eyes of Th1 recipients, significant increases
were also observed in the mRNA levels of IL-12p40 and IL-12p35 (Table I), but the peak normalized units of these cytokines (240 and 416,
respectively, on day 4) were quite low and, therefore, were not
included in Fig. 3. Considerably fewer molecules were preferentially
expressed in eyes of Th2 cell recipients (Fig. 3b). These
included transcripts of four cytokines (IL-4, IL-5, IL-9, and IL-13),
two chemokines (eotaxin/CCL11 and TARC/CCL17), and one chemokine
receptor (CCR-3). Molecules that were similarly expressed in recipients
of Th1 or Th2 cells (Fig. 3c) included transcripts of four
cytokines (TGF-2, TGF-3, platelet-derived growth factor
(PDGF)-, and PDGF-R), six chemokines (C-10/CCL6, MIP-1/CCL9,
MDC/CCL22, CTACK/CCL27, BRAK/CXCL14, and fractalkine/CX3CL1),
and two chemokine receptors (CXCR6 and CX3CR1).
Examination of the data shown in Fig. 3 and Table I reveals several
points of interest. 1) In general, cytokine mRNA expression patterns in
this model fit known Th1 and Th2 associations. 2) Chemokine mRNA
expression patterns were consistent with the type of inflammatory
infiltrate seen in each disease (i.e., eyes with Th1 disease expressed
transcripts of T cell and monocyte chemoattractants, whereas eyes with
Th2 disease up-regulated the mRNA of eosinophil attractant
eotaxin/CCL11). 3) Chemokine mRNA expression patterns seen in this
study contribute new information regarding associations with Th1- or
Th2-induced inflammation (see Discussion). 4) Minimal or no
expression of cytokine or chemokine transcripts was detected in
recipient eyes on day 2 post cell transfer, whereas steep increases in
mRNA for these mediators were observed by day 4. 5) Peak mRNA
expression was observed on day 4 for most mediators in Th1 recipient
eyes, but on day 7 for mediators preferentially expressed in Th2
recipient eyes. 6) Chemokine receptor transcripts seen in Th1- or
Th2-induced disease states matched the chemokine ligand mRNAs that were
present. Furthermore, all chemokine receptor transcripts expressed in
eyes of either Th1 or Th2 recipients reached their highest expression
level on day 7, i.e., after peak mRNA expression of their chemokine
ligands (in most cases). 7) mRNA of several inflammatory molecules were
expressed at significant levels in uninflamed control eyes. These
included six cytokines (IL-18, TGF-2, TGF-3, PDGF-, PDGF-R,
and osteopontin), five chemokines (C-10/CCL6, MIP-1/CCL9,
Eotaxin/CCL11, BRAK/CXCL14, and fractalkine/CX3CL1), and two chemokine
receptors (CXCR6 and CX3CR1). Fig. 3 and Table I show the measured
levels of these constitutively expressed mRNA molecules in mice on day
0. Similar constitutive expression of these thirteen molecules was
observed in eyes of two other control groups, namely, intact
(nonirradiated) HEL-Tg mice (data not shown) and naive B10.A mice (see
below). Interestingly, the expression levels of nine of these thirteen
molecules did not change during the inflammatory process in the eyes of
Th1 or Th2 recipients (Fig. 3).
Chemokines are produced by both infiltrating cells and resident ocular cells
Multiple studies have indicated that ocular cells have the
potential to produce chemokines (7, 9, 10, 11). To define the
origin of abundantly expressed mRNA transcripts in our model, we
isolated both infiltrating leukocytes and RPE from frozen sections of
inflamed eyes using laser capture microdissection. Fig. 4A shows a representative
microdissection of an eye section with Th1-induced inflammation.
Microdissected samples from this and other sections of inflamed eyes of
Th1 or Th2 recipients were used to detect specific chemokine
transcripts, employing conventional RT-PCR (Fig. 4B). As
expected, MIG/CXCL9 and RANTES/CCL5 transcripts were detected in Th1
disease samples only, eotaxin/CCL11 transcripts were detected in Th2
samples only, and C-10/CCL6 transcripts were detected in all samples.
