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Photochemistry and Photobiology, 20**, **: **

Invited Review
Damaging Effects of Ultraviolet Radiation on the Cornea
Naomi C. Delic1,2, J. Guy Lyons1,2,3, Nick Di Girolamo4 and Gary M. Halliday*1
1
Discipline of Dermatology, Bosch Institute, University of Sydney, Camperdown, NSW, Australia
2
Immune Imaging Program, Centenary Institute for Cancer Medicine and Cell Biology, Camperdown, NSW, Australia
3
Sydney Head and Neck Cancer Institute, Cancer Services, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
4
Department of Pathology, School of Medical Sciences, University of New South Wales, Randwick, NSW, Australia
Received 12 September 2016, accepted 18 October 2016, DOI: 10.1111/php.12686

ABSTRACT membrane, and (e) a monolayer of specialized corneal endothe-


lial cells. Located at the periphery of the cornea is a narrow tran-
The cornea sits at the anterior aspect of the eye and, like the sitional band of tissue approximately 1 mm wide, known as the
skin, is highly exposed to ultraviolet radiation (UVR). The limbus, which separates the cornea from the conjunctiva. The
cornea blocks a signicant proportion of UVB from reaching corneal epithelium is perpetually self-renewing, whereby the pro-
the posterior structures of the eye. However, UVA can pene- geny of stem cells residing in the limbus replaces terminally dif-
trate the full thickness of the cornea, even reaching the ante- ferentiated cells that are constantly shed from the corneal surface
rior portion of the lens. Epidemiological data indicate that into the tear pool (14). These limbal epithelial stem cells
UVR is a contributing factor for a multitude of diseases of (LESCs) are unipotent as they give rise to corneal, but not con-
the cornea including pterygium, photokeratitis, climatic dro- junctival epithelia. In vivo studies combined with mathematical
plet keratopathy and ocular surface squamous neoplasia modeling have described a striking spoke-like pattern that forms
(OSSN), although the pathogenic mechanisms of each require as corneal epithelia migrate from the limbus, moving cen-
further elucidation. UVR is a well-known genotoxic agent, tripetally toward the apex of the cornea (Fig. 1c,d) (1,5). Mathe-
and its effects have been well characterized in organs such as matical modeling of this process has demonstrated that the
the skin. However, we are only beginning to identify its development of this pattern requires no external cues; rather, it is
effects on the cornea, such as the UVR signature C ? T and purely population pressure-driven (5). Lineage tracing experi-
CC ? TT transversions identied by sequencing and ments using a mosaic mouse model have recently shown that a
increased proliferative and shedding rates in response to single precursor cell is responsible for a unique spoke that forms,
UVR exposure. Alarmingly, a single low-dose exposure of conrming the role of stem cells in the maintenance of the cor-
UVR to the cornea is sufcient to elicit genetic, molecular neal epithelium (1). The mosaic model (Confetti) used a tamox-
and cellular changes, supporting the consideration of using ifen-inducible system so that any one or two of four uorescent
protective measures, such as wearing sunglasses when out- proteins (mCFP, nucGFP, cytoYFP and cyto-tDimer2) could be
doors. The aim of this review was to describe the adverse expressed in K14-expressing cells, including the limbal epithelial
effects of UVR on the cornea. stem cells, giving each of these cells a distinct color. These lim-
bal stem cells give rise to progeny (stem cell and/or corneal
THE CORNEA epithelial cell) that inherit the same uorescent protein therefore
making this system ideal for lineage tracing.
The human eye is a roughly spherical organ that measures
approximately 2.5 cm in diameter and contains two internal
cavities: the anterior and posterior chambers. The cornea is EXPOSURE OF THE EYE TO ULTRAVIOLET
located at the most anterior aspect of the eye, overlying the iris, RADIATION (UVR)
pupil and anterior chamber, and its health is essential for exqui-
The sun is the main source of UVR emitting a wide spectrum of
site vision (Fig. 1a). Structurally, the mammalian cornea consists
electromagnetic radiation. However, only the ultraviolet (UV),
of ve layers (Fig. 1b): beginning at the most anterior position
visible and infrared bands reach the Earths surface to make up
(a) a multilayered corneal epithelium, (b) a basement membrane-
what is known as terrestrial sunlight, with UVR constituting
like structure known as Bowmans layer, (c) the corneal stroma,
approximately 5% (6) (Fig. 2a). UVR can be further divided into
maintained by resident mesenchymal cells known as keratocytes,
three spectral regions; UVC (100290 nm), UVB (290320 nm)
(d) a second basement membrane, known as Descemets
and UVA (320400 nm). UVC is absorbed by ozone in the earths
atmosphere(7), and thus, the UVR component of terrestrial light
*Corresponding author email: gary.halliday@sydney.edu.au (Gary M. Halliday) consists of approximately 4% UVB and 96% UVA with irradi-
This article is part of the Special Issue honoring Dr. Hasan Mukhtars 70th birth- ances differing depending on cloud cover (Fig. 2b) (8).
day and his outstanding contributions to various aspects of photobiology research,
including photocarcinogenesis and chemoprevention. The eyes and skin are the organs most highly exposed to
2016 The American Society of Photobiology UVR and its potentially damaging effects. When discussing

