Amelioration of Murine Dry Eye
Disease by Topical Antagonist
to Chemokine Receptor 2
The Harvard community has made this
article openly available.
Please share
how
this access benefits you. Your story matters
Citation
Goyal, Sunali. 2009. “Amelioration of Murine Dry Eye
Disease by Topical Antagonist to Chemokine Receptor 2.”
Archives of Ophthalmology 127 (7) (July 13): 882. doi:10.1001/
archophthalmol.2009.125.
Published Version
doi:10.1001/archophthalmol.2009.125
Citable link
http://nrs.harvard.edu/urn-3:HUL.InstRepos:34388142
Terms of Use
This article was downloaded from Harvard University’s DASH
repository, and is made available under the terms and conditions
applicable to Other Posted Material, as set forth at
http://
LABORATORY SCIENCES
Amelioration of Murine Dry Eye Disease
by Topical Antagonist to Chemokine Receptor 2
Sunali Goyal, MD; Sunil K. Chauhan, PhD; Qiang Zhang, MD; Reza Dana, MD, MSc, MPH
Objective:To determine the effect of a topical antago-nist to the chemokine receptor 2 (CCR2) in a murine model of dry eye disease.
Methods:The effects of a topical CCR2 antagonist and a vehicle control treatment were studied in murine dry eyes. A controlled environment chamber induced dry eye by exposing mice to high-flow desiccated air. Corneal
fluo-rescein staining and enumeration of corneal CD11b⫹and
conjunctival CD3⫹T cells were performed in the
differ-ent groups. Real-time polymerase chain reaction was per-formed to quantify expression of different inflamma-tory cytokine transcripts in the cornea and conjunctiva.
Results:Eyes receiving the formulation containing CCR2 antagonist showed a significant decrease in corneal fluo-rescein staining and decreased infiltration of corneal
CD11b⫹cells and conjunctival T cells compared with the
vehicle-treated and untreated dry eye groups. The CCR2 antagonist also significantly decreased messenger RNA
expression levels of interleukins 1␣and 1in the
cor-nea, and tumor necrosis factor␣and interleukin 1in
the conjunctiva.
Conclusion:Topical application of CCR2 antagonist is associated with significant improvement in dry eye dis-ease and is reflected by a decrdis-ease in inflammation at the clinical, molecular, and cellular levels.
Clinical Relevance:Topical application of CCR2 an-tagonist may hold promise as a therapeutic modality in dry eye disease.
Arch Ophthalmol. 2009;127(7):882-887
D
RY EYE SYNDROME OR KERA -toconjunctivitis sicca is one of the most common problems facing patients seeking ophthalmologi-cal care.1In dry eye disease (DED), the ho-meostatic balance between the tear film and the ocular surface epithelium is impaired, causing the ocular surface to respond ab-normally to environmental challenges. The clinical presentation can be highly vari-able, ranging from mild ocular discomfort to sight-threatening corneal complications such as persistent epithelial defects or ster-ile stromal ulceration. Epidemiological stud-ies report that more than 6% of the popu-lation older than 40 years and 15% of thepopulation older than 65 years have DED.2
Clinically significant DED is associ-ated with ocular surface inflammation, al-though the exact immunopathogenesis is
not known.3It is now recognized that the
ocular surface (cornea and conjunctiva) and the lacrimal glands function as an
in-tegrated unit called the lacrimal
func-tional unit, and inflammation in any com-ponent can compromise the function of the others through soluble mediators, genera-tion of autoreactive T cells, and
inhibi-tion of neural transmission.4
Chemokines are small cytokines with chemoattractant properties that
coordi-nate leukocyte migration in immunity and inflammation and hence play a role in the
pathogenesis of many human diseases.5
In-flammatory macrophages, derived from peripheral blood monocytes, are well-characterized cell mediators of tissue de-struction in various chronic inflamma-tory diseases such as rheumatoid arthritis
and multiple sclerosis.6Once inside
tis-sues, they orchestrate tissue destruction by secreting various proinflammatory
cy-tokines (tumor necrosis factor␣[TNF-␣]
and interleukin 1 [IL-1]) and chemo-kines (CXCL8, CCL5, and CCL2) that fur-ther cause influx of ofur-ther inflammatory cells. Our laboratory has previously shown that DED is associated with ingress and
ac-tivation of corneal CD11b⫹cells.7It is
known that desiccating stress upregu-lates chemokines and their receptors in DED, and monocyte chemotactic protein 1 (MCP-1)/CCL2 has been identified as the key molecule for the chemotaxis and in-flux of mononuclear leukocytes into sites
of inflammation.8Monocyte chemotactic
protein 1 is secreted by monocytes, memory T cells, macrophages, fibroblasts, endothe-lial cells, and mast cells, and it stimulates the movement of leukocytes along a che-motactic gradient after binding to its cell sur-face receptor CCR2.9The critical role of the MCP-1/CCR2 pair in inflammation has been
Author Affiliations:Schepens Eye Research Institute (Drs Goyal, Chauhan, Zhang, and Dana), Department of Ophthalmology, Harvard Medical School (Drs Goyal, Chauhan, Zhang, and Dana), and Massachusetts Eye and Ear Infirmary (Dr Dana),
demonstrated using MCP-1 and CCR2 knockout mice, sug-gesting that the inhibition of migration of CCR2-bearing mononuclear cells may be an effective mechanism to modu-late disease progression in chronic inflammation.10Herein, we hypothesized that a topical antagonist to CCR2 could have therapeutic value for the treatment of dry eyes in hu-mans.