Notably, RANTES/CCL5 transcripts were detected in RPE extracts only,
whereas MIG/CXCL9 transcripts were detected in both RPE and leukocyte
extracts. Eotaxin/CCL11 mRNA was also detected in both RPE and
leukocyte extracts, with a larger signal from the RPE extracts.
C-10/CCL6 transcript was detected in both leukocyte and RPE extracts at
approximately equal levels.
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/CCL9 transcripts were detected only in eye
samples with Th2-induced disease, and only in the infiltrating
leukocytes. These data indicate that the predominantly eosinophilic
infiltrate seen in Th2-induced inflammation was a source of
MIP-1/CCL9. Furthermore, MIP-1/CCL9 mRNA was present in whole eye
extracts from Th1-induced inflammation (Table I), but this mRNA was not
detected in leukocytes or RPE from these eyes. This remained true in
four replicate experiments (two shown). This observation suggests that
another cell type within the eye was producing MIP-1/CCL9 mRNA in
Th1-induced inflammation (and possibly in Th2 disease as well). In EAU, eyes express a predominantly Th1-like pattern of inflammatory mediators
EAU is a well-characterized model of human autoimmune eye disease, in which susceptible animals develop ocular inflammation when immunized with eye-specific proteins (26). To determine which inflammatory mediators play a role in the development of EAU, we studied the mRNA complement in eyes of B10.A mice in which EAU was induced by immunization with IRBP. These eyes showed the characteristic inflammation and tissue damage, with an inflammatory infiltrate comprised mainly of MNLs and a small number of PMNs (27).
The measured mRNA levels of cytokines, chemokines, and chemokine
receptors in eyes with EAU are compared in Fig. 5 with those in control naive B10.A mice.
Individual molecules of each of the three groups of inflammatory
mediators are shown in this figure in order of their detected mRNA
level. In addition, the calculated fold increase from baseline for all
tested molecules in EAU eyes is recorded in Table I, allowing
comparison among the molecule expression pattern in EAU with that in
the Th1- and Th2-adoptive transfer models. The mediator transcript
expression pattern in EAU eyes resembles that seen in Th1 recipient
eyes, but enhanced expression of several molecules that characterize
Th2-induced inflammation was also seen in EAU (Table I, Fig. 5).
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, IL-1, IL-1R
antagonist, IL-6, TNF-, and osteopontin. The expression of
IFN- mRNA in the EAU eyes was also elevated, although to a lesser
degree than in Th1 recipient eyes. On the other hand, eyes with EAU
resembled Th2 recipient eyes in showing increased expression of IL-5
transcript (Table I).
A close similarity was clearly seen between the chemokine transcript
expression patterns in EAU eyes and in Th1 recipient eyes. The most
highly expressed chemokine transcripts in EAU eyes were the same as
those seen in Th1 recipient eyes, namely, MIG/CXCL9, RANTES/CCL5, and
IP-10/CXCL10, which characterized Th1-induced disease, and C-10/CCL6,
BRAK/CXCL14, MIP-1/CCL9, and fractalkine/CX3CL1, which were
expressed in both Th1- and Th2-induced ocular inflammation. Other
chemokine transcripts found in Th1-induced inflammation were detected
in EAU eyes at moderate levels. These include molecules such as
MIP-1/CCL3, MIP-1/CCL4, or MIP-2/CXCL1–3. In contrast, no
increase in expression of Th2-specific chemokine transcripts was
observed in eyes with EAU.