1
2 Naomi C. Delic et al.

(a) (b) UV Penetrance


C B A
Pupil Sclera
Retina

Conjunctiva
Corneal epithelium

Bowman s Layer

Corneal stroma

Lens Descemet s Membrane


Iris Endothelium

Cornea

(c) (d)
Limbus

Cornea

Figure 1. Schematic of the human eye (a) the limbus is a narrow area, located at the periphery of the cornea, where the stem cells reside; (b) cellular
structure of the cornea, depicting the ve distinct layers and the depth of penetration of each ultraviolet (UV) wavelength; (c) colored arrows depict the
direction that the epithelial progeny move from the limbus into the epithelium; (d) A K14CreERT2-Confetti murine eye, 50-week post-tamoxifen injec-
tion (1,5) with uorescently labeled corneal epithelial cells, derived from stem cells in the limbus. The bright halo in the center is autouorescence of
the crystalline lens. Scale bar is 200 lm.

exposure to solar radiation, it must be acknowledged that there reectivity of surfaces in the immediate environment and the
are two components: direct and diffuse. Direct UVR exposure extent of eyelid opening, when estimating the dose of UVR that
involves looking directly at the sun, something that humans natu- was received by the ocular surface. The use of a mannequin
rally avoid. However, diffuse (or albedo) UVR exposure is mul- dosimetry system allowed for the evaluation that sunglasses
tidirectional due to photoscattering and reectance by gas or could attenuate UVR irradiance to the corneal surface by 80%,
aerosol molecules in the atmosphere. Due to the directional inci- compared to the absence of sunglasses (10). Most impressively,
dence of diffuse UVR, most ocular UVR exposure is due to this UVB exposure was reduced to 1% when wrap-around style sun-
component, which poses a more difcult task in minimizing its glasses were employed (10), thus conrming the importance of
exposure. In Toowoomba, Australia (27.56 S, 151.95E), the choosing the appropriate eyewear when in a sunny environment.
proportion of diffuse UVB varies from 23% in spring to 59% in In recent years, UVA exposure has also been developed for
winter, whereas diffuse UVA varies from 17% in spring to 31% clinical treatments. UVA in conjunction with riboavin has been
in winter (9). This change in proportion of diffuse UVR can be approved for cross-linking stromal collagen bers as a therapy for
affected not only by the season, but also by the time of day. For keratoconus, a degenerative eye disease that is often characterized
example, Parisi et al. (9) noted that cloud cover increases in the by coning of the cornea due to thinning of stromal collagen (11).
afternoon, and therefore, diffuse UVR readings are often higher In general, shorter wavelengths of UVR and visible light pen-
during this time for both UV wavelengths. In addition to the etrate tissues less than longer wavelengths. Thus, UVB is primar-
atmosphere and clouds, the physical environment (such as snow, ily absorbed by the corneal epithelium, whereas UVA can
buildings, ground coverings) contributes to the scattering effects penetrate the entire thickness of the cornea (12) (Fig. 1b). The
observed in diffuse UV. Furthermore, eyelid opening must be age of the eye is important to its UVR penetrance. In humans, a
considered when estimating the dose of UVR reaching the ocular cornea from a 24-year-old blocks over 90% of UVB (290
surface. Sliney (10) performed extensive measurements which 315 nm) and 45% of UVA (315400 nm) (12). However, cor-
were used to design an algorithm that took into account the neas from the elderly (80+ years) are more efcient at blocking
Photochemistry and Photobiology 3