METHODS
ANIMALS
We used female C57BL/6 mice (Charles River Laboratories, Wilmington, Massachusetts) aged 8 to 12 weeks for this study. The research protocol was approved by the Schepens Eye Re-search Institute Animal Care and Use Committee, and it con-formed to the standards in the Association for Research in Vi-sion and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
INDUCTION OF EXPERIMENTAL DRY EYE
We induced dry eye in the mice by placing them in a con-trolled environment chamber (CEC), which allows continu-ous regulation of relative humidity below 30%, a constant tem-perature of 21°C to 23°C, and airflow of 15 L/min, 24 hours a
day.11To achieve maximum ocular surface dryness, the
con-ditions in the CEC were supplemented with topical applica-tion of atropine sulfate, 1% (Falcon Pharmaceuticals, Fort Worth, Texas), twice daily for the first 48 hours. In addition, the mice also received subcutaneous 0.1-mL injections of 5-mg/mL sco-polamine hydrobromide (Sigma-Aldrich Corp, St Louis,
Mis-souri) 3 times a day (9AM, 1PM, and 5PM) on their dorsal
sur-face for the duration of the experiment.7
TOPICAL CCR2 ANTAGONIST FORMULATION AND TREATMENT REGIMEN
The topical CCR2 antagonist and the vehicle control treat-ment were provided by Johnson & Johnson Vision Care, Inc, Jacksonville, Florida. The CCR2 antagonist at a concentration of 5.0 mg/mL ( J&J-17166864-AAG) was the tested formula-tion. Physiological phosphate-buffered saline ( J&J-17166864-AAG) consisting of purified water, sodium phosphate mono-basic monohydrate, sodium phosphate dimono-basic dihydrate, and sodium chloride was used as the vehicle control.
Forty-eight hours after the induction of dry eye, the mice in the CEC were randomly divided into the following 3 different groups (n=5 in each group): an untreated group, a group receiv-ing topical vehicle control treatment, and a group receivreceiv-ing the CCR2 antagonist formulation. Three microliters of the topical for-mulation was applied to both the eyes of the unanesthetized mice
twice a day (9AMand 5PM) from days 3 to 9. The untreated group
received no eyedrops. Ocular signs of dry eye were evaluated using fluorescein staining at days 2, 5, and 9. Mice were then eutha-nized on day 10 for cellular and molecular studies.
CORNEAL SURFACE STAINING
Fluorescein staining of the corneal epithelium was used as a di-agnostic tool to study the effect of desiccating stress on the ocular surface of the mice. Corneal fluorescein staining was performed at baseline (day 0, before placing the mice in the CEC), day 2 (be-fore instillation of topical therapy), day 5, and finally at day 9. A 0.7-µL quantity of fluorescein, 2.5% (Sigma-Aldrich Corp), was applied into the inferior conjunctival sac of each eye (n=10 eyes per
group) by using a micropipette as previously described.11After 5
minutes, punctate staining on the corneal surface was evaluated in a masked fashion with the help of a slitlamp biomicroscope un-der a cobalt blue light. The National Eye Institute grading system
was used to assess the corneal surface staining.12
RNA ISOLATION AND MOLECULAR ANALYSIS USING REAL-TIME POLYMERASE
CHAIN REACTION
Total RNA was isolated from the cornea and conjunctival tis-sues using a commercially available kit (RNeasy [catalog No. 74004]; Qiagen, Valencia, California). Two corneas and 3 to 4 conjunctivae per group were isolated, pooled, and stored at −80°C in a reagent (TRIzol [catalog No. 15596026]; Invitro-gen Corporation, Carlsbad, California) until future use.