The pattern of chemokine receptor transcript expression in eyes with EAU also showed similarities to that seen in Th1-induced inflammation, with increased expression of CCR1, CCR5, CCR7, CXCR3, and CXCR2 transcripts. Interestingly, CCR6 mRNA was expressed in EAU at remarkably higher levels than in Th1 or Th2 recipient eyes. CCR3 mRNA was only mildly elevated in EAU, in contrast to its elevation in Th2 recipient eyes. Transcripts of two receptors, CX3CR1 and CXCR6, were highly expressed in eyes with EAU as well as in eyes of naive control mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the Th1- and Th2-adoptive transfer models, analysis of inflammatory
mediator transcripts revealed three patterns of expression, i.e.,
preferential elevation in recipients of Th1 or of Th2 cells, or similar
expression in eyes of both recipient groups. Notably, the number of
chemokines and chemokine receptor transcripts that were preferentially
expressed in Th1-induced ocular inflammation was much higher than that
in Th2-induced disease (Fig. 3). This difference could be attributed in
part to a greater capacity of Th1 cells to produce or induce chemokines
(29), a capacity that may also explain in part the
observation that these lymphocytes are profoundly more efficient than
Th2 cells in inducing ocular inflammation in this experimental system
(Fig. 1) (16).
The preferential expression of cytokine transcripts that we see in Th1- or Th2-mediated ocular inflammation is generally in line with well-established Th1/Th2 associations in other systems (30, 31, 32). Less is known, however, about the Th1/Th2 specificity of chemokines, and our results thus contribute new information to this issue (6).
Some of the Th1/Th2 associations we uncovered were expected based on
previous studies. Seven chemokines that were found in the present study
to be strongly associated with Th1-induced inflammation include
MIG/CXCL9, IP-10/CXCL10, monocyte chemoattractant protein-1
(MCP-1)/CCL2, MCP-3/CCL7, RANTES/CCL5, MIP-1/CCL3, and
MIP-1/CCL4. The affiliation of these seven chemokines with Th1 cells
has been indicated in other studies by the findings that these
chemokines 1) are induced by Th1 cytokines (33, 34, 35, 36), 2)
are ligands for chemokine receptors specific to Th1 cells
(37, 38, 39), and 3) are associated with Th1-induced
inflammatory processes such as experimental autoimmune
encephalomyelitis (EAE) (24, 40, 41, 42) or inflammatory bowel
disease (43). Similarly, the two chemokines whose
transcripts were preferentially expressed in Th2 recipient eyes are
known to be Th2 associated. Both eotaxin/CCL11 and TARC/CCL17
are associated with other Th2 disease states (44, 45, 46, 47, 48) and
are ligands for receptors found on Th2 cells (37, 38, 39).
Furthermore, eotaxin/CCL11 has been shown to be induced by Th2
cytokines (45, 49, 50, 51).
Other chemokines characterized in our study have exhibited more
equivocal Th1/Th2 associations. These include four chemokines that were
preferentially associated with Th1-induced eye disease. MIP-2/CXCL1–3,
a neutrophil chemoattractant, is expressed in other Th1-associated
disease states (25, 40, 52, 53), but can also be
up-regulated by Th2-specific cytokines IL-4 or IL-5 and inhibited by
the Th1 cytokine IFN- (49, 54). TCA-3/CCL1 has been
found in both Th1- and Th2-associated disease models (24, 25, 53), and the TCA-3/CCL1 receptor, CCR4, is selectively expressed
on Th2 cells. In our model, mRNA of this chemokine was highly expressed
in Th1 recipients on day 4, but its expression rose considerably in Th2
recipients on day 7. MIP-3/CCL19 is known for its role in lymphocyte
homing to lymph nodes (55), but little is known about its
role in Th-induced inflammation. Lymphotactin/XCL1 was clearly Th1
associated in the present study (Fig. 3) and other systems (25, 29, 53), but it has been reported to down-regulate Th1 responses
in some situations (56).
The three chemokine transcripts up-regulated in both Th1 and Th2 cell
recipients are also not clearly Th1 or Th2 associated. Two of these,
C-10/CCL6 and MIP-1/CCL9, have been proposed to play a role in
Th2-polarized responses, but C-10/CCL6 may also be involved in EAE
development (57, 58, 59). MDC/CCL22, a chemokine whose
transcript was up-regulated in both Th1 and Th2 recipient eyes,
although with different kinetics, has been affiliated mainly with
Th2-mediated responses (46).