(a)
Radio Microwave Infrared Visible Ultraviolet X-Ray Gamma

Wavelength 103 10-8


10-2 10-5 10-6 10-10 10-12
(meters)

UVC UVB UVA


100-290nm 290-320nm 320-400nm

Frequency 104 108 1012 1015 1018 1020


(Hz)

(b)

Figure 2. (a) Schematic demonstrating the wide spectrum of electromagnetic radiation (EM), with ultraviolet radiation (UVR) measured 100400 nm;
(b) an example of solar irradiance measured on a sunny day (red line) compared to an overcast day (black line). Spectrums were measure on the roof of
The Centenary Institute, Camperdown, Australia, using an Optronics OL-754 spectroradiometer. The boundaries of the UVC, UVB, UVA and visible
(VIS) regions are shown.

UVA (60%) but less efcient at ltering shorter wavelengths ocular sensation, followed by photophobia and tearing. These ini-
(80%) (12). Furthermore, corneas from younger individuals tial symptoms are attributed to the loss and damage of epithelial
(<24 years) transmit even more UVA, therefore rendering them cells in the supercial layers of the cornea. Corneal edema
and the ocular medium behind this tissue more vulnerable to results in haze and vision impairment. Further exposure to UVR
UVR damage (12). results in epithelial exfoliation, which leads to agonizing pain. In
1916, Verhoeff and Bell calculated that a threshold dose of
approximately 500 mJ cm 2 of UVR from an articial source
MEDICAL CONDITIONS OF THE CORNEA
was needed to cause pathological changes in the cornea and that
ASSOCIATED WITH UVR EXPOSURE
the main wavelengths responsible within the terrestrial sunlight
band were in the UVB part of the spectrum (13). Ren et al. (14)
Photokeratitis
showed that suprathreshold doses of UVB did in fact disrupt the
UVR has long been known to cause damage to the cornea, the normally ordered process of epithelial desquamation from the
acute reaction to UVR-induced burns being referred to as pho- corneal surface, by elevating it to such a rate that the subepithe-
tokeratitis. The most common accounts of photokeratitis are lial nerve endings underlying the cornea are exposed, thus giving
snowblindness and welders ash. Snowblindness results rise to the characteristic pain associated with photokeratitis.
from excessive exposure to naturally occurring UVB in an envi-
ronment of high reectivity; such conditions are encountered
Climatic droplet keratopathy (CDK)
when skiing, at the beach or at high altitude. Conversely,
welders ash results from exposure to articial UVB (and some- An association of CDK with chronic exposure to UVR has been
times UVC) sources such as those from a welders arc. conrmed by epidemiological studies (15,16). This condition
Photokeratitis remains asymptomatic until several hours post- occurs when soluble plasma proteins photochemically react with
UVR exposure with the earliest symptoms presenting as a gritty UVR and deposit beneath the corneal epithelium, within the
4 Naomi C. Delic et al.

(a) (b)

normal corneal epithelium

x200

(c) (d)
pterygium epithelium

stroma
corneal epithelium elastosis
pterygium epithelium

BV
BV
BV
Bowman s Layer
stroma

x1000 x1000

Figure 3. (a) Clinical image of a patient with a pterygium; (b) a low power histological micrograph demonstrating the encroaching edge of the ptery-
gium (hatched box); (c) magnied micrograph of the hatched box from b, BVblood vessels; (d) demonstrates stromal elastosis, a feature often seen in
tissue chronically exposed to ultraviolet radiation (UVR).

