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`Desiccating Stress Induces T Cell-Mediated
`Sjögren's Syndrome-Like Lacrimal
`Keratoconjunctivitis
`Jerry Y. Niederkorn, Michael E. Stern, Stephen C.
`Pflugfelder, Cintia S. De Paiva, Rosa M. Corrales, Jianping
`Gao and Karyn Siemasko
`
`2006; 176:3950-3957; ;
`J Immunol 
`doi: 10.4049/jimmunol.176.7.3950
`http://www.jimmunol.org/content/176/7/3950
`
`References
`
`cites 20 articlesThis article
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`1451 Rockville Pike, Suite 650, Rockville, MD 20852
`Copyright © 2006 by The American Association of
`Immunologists All rights reserved.
`Print ISSN: 0022-1767 Online ISSN: 1550-6606.
`
`MYLAN - EXHIBIT 1046
`Mylan Pharmaceuticals Inc. et al. v. Allergan, Inc.
`IPR2016-01127, -01128, -01129, -01130, -01131, & -01132
`
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`The Journal of Immunology
`
`Desiccating Stress Induces T Cell-Mediated Sjo¨gren’s
`Syndrome-Like Lacrimal Keratoconjunctivitis1
`
`Jerry Y. Niederkorn,2,3* Michael E. Stern,2† Stephen C. Pflugfelder,2‡ Cintia S. De Paiva,‡
`Rosa M. Corrales,‡ Jianping Gao,† and Karyn Siemasko†
`
`Chronic dry eye syndrome affects over 10 million people in the United States; it is associated with inflammation of the lacrimal
`gland (LG) and in some cases involves T cell infiltration of the conjunctiva. We demonstrate that environmental desiccating stress
`(DS) elicits T cell-mediated inflammation of the cornea, conjunctiva, and LG, but not other organs in mice. The lacrimal kera-
`toconjunctivitis (LKC) was mediated by CD4ⴙ T cells, which, when adoptively transferred to T cell-deficient nude mice, produced
`inflammation in the LG, cornea, and conjunctiva, but not in any other organ. Adoptively transferred CD4ⴙ T cells produced LKC
`even though recipients were not exposed to DS. LKC was exacerbated in euthymic mice depleted of CD4ⴙCD25ⴙforkhead/winged
`helix transcription factorⴙ regulatory T cells. The results suggest that DS exposes shared epitopes in the cornea, conjunctiva, and
`induce pathogenic CD4ⴙ T cells that produce LKC, which under normal circumstances is restrained by
`LG that
`CD4ⴙCD25ⴙforkhead/winged helix transcription factorⴙ regulatory T cells. The Journal of Immunology, 2006, 176: 3950 –3957.
`
`D ry eye is a common ocular disease and is one of the
`
`leading causes of patients seeking ophthalmic care. Dry
`eye or lacrimal keratoconjunctivitis (LKC)4 has a tre-
`mendous impact on quality of life and can cause debilitating eye
`pain and loss of vision. In animal models and humans, chronic dry
`eye disease is often associated with T cell infiltration of the con-
`junctiva (1–3). T cell infiltration has also been observed in dry eye
`patients with or without a systemic autoimmune disease, such as
`Sjogren’s syndrome (SS) (3). It has been proposed that in some
`forms of dry eye disease, autoreactive T cells alter the function of
`the ocular surface epithelium and also cause reduced tear secretion
`(4). The presence of CD4⫹ T cells in the epithelium and stroma of
`the conjunctiva and the salutary effect of the T cell-immunosup-
`pressive drug, cyclosporine, in some patients support the proposi-
`tion that dry eye is an immune-mediated disease (2, 5). However,
`others have argued that inflammation of the cornea and conjunc-
`tiva is the result, not the cause, of dry eye disease.
`It is now recognized that the ocular surface and lacrimal glands
`function as an integrated unit, termed the lacrimal functional unit
`(LFU), which is linked by their sensory and autonomic nerves (4).
`Inflammation in any component of the LFU has the capability to
`compromise the function of other components through soluble me-
`
`*Department of Ophthalmology, University of Texas Southwestern Medical Center,
`Dallas, TX 75390; †Department of Biological Sciences, Allergan, Irvine, CA 92623;
`and ‡Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030
`
`Received for publication December 8, 2005. Accepted for publication January
`19, 2006.
`
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance
`with 18 U.S.C. Section 1734 solely to indicate this fact.