Conflicting evidence regarding the Th1/Th2 associations of many
chemokines suggests that the elaboration of these molecules may be
tissue and context dependent. Indeed, previous work has shown that
different cell types can produce different chemokines in response to
the same extracellular signal (60). In the eye, RPE cells
have been shown to elaborate chemokines in vitro (7, 9, 10, 11). Laser capture microdissection enabled us to demonstrate,
for the first time, chemokine transcript expression by these cells in
vivo, during an ongoing inflammatory response. Furthermore, we found
that RPE and infiltrating leukocytes differed partially in their
chemokine expression patterns, even though the context (the inflamed
eye) was the same (Fig. 4). This finding supports a role for RPE in
shaping ocular inflammatory responses in an eye-specific manner. It is
also of interest that different chemokine transcripts were expressed by
RPE cells in eyes developing inflammation induced by Th1 or Th2 cells
(Fig. 4).
It is particularly noteworthy that in addition to the mediator
transcripts up-regulated during the inflammatory response, we found
multiple mediator transcripts that were expressed constitutively in
intact mouse eyes and whose expression levels did not change much
during the inflammatory process. These included trancripts of three
chemokines (fractalkine/CXC3CL1, BRAK/CXCL14, and CTACK/CCL27),
two chemokine receptors (CXCR-6 and CXC3R1), and five cytokines
(TGF-2, TGF-3, PDGF-, osteopontin, and PDGF-R). Constitutive
expression of TGF-2, TGF-3, and PDGF- in the eye is well known
(61, 62), but the expression of chemokines and chemokine
receptors in the normal eye has not previously been described. While
the role of these chemokines is not known, it is possible that they
participate in the physiologic process of immune surveillance, i.e.,
lymphocyte migration through eye tissues. Previous analyses of
adoptively transferred T cells inducing EAE (63, 64) or
EAU (65) demonstrated that small numbers of activated
lymphocytes migrate into the target organ shortly after injection. This
primary wave of infiltrating cells is postulated to release mediators
that initiate the massive cell invasion that begins 2–3 days later
(64). It is proposed, therefore, that the constitutively
expressed chemokines we identified help mediate the poorly defined
primary process in the eye, and that similar batteries of chemokines
carry out this function in other organs. The need for constitutively
expressed chemokines for the invasion of activated lymphocytes was
indicated by the recent finding that this process is inhibited by
treatment with pertussis toxin, an agent that inhibits chemotaxis
(66). In addition to the aforementioned functions, some of
the constitutively expressed mediators may play a role in ocular immune
privilege. TGF-2 is well known for its immunosuppressive activity
and presumed role in protecting the eye from damaging immune responses
(62). Interestingly, fractalkine/CXC3CL1 is constitutively
expressed in the brain and has recently been implicated in limiting CNS
inflammation (67, 68).
Also of interest are our findings with osteopontin. This molecule, also
known as early T cell activation gene-1, exerts both cytokine and
chemoattractant activities, and was recently shown to play a role in
immunopathogenic processes of cell-mediated diseases (69, 70). Exceedingly high levels of osteopontin mRNA were found in
intact eyes, and these levels increased considerably in EAU (Fig. 5)
and in Th1-mediated inflammation, but not in Th2-induced disease (Table I and Fig. 3).
A major goal of the present study was to better understand the
pathogenesis of ocular inflammation by comparing the inflammatory
molecule expression pattern in recipients of Th1 or Th2 cells with that
in mice developing EAU. EAU serves as an animal model for ocular
inflammatory conditions with presumed autoimmune etiology, such as
sympathetic ophthalmia, birdshot retinochoroidopathy, Behcet’s
disease, or the Vogt-Koyanagi-Harada syndrome (2, 26, 28).