The first strand of complementary DNA (cDNA) was syn-thesized with random hexamers using reverse transcriptase (SuperScript III [catalog No. 18080]; Invitrogen Corporation) according to the manufacturer’s recommendations. Real-time polymerase chain reaction was performed using dye-labeled predesigned primers (FAM-MGB; Applied Biosystems, Foster
City, California) for TNF-␣(assay Mm99999068_m1),
glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH) (assay
Mm99999915_g1), IL-1␣(assay Mm00439620_m1), and IL-1
(assay Mm00434228_m1). One microliter of cDNA was loaded in each well, and the assays were performed in duplicate. A non-template control was included in all of the experiments to
evalu-ate DNA contamination of the reagent used. TheGAPDHgene
was used as the endogenous reference for each reaction. The results were normalized by the cycle threshold of GAPDH, and the relative messenger RNA (mRNA) level in the untreated group was used as the normalized control for the other 2 groups.
ANALYSIS OF CELLULAR INFILTRATION BY IMMUNOHISTOCHEMICAL STAINING
The following primary antibodies were used for immunohis-tochemical staining: fluorescein isothiocyanate–conjugated rat anti–mouse CD11b (monocyte/macrophage marker [catalog No. 557396; BD Pharmingen, San Diego, California]; isotype fluo-rescein isothiocyanate–conjugated rat anti–mouse IgG2bk [cata-log No. 553988BD; Pharmingen]) and purified hamster anti– mouse CD3e (T-cell marker [catalog No. 553057; BD Pharmingen]; isotype purified hamster IgG1 [catalog No. 553969BD; Pharmingen]). The secondary antibody used was the Cy3-conjugated goat anti–Armenian hamster antibody (code 127165-160; Jackson Laboratories, Bar Harbor, Maine).
For whole-mount immunofluorescence corneal staining, freshly excised corneas were washed in phosphate-buffered sa-line and fixed in acetone for 15 minutes. The immunostaining
was performed as described previously.7Three eyeballs from
3 mice per group were taken, and cells were counted in 5 to 6 areas in the periphery (0.5-µm area from the limbus) and 1 to 2 areas in the center (central 2-µm area) of each cornea in a masked fashion by using an epifluorescence microscope (model
E800; Nikon, Melville, New York) at magnification⫻40. The
mean number of cells was obtained by averaging the total num-ber of cells in all the areas studied, and the result was ex-pressed as the number of positive cells per square millimeter. For cross-sectional staining of the conjunctiva, whole eye-balls were excised, frozen in optimal cutting temperature com-pound, cut into 7-µm frozen sections, and fixed in acetone for
15 minutes at room temperature.13Three eyeballs from 3 mice
epifluores-cence microscope using an objective lens with magnification
⫻40. The mean number of cells was obtained by averaging the
cell number per cross section studied.
STATISTICAL ANALYSIS
We performed a 2-tailedttest, andP⬍.05 was deemed
statis-tically significant. Results are presented as the mean (SEM) of at least 3 experiments.
RESULTS
EVALUATION OF CLINICAL SIGNS OF DED USING CORNEAL FLUORESCEIN STAINING
On day 0, mice were placed in the CEC. As validated
pre-viously,7,1148 hours after placing mice in the CEC, all
mice showed an increase in corneal staining correspond-ing to dry eye induction. Treatment with the CCR2 an-tagonist and the vehicle control was started on day 3, and corneal fluorescein staining scores were measured at days 5 and 9. Treatment with the topical CCR2 antagonist showed a significant decrease in corneal fluorescein
stain-ing compared with the untreated group (P⬍.001) but
was comparable to the vehicle-treated group at day 5
(P= .07). However, by day 9, the CCR2 antagonist–
treated eyes showed significantly decreased corneal stain-ing compared with the untreated and vehicle-treated
groups (P⬍.001 for both groups;Figure 1). An
over-all reduction of 45% was seen in the corneal fluorescein staining score from baseline (day 2) in the CCR2 antago-nist–treated eyes compared with only an 8% reduction in the vehicle-treated group.