`1 This work was supported by Grant EY11915 and unrestricted grants from Research
`to Prevent Blindness (New York, NY) and Allergan.
`2 J.Y.N., M.E.S., and S.C.P. contributed equally to this study.
`3 Address correspondence and reprint requests to Dr. Jerry Y. Niederkorn, Depart-
`ment of Ophthalmology, University of Texas Southwestern Medical Center, 5323
`Harry Hines Boulevard, Dallas, TX 75390. E-mail address:
`jerry.niederkorn@
`utsouthwestern.edu
`4 Abbreviations used in this paper: LKC, lacrimal keratoconjunctivitis; CLN, cervical
`lymph node; DS, desiccating stress; FoxP3, forkhead/winged helix transcription fac-
`tor; GC, goblet cell; LFU, lacrimal functional unit; LG, lacrimal gland; NS, non-
`stressed; OGD, Oregon Green dextran; SS, Sjogren’s syndrome.
`
`diators, generation of autoreactive T cells to shared Ags, and inhibi-
`tion of neural transmission (4). SS causes profound dysfunction of the
`LFU, which includes decreased tear secretion, loss of conjunctival
`goblet cells (GC) transformation of the ocular surface epithelium to a
`keratinized surface. Immunopathological changes, including immune-
`mediated inflammation and increased production of inflammatory cy-
`tokines and chemokines, have been detected in the dysfunctional lac-
`rimal glands and ocular surface tissues in SS (6–8).
`It has been proposed that a wide range of factors that alter the
`integrity of tissues create danger signals that activate APCs and
`promote the generation of immune responses to self Ags (9). The
`danger hypothesis has been implicated in the pathogenesis of SS
`(10) and is consistent with the observation that desiccating stress
`(DS) to the ocular surface induces the expression of proinflamma-
`tory cytokines (11). With this in mind, we tested the hypothesis
`that environmental DS to the ocular surface would break self-tol-
`erance and induce the development of T cell-mediated inflamma-
`tion directed against epitopes expressed on the ocular surface and
`the lacrimal gland (LG).
`
`Materials and Methods
`Mice
`
`(BALB/cByJ-
`nude BALB/c
`Female BALB/c, T cell-deficient
`HfH11⬍␯⬎), and C57BL/6 mice, 6 – 8 wk old, were purchased from The
`Jackson Laboratory or Charles River Laboratories. All animal experiments
`were approved by the institutional animal care and use committees at Al-
`lergan and Baylor College of Medicine. All studies adhered to the Asso-
`ciation for Research in Vision and Ophthalmology statement for the Use of
`Animals in Ophthalmic and Vision Research.
`
`Induction of DS in mice
`
`DS was induced by s.c. injection of scopolamine hydrobromide (0.5 mg/0.2
`ml; Sigma-Aldrich) four times a day (0800, 1200, 1400, and 1700 h),
`alternating between the left and right flanks of 4- to 6-wk-old mice, as
`previously described (12). Up to five mice were placed in a cage with a
`perforated plastic screen on one side to allow airflow from a fan (Cafrano)
`placed 6 in. in front of it for 16 h/day. Room humidity was maintained at
`30 –35% and temperature at 80°F. DS was induced for either 5 or 12 con-
`secutive days. This model of dry eye disease has been used previously with
`no discernible ill effects from the scopolamine treatment or low humidity
`(11, 12). Changes in corneal permeability were assessed by measuring
`corneal staining by Oregon Green dextran (OGD; 70,000 m.w.; Molecular
`Probes) as previously described (13). Aqueous tear production was assessed
`
`Copyright © 2006 by The American Association of Immunologists, Inc.
`
`0022-1767/06/$02.00
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`The Journal of Immunology
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`using cotton thread (Quick Thread; FCI Ophthalmics) as previously described
`(14). Control mice were maintained in a nonstressed (NS) environment con-
`taining 50–75% relative humidity without exposure to forced air. Tear pro-
`duction was measured with phenol red-impregnated cotton threads (Zone-
`Quick; Oasis) placed into the tear meniscus of the lateral canthus for 30 s.
`
`Measurement of GC density
`
`Surgically excised eyes were fixed in 10% formalin and embedded in paraffin.