IRBP-induced EAU in the mouse has been thought to be a T cell-mediated
disease, with prominent involvement of Th1 cells, based on an
association with Th1-specific cytokines (71, 72), as well
as on the direct relationship between the susceptibility to the disease
and the lymphokine profile of the animal (73). Data
collected in this study confirmed these observations and profoundly
extended the scope of the investigation by including characterization
of an extensive battery of Th1- and Th2-specific cytokines, as well as
chemokines and chemokine receptors. The pattern of increased expression
of transcripts of the three families of inflammatory molecules in the
EAU eyes closely resembled that of Th1 recipient eyes (Table I). The
elevated expression of IL-5 mRNA and marginal up-regulation of
eotaxin/CCL11 mRNA may indicate a minor involvement of Th2 cells in the
pathogenic process.
The data reported in this work underscore the multiplicity of mediators
that participate in the ocular inflammatory process. Analysis of these
data shows that significant levels of up-regulated transcripts of
cytokines and chemokines are first detected on day 4. It is probable,
however, that the inflammatory process is triggered earlier by low
levels of cytokines released by the small number of HEL-specific T
cells that invade the eye immediately following cell injection. In this
model, the primary cytokines initiate a cycle in which cytokines,
chemokines, and adhesion molecules are up-regulated, leading to
additional cell recruitment, leading to additional inflammatory
mediator production, and so on. An important implication of this model
is that inhibition of sight-damaging ocular inflammation should be
targeted at upstream cytokines. This approach has been successful in
inflammatory diseases such as Crohn’s disease and rheumatoid
arthritis, which can be ameliorated by blocking TNF-, an upstream
cytokine (3, 74, 75, 76). It is of interest, however, that the
anti-TNF- agents effective in these two diseases were
considerably less effective in treatment of ocular inflammation
(76). Furthermore, an anti-TNF- Ab was recently
found to exacerbate EAU in mice when administered during the efferent
phase of the disease (77). Our data point to other
upstream cytokines that are potential targets for therapeutic
inhibition. For example, both IL-1 and IL-6 were greatly up-regulated
in eyes with Th1-induced inflammation and in EAU. Agents that block
these two cytokines are already under development for use in humans
(3). This study provides an impetus for characterizing the
efficacy of these and other agents in combating ocular inflammatory
disease.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: Tg, transgenic; EAE, experimental autoimmune encephalomyelitis; EAU, experimental autoimmune uveoretinitis; HEL, hen egg lysozyme; IP-10, IFN--inducible protein-10; IRBP, interphotoreceptor retinoid-binding protein; MCP, monocyte chemoattractant protein; MIG, monokine induced by IFN-; MIP, macrophage-inflammatory protein; MNL, mononuclear leukocyte; PDGF, platelet-derived growth factor; PMN, polymorphonuclear; RPE, retinal pigment epithelium; CCL, CC chemokine ligand.
Received for publication September 26, 2001. Accepted for publication December 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF-stimulated IL-8 and MCP-1 protein production in human retinal pigmented epithelial cells. Curr. Eye Res. 18:349.[Medline]
, MCP-1 and RANTES in experimental autoimmune uveitis. Invest. Ophthalmol. Visual Sci. 42:1547. interferon-mediated resolution of replicative Legionella pneumophila lung infection. Infect. Immun. 68:6567.-interferon encodes a member of the platelet factor 4 family of cytokines. Proc. Natl. Acad. Sci. USA 87:5238.-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315:672.[Medline]
chemokine C10 by IL-4 in mouse macrophages. J. Immunol. 152:5084.[Abstract]
and 8-isoprostane production induced by intracerbeoventricular injection of LPS. J. Neuroimmunol. 115:135.[Medline]