ENUMERATION OF CD11bⴙ
MONOCYTES IN CORNEAS
Ithasbeenshownthatthenormalcorneahasaresidentpopu-lation of bone marrow–derived immature CD11b⫹
antigen-presenting cells, and the induction of dry eye increases the number of CD11b⫹cells in the cornea.7,14Therapy with the
CCR2antagonist,comparedwiththeuntreateddryeyegroup, significantly decreased the total number of CD11b⫹cells in the periphery (11.6 [0.3] vs 19.3 [0.3] cells/mm2[P⬍.001])
andinthecenter(7.4[0.3]vs14.0[0.3]cells/mm2[P⬍.001])
of the cornea. Similarly, the CCR2 antagonist–treated eyes showed a significant decrease in the number of CD11b⫹cells in the cornea in the periphery and in the center compared with the vehicle-treated group (16.9 [0.3] cells/mm2in the periphery [P⬍.001] and 12.4 [0.3] cells/mm2in the cen-ter [P⬍.001]) (Figure 2).
ENUMERATION OF CD3ⴙT CELLS
IN THE CONJUNCTIVA
Studies in various animal models and humans have made it clear that desiccating stress is often associated with T-cell infiltration of the conjunctiva.15,16The T cells mi-grate into the peripheral tissues along a chemokine gra-dient and can mediate chronic ocular surface inflammation in a variety of dry eye states.15The effect of topical CCR2 antagonist application on dry eyes was studied, and we found that the treatment significantly decreased the total
number of CD3⫹T cells in the conjunctiva compared with
the untreated and the vehicle-treated groups (P⬍.001
for both groups) (Figure 3).
mRNA EXPRESSION LEVELS OF TNF-␣, IL-1␣,
AND IL-1IN CORNEA AND CONJUNCTIVA
The levels of mRNA encoding different proinflammatory cytokines, such as TNF-␣, IL-1␣, and IL-1, are elevated in the cornea and conjunctiva in DED.16-18Real-time poly-merase chain reaction was used to quantify the transcripts encoding TNF-␣, IL-1␣, and IL-1in the cornea and the conjunctiva of different groups (4-6 per group). Among-group comparisons showed that treatment with the CCR2 antagonist significantly decreased the mRNA expression levels of IL-1␣(P=.04) and IL-1(P=.01) in the cornea
compared with the vehicle-treated group (Figure 4A).
Al-though a similar downward trend was seen in levels of
TNF-␣, statistical significance could not be reached
be-cause of the high variance.
In the conjunctiva, topical therapy with the CCR2
an-tagonist significantly decreased mRNA levels of TNF-␣
(P=.02) and IL-1(P=.04) compared with the
vehicle-treated eyes (Figure 4B). The levels of IL-1␣, although de-creased, were not statistically significant.
COMMENT
Chemokines are a group of small-molecular-weight basic proteins (8-10 kDa) that represent the largest family of cy-tokines encoded in the human genome, constituting a group of more than 50 distinct chemokines and 20 chemokine re-ceptors.5They are secreted at sites of inflammation by resi-dent tissue cells, resiresi-dent and recruited leukocytes, and cytokine-activatedendothelialcells,andtheyexerttheirfunc-tions by binding to specific G-protein–coupled cell surface receptors on target cells.19The main stimuli for chemokine production are proinflammatory cytokines such as IL-1 and TNF-␣and bacterial products such as lipopolysaccharides.20
12
10
8
6
4
2
0
Day 2 Day 5 Day 9 Measurement Time
Corneal Fluorescein Staining Score
Untreated Vehicle treated CCR2 antagonist treated
P < .001
P < .001
[image:4.612.67.295.45.213.2]P < .001
The chemokines are divided into the CXC subfamily, which is primarily responsible for recruitment of neutro-phils, and the CC subfamily, which preferentially attracts monocytes, eosinophils, basophils, and lymphocytes with variable selectivity. Monocyte chemoattractant protein 1, also known as CCL2, an important member of the CC chemokine subfamily, is a potent chemoattractant for mono-cytes and binds solely to CCR2, a 7-transmembrane– spanning protein that is linked to the downstream
signal-ing pathways.21Wide expression of CCR2 occurs on
peripheral blood monocytes, activated T cells, natural killer
cells, dendritic cells, and basophils,22 and the MCP-1/
CCR2 pair has been implicated in a number of chronic in-flammatory diseases such as multiple sclerosis, atheroscle-rosis, and experimental autoimmune uveitis. Studies have shown that, when exposed to appropriate stimulus, hu-man corneal keratocytes and endothelial cells express a high level of MCP-1,23,24suggesting that, in inflammatory con-ditions under the regulation of MCP-1, CCR2-expressing cells can migrate into all layers of the cornea. Given these observations, we hypothesized that disruption of this chemokine-chemokine receptor axis with topical CCR2 an-tagonist could be used as a therapeutic target for control-ling pathological inflammation in DED.