`Sections (6 ␮m) were stained with periodic acid-Schiff reagent. Sections from
`three eyes in each group (in two different sets of experiments) were examined
`and photographed with a Nikon Eclipse E400 microscope equipped with a
`Nikon DXM 1200 digital camera. GC density was measured in the superior
`and inferior bulbar and tarsal conjunctiva and expressed as number per 100 ␮m
`using Metavue 6.24r software (Molecular Devices).
`
`In vivo analysis of adoptively transferred CFSE-labeled
`lymphocytes
`Spleen cells (1 ⫻ 107/ml) were collected from BALB/c mice subjected to
`DS and were labeled with 10 ␮M CFSE (Molecular Probes) for 15 min at
`room temperature. Cells were washed three times in RPMI 1640 and were
`injected i.p. into BALB/c mice (1 ⫻ 107 spleen cells/0.1 ml/mouse).
`
`Immunohistochemistry
`
`OCT-embedded globes with attached lids were sectioned at 10-␮m thick-
`ness and stained for the expression of mouse CD4 (rat anti-mouse CD4; rat
`IgG2a, k; clone H129.19; BD Pharmingen) using Vectastain Elite ABC
`reagents (Vector Laboratories).
`
`Adoptive cell transfer and anti-Thy 1.2 Ab treatment
`
`Spleens and cervical lymph nodes (CLN) were collected from mice sub-
`jected to DS and NS, and one donor-equivalent of either spleen or CLN
`cells was transferred i.p. to syngeneic nude mice. One donor-equivalent is
`defined as the number of cells remaining after the respective in vitro ma-
`nipulation (e.g., anti-Thy 1.2 Ab treatment or CD4⫹ T cell enrichment) of
`a single spleen or CLN from a single donor. The remaining cells represent
`the total lymphocyte population for that spleen cell category for a single
`donor. One splenic equivalent of T cells was equal to ⬃5 ⫻ 107 cells.
`Spleen cells were depleted of T cells by in vitro treatment with anti-Thy 1.2
`Ab (BD Pharmingen) in the presence of complement. Control aliquots of
`spleen cells were treated with complement alone. Cells were washed with
`RPMI 1640 medium before being adoptively transferred in 0.1 ml of RPMI
`1640 medium.
`CD4⫹ and CD25⫹ T cell enrichment
`BALB/c CLN cell suspensions were enriched for CD4⫹ T cells by positive
`selection using rat anti-mouse CD4-conjugated magnetic microbeads
`(MACS system; Miltenyi Biotec) as described previously (15). The CD4-
`enriched cell suspensions contained ⬎87% CD4⫹ T cells as determined by
`flow cytometry. CD4⫹CD25⫹ T cells were enriched using a mouse
`CD4⫹CD25⫹ regulatory T cell magnetic isolation kit (Miltenyi Biotec)
`according to the manufacturer’s instructions.
`Permeabilized CD4⫹CD25⫹ cells were tested for the expression of cy-
`toplasmic forkhead/winged helix transcription factor (Foxp3) protein.
`Briefly, CD4⫹CD25⫹ cells were washed three times in HBSS, resuspended
`in Cytofix/Cytoperm solution (BD Pharmingen), and incubated for 30 min
`at room temperature. Cells were washed three times in Perm/Wash buffer
`and resuspended with an FITC-labeled rat anti-mouse Foxp3 Ab (eBio-
`sciences) at a concentration of 1 ␮g/ml for 30 min on ice. Cells were
`washed in Perm/Wash buffer three times, incubated with PE-labeled sec-
`ondary Ab for 20 –30 min at 4°C, washed three additional times in Perm/
`Wash buffer, fixed in 1% paraformaldehyde, and assessed for fluorescence
`in a FACScan flow cytometer (BD Biosciences). The results were analyzed
`using CellQuest version 3.1f software (BD Biosciences). CD4⫹CD25⫹
`cells were found to be 89% Foxp3⫹ (data not shown).
`In vivo depletion of CD25⫹ cells
`In vivo depletion of CD25⫹ regulatory T cells was achieved as described
`previously (16). Briefly, anti-CD25 Ab (hybridoma PC61; American Type
`Culture Collection) was administered i.p. to normal BALB/c mice 7 days
`before and on the day DS was initiated.
`
`Statistics
`
`Values for cell counts in tissue specimens and tear production were eval-
`uated by either Student’s t test or ANOVA.