antagonists for the treatment of Crohn’s disease. Expert Opin. Pharmacother. 1:615.[Medline]
) in pathogenesis of experimental autoimmune uveitis in IFN()-deficient and wild type mice. FASEB J. 15:A355.This article has been cited by other articles:
|
|
 |
|
  A. Dagkalis, C. Wallace, H. Xu, S. Liebau, A. Manivannan, M. A. Stone, M. Mack, J. Liversidge, and I. J. Crane Development of Experimental Autoimmune Uveitis: Efficient Recruitment of Monocytes Is Independent of CCR2 Invest. Ophthalmol. Vis. Sci., September 1, 2009; 50(9): 4288 - 4294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Shi, C. A. Cox, B. P. Vistica, C. Tan, E. F. Wawrousek, and I. Gery Phenotype Switching by Inflammation-Inducing Polarized Th17 Cells, but Not by Th1 Cells J. Immunol., November 15, 2008; 181(10): 7205 - 7213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Shi, A. Maminishkis, T. Banzon, S. Jalickee, R. Li, J. Hammer, and S. S. Miller Control of Chemokine Gradients by the Retinal Pigment Epithelium Invest. Ophthalmol. Vis. Sci., October 1, 2008; 49(10): 4620 - 4630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Cox, G. Shi, H. Yin, B. P. Vistica, E. F. Wawrousek, C.-C. Chan, and I. Gery Both Th1 and Th17 Are Immunopathogenic but Differ in Other Key Biological Activities J. Immunol., June 1, 2008; 180(11): 7414 - 7422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Su, R. S. Grajewski, D. Luger, R. K. Agarwal, P. B. Silver, J. Tang, J. Tuo, C.-C. Chan, and R. R. Caspi Altered Chemokine Profile Associated with Exacerbated Autoimmune Pathology under Conditions of Genetic Interferon-{gamma} Deficiency Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4616 - 4625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tuo, C. M. Bojanowski, M. Zhou, D. Shen, R. J. Ross, K. I. Rosenberg, D. J. Cameron, C. Yin, J. A. Kowalak, Z. Zhuang, et al. Murine Ccl2/Cx3cr1 Deficiency Results in Retinal Lesions Mimicking Human Age-Related Macular Degeneration Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3827 - 3836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Rajendram, S. Saraswathy, and N. A Rao Photoreceptor mitochondrial oxidative stress in early experimental autoimmune uveoretinitis Br J Ophthalmol, April 1, 2007; 91(4): 531 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Oh-i, H. Keino, H. Goto, N. Yamakawa, K. Murase, Y. Usui, T. Kezuka, J.-i. Sakai, M. Takeuchi, and M. Usui Intravitreal injection of Tacrolimus (FK506) suppresses ongoing experimental autoimmune uveoretinitis in Rats Br J Ophthalmol, February 1, 2007; 91(2): 237 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Fujimoto, C.-R. Yu, G. Shi, B. P. Vistica, E. F. Wawrousek, D. M. Klinman, C.-C. Chan, C. E. Egwuagu, and I. Gery Pertussis Toxin Is Superior to TLR Ligands in Enhancing Pathogenic Autoimmunity, Targeted at a Neo-Self Antigen, by Triggering Robust Expansion of Th1 Cells and Their Cytokine Production J. Immunol., November 15, 2006; 177(10): 6896 - 6903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Hikita, B. P. Vistica, H. R. Jones, J. R. Keswani, M. M. Watson, V. R. Ericson, G. S. Ayoub, I. Gery, and D. O. Clegg Osteopontin is proinflammatory in experimental autoimmune uveitis. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4435 - 4443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wang, F. Bai, M. Pries, S. Buus, J. U. Prause, and M. H. Nissen Identification of MHC class I h-2 kb/db-restricted immunogenic peptides derived from retinal proteins. Invest. Ophthalmol. Vis. Sci., September 1, 2006; 47(9): 3939 - 3945. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Takase, C.-R. Yu, D.-I. Ham, C.