Dry eye disease causes ocular surface inflammation evi-denced by increased levels of inflammatory cytokines (IL-1,
IL-6, IL-8, and TNF-␣) and T lymphocytes at the ocular
surface.16-18,25Despite continuous exposure to desiccating stress and rigorous anticholinergic treatment, CCR2 an-tagonist–treated eyes showed reversal in corneal epithe-lial damage as seen by a decrease in corneal fluorescein up-take. This could have been caused by a decrease in the expression of proinflammatory cytokines because studies have shown that elevated cytokine levels in the tear film create an environment in which terminal differentiation of the ocular surface epithelium is impaired, thereby impair-ing the epithelial surface production of mature
surface-protective molecules.26Topical therapy with the CCR2
an-tagonist decreased the corneal and conjunctival expression
of IL-1 and TNF-␣. These cytokines have been implicated
in the pathogenesis of corneal ulceration, uveitis, and cor-neal transplant rejection.27-29These cytokines also are re-leased by activated macrophages; hence, decreased mac-rophage infiltration in the CCR2 antagonist–treated eyes may account for decreased expression of these cytokines. Our laboratory has previously shown corneal infiltration by mature major histocompatibility complex class II– expressing antigen-presenting cells in DED.7Consistent with
350
300
250
200
150
100
50
0
Periphery Center
Cornea Area
CD 11b
+ Cells/mm 2 of Cornea
Untreated Vehicle treated CCR2 antagonist treated A
B
Untreated Vehicle Treated CCR2 Antagonist Treated
Groups
P < .001
P < .001
P < .001
[image:5.612.69.545.46.404.2]P < .001
our hypothesis, blockade of MCP-1/CCR2 interaction by
using CCR2 antagonist resulted in decreased CD11b⫹
monocyte infiltration into the cornea. This is important be-cause the corneal antigen–presenting cell mobilization can significantly affect the pathogenesis and severity in vari-ous corneal immune reactions such as graft rejection and herpes keratitis.30
Studies in various animal models and in humans have shown that chronic DED is associated with T-cell
infil-tration in the conjunctiva.16Chemokines are not only
ca-pable of attracting and activating T cells, but the chemo-kine-receptor ligation is also involved in T-cell
differentiation.31,32Our study showed significantly
re-duced T-cell infiltration in the conjunctiva in the eyes treated with the CCR2 antagonist. The blockade of CCR2 receptor-ligand interaction possibly modulates the im-munoinflammatory response at the ocular surface by downregulating RNA transcripts for inflammatory cyto-kines in the conjunctival epithelium and ultimately caus-ing decreased T-cell homcaus-ing.
The chemokine system has emerged as a critical regu-lator of dendritic cell and lymphocytic trafficking.33 Che-mokines arising under inflammatory conditions act on various chemokine receptors and attract immature den-dritic cells to the affected sites. In the inflamed tissue, these immature cells pick up antigen and mature with time. During maturation, chemokine receptor
expres-sion changes; this switch results in the dendritic cells leav-ing the tissues and beleav-ing drawn into lymphatics and,
ul-timately, into the T-cell–rich regions of lymph nodes.34
Our results strongly suggest that MCP-1/CCR2 dynam-ics are critical in the pathogenesis of DED. Beneficial ef-fects of CCR2 blockade in DED may be primarily caused
by decreased recruitment of CCR2⫹mononuclear cells
in response to local MCP-1 (CCL2) to the ocular sur-face, which in turn reduces the trafficking of antigen-presenting dendritic cells to the draining lymph nodes, hence decreasing the T-cell effector responses. In addi-tion, blockade of the MCP-1/CCR2 axis may inhibit
re-cruitment of CCR2⫹T cells to the eye in response to
lo-cal MCP-1 and therefore may disrupt the ongoing cycle of T-cell recruitment, reactivation, and effector re-sponses at the ocular surface.