`
`Results
`DS induces immune-mediated inflammation of the ocular surface
`and lacrimal gland
`
`C57BL/6 mice exposed to DS displayed altered corneal epithelial
`barrier function, which was reflected in an 85% increase in the
`uptake of the OGD label that was used to measure the integrity of
`the corneal epithelial barrier (Fig. 1). Histopathological analysis
`revealed reduced numbers of conjunctival GC, decreased tear pro-
`duction, and CD4⫹ T cell infiltration of the conjunctival basal
`
`FIGURE 1.
`Increased corneal permeability induced by DS. C57BL/6
`mice were subjected to 5 days of DS or NS conditions. Mouse corneas were
`viewed and photographed under cobalt blue light 10 min after application
`of 1 ␮l of the fluorescent molecule OGD to the ocular surface. A, Ocular
`surface of mouse subjected to 5 days of DS. B, Ocular surface of untreated,
`NS mouse. C, Quantification of OGD uptake by corneas of NS normal
`mice and mice subjected to 5 days of DS. There were five animals in each
`group in three independent experiments.
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`3952
`
`DS INDUCES T CELL-MEDIATED KERATOCONJUNCTIVITIS
`
`epithelium (Fig. 2, A–E). The greater severity of keratoconjuncti-
`vitis in C57BL/6 mice than BALB/c mice is consistent with the
`observation that BALB/c mice have a larger
`repertoire of
`CD4⫹CD25⫹ regulatory T cells compared with C57BL/6 mice
`(17). The role of CD4⫹CD25⫹ regulatory T cells was tested by
`treating BALB/c mice with anti-CD25 Ab to deplete natural reg-
`ulatory T cells before subjecting the mice to DS. Anti-CD25-
`treated BALB/c mice developed significant keratoconjunctivitis
`and displayed a steep reduction in the density of conjunctival GC
`compared with the isotype control mice and mice subjected to DS
`without Ab treatment (Fig. 2F).
`
`DS-induced lacrimal keratoconjunctivitis can be adoptively
`transferred with CD4⫹ T cells
`Adoptive transfer experiments were performed to determine the
`role of T cells in the ocular inflammation elicited by DS. Draining
`
`CLN cells were collected from BALB/c mice after a 5-day expo-
`sure to DS and were adoptively transferred to athymic (nude)
`BALB/c mice that were maintained under NS conditions (50 –75%
`relative humidity). Even though the adoptive cell transfer recipi-
`ents were NS, they had reduced tear production and developed
`LKC after receiving CLN cells from donors treated with DS (Fig.
`3). Inflammation was immune mediated, because the nude mouse
`recipients of CLN cells from NS donors maintained normal tear
`production and did not develop inflammation of the conjunctiva
`(Fig. 3, B and C). Importantly, inflammation in recipients of CLN
`cells from DS donors was restricted to the lacrimal functional unit
`(cornea, conjunctiva, and LG), and no significant inflammatory
`cell infiltrates were found in the salivary gland, oral mucosa, thy-
`roid, heart, lung, colon, spleen, adrenal gland, or vagina (data not
`shown). Histopathological analysis revealed significant LG inflam-
`mation and loss of conjunctival GC, which coincided with a steep
`
`FIGURE 2. DS induces LKC. C57BL/6 mice (A–E) and BALB/c mice (A, B, and F) were maintained in either NS conditions (50 –75% relative humidity)
`or DS (30 –35% relative humidity) for either 5 days (A–E) or 12 days (A and C). Five days of DS consistently produced LKC in C57BL/6 mice. A, Reduced
`numbers of conjunctival GC; B, diminished tear production; C, CD4⫹ cell density in conjunctivae of NS mice. D, Mice treated with DS; CD4⫹ T cells
`(brown stain) in basal conjunctival epithelium. E, CD4⫹ T cell density in conjunctivae of DS and NS mice. F, Reduced conjunctival GC density in BALB/c
`mice treated with anti-CD25 before exposure to 5 days of DS. There were three animals in each group in two independent experiments (i.e., n ⫽ 6
`mice/group).
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`reduction in tear production (Fig. 3A) and the appearance of
`ocular surface inflammation (Fig. 3, B and C). Immunohisto-
`chemical staining demonstrated a significant infiltrate of CD4⫹
`T cells in the LG in the adoptive cell transfer recipients (Fig. 4).