-C. Chan, J. Chen, B. P. Vistica, E. F. Wawrousek, S. K. Durum, C. E. Egwuagu, and I. Gery Inflammatory processes triggered by TCR engagement or by local cytokine expression: differences in profiles of gene expression and infiltrating cell populations J. Leukoc. Biol., September 1, 2006; 80(3): 538 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chen, C. Fujimoto, B. P. Vistica, E. F. Wawrousek, B. Kelsall, and I. Gery Active participation of antigen-nonspecific lymphoid cells in immune-mediated inflammation. J. Immunol., September 1, 2006; 177(5): 3362 - 3368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H Takase, S Sugita, C Taguchi, Y Imai, and M Mochizuki Capacity of ocular infiltrating T helper type 1 cells of patients with non-infectious uveitis to produce chemokines Br J Ophthalmol, June 1, 2006; 90(6): 765 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Crane, H. Xu, C. Wallace, A. Manivannan, M. Mack, J. Liversidge, G. Marquez, P. F. Sharp, and J. V. Forrester Involvement of CCR5 in the passage of Th1-type cells across the blood-retina barrier in experimental autoimmune uveitis J. Leukoc. Biol., March 1, 2006; 79(3): 435 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Hashida, N. Ohguro, K. Nakai, M. Kobashi-Hashida, S.-i. Hashimoto, K. Matsushima, and Y. Tano Microarray Analysis of Cytokine and Chemokine Gene Expression after Prednisolone Treatment in Murine Experimental Autoimmune Uveoretinitis Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4224 - 4234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-y. Zeng, X.-a. Zhu, C. Zhang, L.-P. Yang, L.-m. Wu, and M. O. M. Tso Identification of Sequential Events and Factors Associated with Microglial Activation, Migration, and Cytotoxicity in Retinal Degeneration in rd Mice Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 2992 - 2999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-K. Lim, C. Fujimoto, R. Ursea, S. P. Mahesh, P. Silver, C.-C. Chan, I. Gery, and R. B. Nussenblatt Suppression of Immune-Mediated Ocular Inflammation in Mice by Interleukin 1 Receptor Antagonist Administration Arch Ophthalmol, July 1, 2005; 123(7): 957 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Zheng, M. J. Kang, K. Crothers, Z. Zhu, W. Liu, C. G. Lee, L. A. Rabach, H. A. Chapman, R. J. Homer, D. Aldous, et al. Role of Cathepsin S-Dependent Epithelial Cell Apoptosis in IFN-{gamma}-Induced Alveolar Remodeling and Pulmonary Emphysema J. Immunol., June 15, 2005; 174(12): 8106 - 8115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Yang, I-M. Fang, C.-P. Lin, C.-M. Yang, and M.-S. Chen Effects of the NF-{kappa}B Inhibitor Pyrrolidine Dithiocarbamate on Experimentally Induced Autoimmune Anterior Uveitis Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1339 - 1347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Kim, Z. Li, R. N. Fariss, D. F. Shen, S. P. Mahesh, C. Egwuagu, C.-R. Yu, C. N. Nagineni, C.-C. Chan, and R. B. Nussenblatt Constitutive and Cytokine-Induced GITR Ligand Expression on Human Retinal Pigment Epithelium and Photoreceptors Invest. Ophthalmol. Vis. Sci., September 1, 2004; 45(9): 3170 - 3176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Curnow, K. Wloka, J. M. Faint, N. Amft, C. M. G. Cheung, V. Savant, J. Lord, A. N. Akbar, C. D. Buckley, P. I. Murray, et al. Topical Glucocorticoid Therapy Directly Induces Up-Regulation of Functional CXCR4 on Primed T Lymphocytes in the Aqueous Humor of Patients with Uveitis J. Immunol., June 1, 2004; 172(11): 7154 - 7161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Greiner, C. C. Murphy, F. Willermain, L. Duncan, J. Plskova, G. Hale, J. D. Isaacs, J. V. Forrester, and A. D. Dick Anti-TNF{alpha} Therapy Modulates the Phenotype of Peripheral Blood CD4+ T Cells in Patients with Posterior Segment Intraocular Inflammation Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 170 - 176. [Abstract] [Full Text] [PDF]
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), or control
mice, tested after irradiation, but before Th cell injection (
). For
both recipient groups, recorded values represent means of three
replicate mice per group ± SEM.
),
or mean ± range in two untreated control mice (
). See
Materials and Methods for additional details.