In conclusion, treatment with the topical CCR2 an-tagonist in our DED mouse model led to significant im-provement in the dry eye state at the clinical, molecular, and cellular levels. The reversal of clinical signs and un-derlying inflammatory changes by using a topical CCR2 antagonist in the present study suggests that CCR2 an-tagonism could hold promise as a therapeutic modality in DED. However, the chemokine system is character-ized by significant redundancy, pleiotropy, and differ-ences among different species, which complicates devel-opment of new therapeutics. It is hoped, however, that
60
50
40
30
20
10
0
Untreated Vehicle CCR2 Antagonist Treatment
No. of T Cells per Cross Section
A
B
Untreated Vehicle Treated CCR2 Antagonist Treated
Groups
P < .001
P < .001
St St St
St St
St
[image:6.612.69.546.48.369.2]Ep Ep Ep
more effective therapeutics will be found in the near fu-ture as the intricacies of the chemokine system are bet-ter understood.
Submitted for Publication:December 27, 2008; final re-vision received February 19, 2009; accepted February 20, 2009.
Correspondence:Reza Dana, MD, MSc, MPH, Schepens Eye Research Institute, 20 Staniford St, Boston, MA 02114 (reza.dana@schepens.harvard.edu).
Financial Disclosure:Dr Dana has received research sup-port from Johnson & Johnson Pharmaceutical Research and Development.
Funding/Support:This study was supported by Johnson & Johnson Vision Care, Inc.
REFERENCES
1. Schaumberg DA, Sullivan DA, Buring JE, Dana MR. Prevalence of dry eye syn-drome among US women.Am J Ophthalmol. 2003;136(2):318-326. 2. Schein OD, Munoz B, Tielsch JM, Bandeen-Roche K, West S. Prevalence of dry
eye among the elderly.Am J Ophthalmol. 1997;124(6):723-728.
3. Stern ME, Pflugfelder SC. Inflammation in dry eye.Ocul Surf. 2004;2(2):124-130. 4. Stern ME, Beuerman RW, Fox RI, Gao J, Mircheff AK, Pflugfelder SC. The pa-thology of dry eye: the interaction between the ocular surface and lacrimal glands. Cornea. 1998;17(6):584-589.
5. Luster AD. Chemokines: chemotactic cytokines that mediate inflammation.N Engl J Med. 1998;338(7):436-445.
6. Gerard C, Rollins BJ. Chemokines and disease.Nat Immunol. 2001;2(2):108-115.
7. Rashid S, Jin Y, Ecoiffier T, Barabino S, Schaumberg DA, Dana MR. Topical omega-3 and omega-6 fatty acids for treatment of dry eye.Arch Ophthalmol. 2008;126(2):219-225.
8. Lagu B, Gerchak C, Pan M, et al. Potent and selective CC-chemokine receptor-2 (CCR2) antagonists as a potential treatment for asthma.Bioorg Med Chem Lett. 2007;17(15):4382-4386.
9. Tylaska LA, Boring L, Weng W, et al. CCR2 regulates the level of MCP-1/CCL2 in vitro and at inflammatory sites and controls T cell activation in response to alloantigen.Cytokine. 2002;18(4):184-190.
10. Oshima T, Sonoda KH, Tsutsumi-Miyahara C, et al. Analysis of corneal inflam-mation induced by cauterisation in CCR2 and MCP-1 knockout mice.Br J Ophthalmol. 2006;90(2):218-222.
11. Barabino S, Shen L, Chen L, Rashid S, Rolando M, Dana MR. The controlled-environment chamber: a new mouse model of dry eye.Invest Ophthalmol Vis Sci. 2005;46(8):2766-2771.
12. Lemp MA. Report of the National Eye Institute/Industry workshop on clinical trials in dry eyes.CLAO J. 1995;21(4):221-232.
13. Ecoiffier T, El Annan J, Rashid S, Schaumberg D, Dana R. Modulation of integrin ␣41(VLA-4) in dry eye disease.Arch Ophthalmol. 2008;126(12):1695-1699.
14. Hamrah P, Liu Y, Zhang Q, Dana MR. Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation.Arch Ophthalmol. 2003;121(8):1132-1140.
15. Niederkorn JY, Stern ME, Pflugfelder SC, et al. Desiccating stress induces T cell– mediated Sjögren’s syndrome–like lacrimal keratoconjunctivitis.J Immunol. 2006; 176(7):3950-3957.