`Inflammation was T cell dependent, because in vitro treatment
`of CLN cell suspensions with anti-Thy 1.2 Ab plus complement
`before adoptive transfer prevented the development of LKC
`(Fig. 5A). Moreover, histopathological examination of the eyes
`and LGs of recipients of CLN cell suspensions depleted of T
`cells demonstrated significantly reduced mononuclear cellular
`infiltrates in the conjunctivae and the sparing of 80% of the
`normal tear production (Fig. 5, B and C). To determine whether
`the CD4⫹ T cells in the conjunctivae and LGs contributed to
`LKC, adoptive transfer experiments were performed in which
`either CD4⫹ T cell-enriched or CD4⫹ T cell-depleted CLN cell
`suspensions were collected from mice subjected to DS and
`transferred to syngeneic BALB/c nude mice. Recipients of
`CD4⫹ T cell-enriched CLN cells developed intense LG inflam-
`mation (Fig. 5D), with significant increases in the number of
`both conjunctival mononuclear cells and neutrophils (data not
`shown). Moreover, CD4⫹ T cells were detected in the LG of the
`adoptive transfer recipients (Fig. 5G). Additional evidence of
`CD4⫹ T cell involvement was demonstrated in experiments in
`which CD4⫹ T cells were collected from mice subjected to DS,
`labeled with CFSE, and adoptively transferred to nude mice that
`were maintained in an NS environment. Accumulations of
`CFSE-labeled CD4⫹ T cells were found in the conjunctival
`epithelium of the nude mice that received adoptive cell transfers
`(Fig. 5H), but were absent
`in nonocular organs (data not
`shown). By contrast, adoptive transfer of CFSE-labeled CD4⫹
`T cells from normal donors did not preferentially localize in the
`corneas or conjunctivae of nude mouse recipients (Fig. 6), sug-
`gesting that mice subjected to DS develop CD4⫹ T cells that
`preferentially localize in the eye and LGs, where they produce
`inflammatory disease, GC loss, and LG dysfunction.
`
`Role of CD4⫹CD25⫹ T cells in mitigating LKC
`The inflammation produced in the adoptive cell transfer recip-
`ients was consistently more severe in nude mice than euthymic
`mice, suggesting the possible mitigating effects of one or more
`regulatory T cell populations in the euthymic host. To examine
`the role of natural regulatory T cells in LKC, euthymic BALB/c
`mice were treated with anti-CD25 mAb to deplete putative nat-
`ural regulatory T cells before receiving adoptively transferred T
`cells from BALB/c donors treated with DS. Euthymic BALB/c
`
`FIGURE 3. Adoptive T cell transfer of LKC. Euthymic BALB/c mice
`(normal) were subjected to either DS or NS conditions for 5 days, and one
`donor-equivalent of CLN cells was adoptively transferred to each athymic
`(nude) BALB/c nude mouse recipient. Nude mice were maintained under NS
`conditions after adoptive cell transfer and were assessed for tear production
`and conjunctival cellular infiltrates 7 days later. A, Reduced tear production. B
`and C, Mononuclear (B) and neutrophil (C) cell infiltrates in conjunctivae of
`adoptive transfer recipients are shown. There were five animals in each group.
`
`FIGURE 4. CD4⫹ T cell infiltrate in lacrimal glands in recipients of
`adoptively transferred CD4⫹ T cells from mice subjected to DS. A, CD4⫹
`T cells (brown) in LG in a recipient of CLN cells from a DS donor. B,
`Immunohistochemistry of the same specimen as that in A, but tested with
`an isotype control Ab. There were five animals in each group.
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`3954
`
`DS INDUCES T CELL-MEDIATED KERATOCONJUNCTIVITIS
`
`FIGURE 5. DS-induced LKC is mediated by T cells. BALB/c mice were placed in DS for 5 days, and CLN and spleen cells were collected. CLN cells
`were treated with anti-Thy 1.2 Ab and complement to deplete T cells. One donor-equivalent of unfractionated or CD4⫹ T cell-enriched CLN or spleen cells
`was injected i.p. into each BALB/c nude mouse. Nude mice were kept under NS conditions. Tissues were collected for histology 48 h later. A, Histopa-
`thology of conjunctiva, cornea, and LG after adoptive CLN cell transfer. Note the absence of GC in conjunctivae in recipients of untreated or complement-
`treated CLN cells. Normal GC (short arrows) in hosts receiving T cell-depleted CLN cells. Corneal inflammation (long arrow) is absent in recipients of
`anti-Thy 1.2-treated CLN cells. B, Mononuclear cells infiltrate the conjunctivae of CLN cell recipients. C, Reduced tear production in CLN cell recipients.