Bowmans layer and the supercial stroma, causing opacication commonly arise from the nasal limbus and form characteristic
of the cornea and visual impairment (17,18). Recent advances in wing-shaped lesions that can grow bilaterally and, if left
confocal laser scanning microscopy for studying CDK described untreated, restrict eye movement and vision (22). The Coroneo
further abnormalities in addition to those already dened. Effect has aided in explaining the predominance of nasally
Patients suffering from moderate-to-advanced CDK display an located pterygia (and cortical cataracts), by showing that the ante-
increased reectivity of the supercial corneal epithelium as well rior chamber is capable of focusing light that enters the eye tem-
as alterations in the density and morphology of the sub-basal porally and is internally reected onto the opposing nasal side of
nerve plexus (19). Tear uid from patients with CDK has been the corneolimbal region. Epidemiological studies have indicated
analyzed and found to contain a myriad of inammatory cytoki- that UVR plays a major role in the pathogenesis of pterygia
nes including interleukin (IL)-1b, IL-5, IL-6, IL-7, IL-8, mono- (23,24). This association is supported by the increase in preva-
cyte chemoattractant protein-1 (MCP-1), macrophage lence in equatorial regions and among individuals that work out-
inammatory protein-1 beta (MIP-1b) and tumor necrosis factor- doors in an environment with high reectance compared to
alpha (TNF-a) (18). The matrix metalloproteinase (MMP) proen- indoor workers (regardless of latitude). This risk was increased
zymes, proMMP-2 and proMMP-9, are found in higher quantity 20-fold when the surface was sealed concrete and several hun-
in the tear uid of CDK patients, while a higher level of active dred-fold when working on sand (23). Further evidence of the
MMP-9 is found in the corneal epithelial basement membrane, role of UVR in pterygia is seen by the formation of elastic tissue
compared to healthy subjects (18). The signicant increase in in the pterygium stroma, similar to the solar elastosis that is often
inammatory cytokines and MMPs in corneas of patients suffer- seen in the dermis after prolonged UVR exposure (Fig. 3d) (25).
ing from CDK indicates that its pathology is attributable to a sig-
nicant chronic inammatory response and disruption of the
Ocular surface squamous neoplasms (OSSN)
extracellular matrix (ECM) (18).
Ocular surface squamous neoplasms (OSSN) are the most com-
mon form of nonmelanocytic lesions in the conjunctiva and cor-
Pterygium
nea and refer to a disease spectrum comprising mildly dysplastic
Pterygia (Fig. 3a) are common benign lesions of the ocular sur- to invasive lesions that originate from squamous epithelium (26
face (20), characterized by local displacement of normal epithe- 28). Patients with these lesions present with a pathological mass
lium by abnormal epithelial cells. Histologically (Fig. 3b,c), a that can be raised and gelatinous, irritation, red eye and leuko-
demarcation zone exists that distinguishes the normal corneal plakia, originating from the limbus before involving the cornea
epithelium from the advancing edge of the pterygium head, which and conjunctiva (27,29). Peak incidence of OSSN occurs at a lat-
often displays regions of elastotic connective tissue (21). Pterygia itude of 16 south (30,31). Two predominant and distinct main
Photochemistry and Photobiology 5