16. Stern ME, Gao J, Schwalb TA, et al. Conjunctival T-cell subpopulations in Sjögren’s and non-Sjögren’s patients with dry eye.Invest Ophthalmol Vis Sci. 2002;43 (8):2609-2614.
17. Pflugfelder SC, Jones D, Ji Z, Afonso A, Monroy D. Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjögren’s syndrome keratocon-junctivitis sicca.Curr Eye Res. 1999;19(3):201-211.
18. Solomon A, Dursun D, Liu Z, Xie Y, Macri A, Pflugfelder SC. Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease.Invest Ophthalmol Vis Sci. 2001;42(10):2283-2292. 19. Premack BA, Schall TJ. Chemokine receptors: gateways to inflammation and
infection.Nat Med. 1996;2(11):1174-1178.
20. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines— CXC and CC chemokines.Adv Immunol. 1994;55:97-179.
21. Charo IF. CCR2: from cloning to the creation of knockout mice.Chem Immunol. 1999;72:30-41.
22. Izikson L, Klein RS, Luster AD, Weiner HL. Targeting monocyte recruitment in CNS autoimmune disease.Clin Immunol. 2002;103(2):125-131.
23. Yamagami H, Yamagami S, Inoki T, Amano S, Miyata K. The effects of proin-flammatory cytokines on cytokine-chemokine gene expression profiles in the hu-man corneal endothelium.Invest Ophthalmol Vis Sci. 2003;44(2):514-520. 24. Yamagami S, Yokoo S, Mimura T, Amano S. Effects of TGF-2 on immune
re-sponse-related gene expression profiles in the human corneal endothelium. In-vest Ophthalmol Vis Sci. 2004;45(2):515-521.
25. Pflugfelder SC. Antiinflammatory therapy for dry eye.Am J Ophthalmol. 2004;137 (2):337-342.
26. Jones DT, Monroy D, Ji Z, Pflugfelder SC. Alterations of ocular surface gene ex-pression in Sjögren’s syndrome.Adv Exp Med Biol. 1998;438:533-536. 27. Dana MR, Yamada J, Streilein JW. Topical interleukin 1 receptor antagonist
pro-motes corneal transplant survival.Transplantation. 1997;63(10):1501-1507. 28. Brito BE, O’Rourke LM, Pan Y, Anglin J, Planck SR, Rosenbaum JT. IL-1 and
TNF receptor-deficient mice show decreased inflammation in an immune com-plex model of uveitis.Invest Ophthalmol Vis Sci. 1999;40(11):2583-2589. 29. Rosenbaum JT, Planck ST, Huang XN, Rich L, Ansel JC. Detection of mRNA for
the cytokines, interleukin-1 alpha and interleukin-8, in corneas from patients with pseudophakic bullous keratopathy.Invest Ophthalmol Vis Sci. 1995;36(10): 2151-2155.
30. McLeish W, Rubsamen P, Atherton SS, Streilein JW. Immunobiology of Langer-hans cells on the ocular surface, II: role of central corneal LangerLanger-hans cells in stromal keratitis following experimental HSV-1 infection in mice.Reg Immunol. 1989;2(4):236-243.
31. Murphy KM, Ouyang W, Farrar JD, et al. Signaling and transcription in T helper development.Annu Rev Immunol. 2000;18:451-494.
32. Bonecchi R, Bianchi G, Bordignon PP, et al. Differential expression of chemo-kine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s.J Exp Med. 1998;187(1):129-134.
33. Charbonnier AS, Kohrgruber N, Kriehuber E, Stingl G, Rot A, Maurer D. Macro-phage inflammatory protein 3␣is involved in the constitutive trafficking of epi-dermal Langerhans cells.J Exp Med. 1999;190(12):1755-1768.
34. Baggiolini M. Chemokines in pathology and medicine.J Intern Med. 2001;250(2): 91-104. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0
TNF-α IL1-α IL1-β
Relative Expression of mRNA in Cornea
Vehicle treated CCR2 antagonist treated A
P = .04
P = .01
1.4 1.2 1.8 1.6 1.0 0.8 0.6 0.4 0.2 0.0
TNF-α IL1-α IL1-β
Relative Expression of mRNA in Conjunctiva
B
P = .02
[image:7.612.67.295.43.341.2]P = .04
Figure 4.Real-time polymerase chain reaction analysis showing transcript levels of tumor necrosis factor␣(TNF-␣), interleukin 1␣(IL-1␣), and IL-1