`D, Intense LG inflammation (arrow) in recipients of CD4⫹ T spleen cells from DS donors. E, Normal LG. F, Isotype control Ab (background) staining
`of LG from a recipient of CD4⫹ spleen cells from DS donors. G, Anti-CD4 immunostaining (brown) of LG from same mouse as that in F. H, Accumulation
`of CD4⫹ T cells in the conjunctiva after adoptive transfer of CFSE-labeled, CD4⫹ T cell-enriched spleen cells. There were five animals in each group.
`
`mice treated with anti-CD25 Ab before receiving adoptively
`transferred CD4⫹ CLN from DS donors developed severe LKC
`and a 38% reduction in tear production (Fig. 7). To confirm that
`the CD4⫹CD25⫹ T cells exerted a mitigating effect on the de-
`velopment of dry eye disease, nude mice were reconstituted
`with one spleen cell equivalent of either CD4⫹CD25⫹ (⬃89%
`Foxp3⫹) T cells or CD4⫹CD25⫺ (⬍1% Foxp3⫹) T cells from
`euthymic, BALB/c donors that had been maintained in an NS
`environment. On the same day, the nude mice also received
`pathogenic, LKC-inducing CD4⫹ T cells from BALB/c donor
`mice that had been maintained for 5 days in a DS environment.
`Nude mice reconstituted with CD4⫹CD25⫹Foxp3⫹ putative
`regulatory T cells resisted the development of LKC and main-
`tained 90% tear production compared with a 28% reduction in
`tears in nude mice not receiving CD4⫹CD25⫹ T cells (Fig. 8A).
`Moreover, CD4⫹CD25⫹ Foxp3⫹ reconstituted mice resisted in-
`flammation and expressed a 60% reduction in the number of
`
`FIGURE 6. Absence of conjunctival and corneal
`inflammation in
`BALB/c nude mice that received CFSE-labeled CD4⫹ spleen cells from
`NS BALB/c donor mice. There were five animals in each group.
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`3955
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`FIGURE 7. Depletion of natural T regulatory cells by systemic treatment with anti-CD25 mAb exacerbates DS-induced LKC. Pathogenic CD4⫹
`T cells were isolated from the CLN of BALB/c mice that were subjected to 5 days of DS and adoptively transferred to euthymic BALB/c mice (1 ⫻
`107 CD4⫹ cells/mouse) that were treated with either anti-CD25 Ab or an isotype control Ab (500 ␮g given i.p. 7 days before and on the day of the
`adoptive transfer of CD4⫹ CLN cells). A, Keratoconjunctivitis in anti-CD25-treated BALB/c mice. Arrows indicate inflammatory cell infiltrates in
`conjunctiva and cornea. B, Reduced tear production in anti-CD25-treated BALB/c mice that received CD4⫹ CLN from DS donors. There were five
`animals in each group.
`
`mononuclear cells and a 97% reduction in the number of neu-
`trophils that infiltrated the conjunctivae (Fig. 8, B and C). His-
`topathological analysis confirmed the mitigating effects of
`adoptively transferred CD4⫹CD25⫹Foxp3⫹ cells. Conjunctival
`GC were virtually eliminated in recipients of CD4⫹ T cells
`from DS donors, but were spared in nude mice that were re-
`constituted with CD4⫹CD25⫹Foxp3⫹ cells from NS donors
`(Fig. 8, D–F). Likewise, corneal and LG inflammation was ex-
`tinguished in CD4⫹CD25⫹Foxp3⫹ cell
`recipients (Fig. 8,
`G–L).
`
`Discussion
`The causes of dry eye diseases, including SS, are multifactorial
`and involve immunogenetic, hormonal, and environmental factors
`(18, 19). In this study we demonstrated the isolated effect of a
`single environmental factor, DS, on the development of immune-
`mediated dry eye disease. Animals subjected to DS developed oc-
`ular surface lesions that mimicked those found in dry eye patients.
`The preponderance of CD4⫹ T cells in LG inflammation bore a
`striking resemblance to the lesions found in SS patients and in
`animal models of SS (19 –21). More important, the results indicate
`that tear insufficiency alters the function of the corneal epithelium
`and elicits inflammation of the ocular surface. The DS-induced
`inflammation elicited the generation of autoreactive CD4⫹ T cells
`that infiltrated all three components of the lacrimal functional unit
`(i.e., cornea, conjunctiva, and LG). Once generated, pathogenic
`CD4⫹ T cells produced ocular surface and LG inflammation, even
`in animals housed in an NS environment.
