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Amelioration of Murine Dry Eye

Disease by Topical Antagonist

to Chemokine Receptor 2

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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

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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 1␤in the

cor-nea, and tumor necrosis factor␣and interleukin 1␤in

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 the

population 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),

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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

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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 CD11b

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 CD3T 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-1␤in 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

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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

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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

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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

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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

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Untreated Vehicle CCR2 Antagonist Treatment

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Untreated Vehicle Treated CCR2 Antagonist Treated

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P < .001

P < .001

St St St

St St

St

[image:6.612.69.546.48.369.2]

Ep Ep Ep

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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.

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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␤

Figure

Figure 1. Corneal fluorescein staining score. Topical chemokine receptor 2(CCR2) antagonist treatment produced a significant decrease in cornealfluorescein staining compared with the untreated group at day 5 and withboth the untreated and vehicle-treated g
Figure 2. Enumeration of corneal CD11buntreated and vehicle-treated eyes and in eyes treated with a topical chemokine receptor 2 (CCR2) antagonist
Figure 3. Enumeration of conjunctival T cells. A, Representative images of conjunctival cross sections immunostained for CD3 (red) and nucleus (blue) ofuntreated and vehicle-treated eyes and eyes treated with a topical chemokine receptor 2 (CCR2) antagonis
Figure 4. Real-time polymerase chain reaction analysis showing transcriptin the cornea and conjunctiva

References

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