`The milder ocular surface inflammation induced by DS in eu-
`thymic BALB/c mice compared with C57BL/6 mice is consistent
`with the hypothesis that DS-induced LKC is mediated by CD4⫹ T
`cells, but is mitigated by CD4⫹CD25⫹ natural regulatory T cells,
`because BALB/c mice have a better developed repertoire of
`CD4⫹CD25⫹ regulatory T cells than C57BL/6 mice (17). More-
`over, elimination of CD25⫹ T cells in euthymic mice increased the
`severity of LKC, whereas reconstitution of athymic mice with
`CD4⫹CD25⫹ regulatory T cells mitigated LKC.
`A phenomenon similar to what we have reported also occurs in
`some patients who are exposed to ocular surface dryness from any
`
`number of mechanisms. It is not unusual for patients who take
`medications with anticholinergic side effects, such as antidepres-
`sants, to develop a permanent dry eye despite cessation of the
`offending medication. These patients frequently respond to immu-
`nomodulatory therapies such as topical corticosteroids and cyclo-
`sporine. This phenomenon is also observed when there is incom-
`plete lid closure after blepharoplasty surgery where there is
`exposure and desiccation of a portion of the ocular surface. Pa-
`tients with well-documented normal tear production before this
`surgery may develop a marked depression in tear production that
`persists after the exposure is treated. However, these patients fre-
`quently respond to immunomodulatory therapies. We believe that
`persistent DS results in the up-regulation of ocular surface Ags that
`initiate an autoimmune LKC. This is influenced by a variety of
`host susceptibility factors, including age, gender, and systemic im-
`mune dysregulation (e.g., rheumatoid arthritis or graft-vs-host
`disease).
`The present findings also suggest that mild inflammation pro-
`duced by transient DS is brought under control by CD4⫹25⫹
`Foxp3⫹ regulatory T cells. However, in systemic autoimmune dis-
`eases, such as SS, ocular surface inflammation occurs in conjunc-
`tion with inflammation in multiple organ systems and might be
`more resistant to immune regulation by CD4⫹CD25⫹Foxp3⫹ reg-
`ulatory T cells. In SS, it is widely believed that inflammation of the
`LG compromises tear production, which culminates in desiccation
`of the ocular surface. The desiccated ocular surface, in turn, pro-
`vokes inflammation and might elicit the generation of a danger
`signal, which breaks tolerance to self epitopes expressed on cells
`on the ocular surface. Our results indicate that desiccation of the
`ocular surface induces an immune response not only to the corneal
`and conjunctival epithelium, but also to elements in the LG, sug-
`gesting that there are shared epitopes among the components of the
`LFU. Identifying these epitopes will be important for devising
`therapeutic strategies that may be useful in the management of
`⬎10 million dry eye patients.
`
`Disclosures
`The authors have no financial conflict of interest.
`
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`DS INDUCES T CELL-MEDIATED KERATOCONJUNCTIVITIS
`
`FIGURE 8. CD4⫹CD25⫹Foxp3⫹ T cells mitigate LKC. BALB/c nude mice were not reconstituted (none) or were reconstituted with CD4⫹
`CD25⫹Foxp3⫹ T cells (putative suppressor cells) from euthymic BALB/c mice maintained in NS conditions. Pathogenic CD4⫹ T cells were isolated from
`euthymic BALB/c mice subjected to DS and were adoptively transferred to nude mice on the same day as the reconstitution with CD4⫹CD25⫹Foxp3⫹ T
`cells. A, Reduced tear production. B, Mononuclear cell infiltrates into the conjunctivae. C, Neutrophil infiltration into conjunctivae. Histopathology of
`conjunctiva (D–F), cornea (G–I), and LG (J–L) is shown. Groups consisted of untreated controls (D, G, and J), recipients of pathogenic CD4⫹ T cells from
`DS donors (E, H, and K), and recipients of pathogenic CD4⫹ T cells from DS donors and CD4⫹CD25⫹ T cells from NS donors. Note the loss of
`conjunctival GC (arrows; E) and intense LG inflammation (K) in a nude mouse that received CD4⫹ T cells from a DS mouse, but was not reconstituted
`with normal CD4⫹CD25⫹Foxp3⫹ T cells. There were two animals in E, H, and K; all other panels had five animals in each group.
`
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