groups of patients are affected. The rst comprises of older specic genes in the cornea have come from Moloney et al. (36)
males, who live in temperate climates and are not associated with and Hassan et al. (37). In these investigations, BRM was found
human immunodeciency virus (HIV) or human papillomavirus to be mutated or have reduced expression in human non-
(HPV) infection (31). The second group includes younger indi- melanoma skin carcinomas (NMSC) (36,38). This gene encodes
viduals (both males and females) that reside in the tropics, but one of two ATPase subunits in the SWI/SNF complex involved
are associated with HIV or HPV infection (31). The association in unraveling DNA for factors requiring access to it to mediate
with HIV suggests that immunosuppression plays a role in pre- their functions, such as repair and transcription. In a photocar-
disposing these patients to UV-induced OSSNs, whereas the nat- cinogenesis study, using a BRM knockout mouse, it was shown
ure of the association with, and identities of specic subtypes of that the absence of this gene resulted in the increased incidence
HPV involved, are still unknown (31). OSSN and UVR are also of both eye and skin tumors, implying that it functions as a
linked via patients suffering from xeroderma pigmentosa, a rare tumor suppressor (39). Both carcinomas and sarcomas of the eye
autosomal-recessive genetic disorder in which the DNA damage were induced by chronic UVR exposure in Brm- and/or Tp53-
repair machinery is faulty (32). These patients often develop decient mice. Subsequent studies (37) examined corneas of
OSSNs as well as skin cancer at a young age (26,32). mice chronically exposed to UVR (i.e. beyond 25 weeks). Cor-
neas of Brm knockout mice displayed increased thickening,
UVR-INDUCED GENETIC CHANGES hyperplasia and decreased atrophy when compared to Brm wild-
type mice, the damage further exacerbated when Brm knockout
It is well established that solar radiation is the main culprit in the mice lost a p53 allele. It was also noticed that irradiated Brm
initiation and progression of several types of skin cancers (squa- knockout mice demonstrated increased proliferation and regener-
mous cell carcinomas, basal cell carcinomas, melanomas) (23). ation of the corneal epithelium, which was conrmed by Ki-67
Specically, UVR has been proven to be a highly genotoxic staining (37). Brm knockouts showed an increased number of
agent (33) by inducing DNA damage in the form of two well- Ki-67+ corneal epithelial cells compared to Brm wild types after
described photoproducts: cyclobutane pyrimidine dimers (CPDs) only 2 weeks of irradiation, which was maintained after
and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). UVB 25 weeks of irradiation (37). Of signicance is that proliferation
photons can be directly absorbed by DNA bases, largely by the in the Brm-decient corneas displayed disorganization, with pro-
pyrimidine constituents. This energetic absorption induces the liferative cells observed in the suprabasal layer of the epithelium,
formation of covalent bonds between two adjacent pyrimidine rather than being conned to the basal layer, which is typical in
bases, with CPDs being the most prevalent photolesion. Because the Brm wild-type corneas with or without irradiation (37).
of their inefcient removal and abundance, CPDs are considered Taken together, the corneal thickening and cellular proliferation
promutagenic. Misincorporation of adenine instead of guanine data rmly suggest that Brm plays a signicant role in prevent-
opposite a cytosine in CPDs results in the UV signature C ? T ing hyperproliferation in response to acute or chronic UVR expo-
and CC ? TT transversions (12). As the cornea is at the air sure, thus conrming its tumor suppressor status in the cornea.
interface and absorbs approximately 90% of the UVB that irradi- As with NMSCs, TP53 mutations are observed in UVR-
ates the surface of the eye, it is expected that it will sustain induced corneal sarcomas. Studies in the opossum found that
DNA damage. Studies exploring DNA damage in the mam- these mutations were the hallmark UVR signature C ? T con-
malian cornea have observed the presence of photolesions. version located in codons equivalent to those found in mutational
Experimental models using human and rabbit eyes described the hot spots of human UVR-induced SCCs (40). Similar studies
rate of CPD formation versus CPD distribution in the cornea, were conducted in human conjunctival SCCs, where a high
according to different UV wavelengths (12,34). UVA penetrates prevalence of CC ? TT transversions, a signature mutation of
as far as the corneal endothelium and supercial layers of the UVR, was found (41). The location of these mutations at sites of
crystalline lens; however, CPDs are induced at a much lower rate DNA photoproduct formation suggested that the TP53 mutations
by UVA than by UVB (12,34). Conversely, although little UVB in these OSSN were a result of UVR directly acting on DNA
penetrates beyond the anterior third of the corneal stroma, this (40,41).
region has a signicantly higher rate of CPD formation (12,34). Although no TP53 mutations were identied, Galor et al. (42)
Although physiologically irrelevant, UVC-induced CPDs are were rst to perform whole-exome sequencing on OSSN. Of the
highly concentrated in the supercial layers of the epithelium seven samples included in their analysis, they identied muta-
and are virtually undetectable in the remaining 90% of the tions in Titin (TTN), Neuron Navigator 2 (NAV2), FAT atypical
stroma (12). Those studies demonstrated a shield-like role the cadherin 2 (FAT2), hepatocyte growth factor (HGF), dynein
cornea plays in protecting the posterior structures of the eye from axonemal heavy-chain 8 (DNAH8) and CREB binding protein
UVR-induced damage. In addition to CPDs, UVB-induced 6-4 (CREBBP)(42). The functional signicance of these mutant genes
PPs were more abundant in the epithelial layers of the rat cornea. remains to be elucidated.
However, when broadband UVR (280380 nm) doses were used,
equating to well over two minimal erythemal doses (i.e. twice
UVR-INDUCED MOLECULAR CHANGES
the dose needed to cause sunburn to exposed skin), evidence of
6-4 PPs was detected throughout all ocular structures, including Multiple molecular factors that have altered activity in response
the corneal stroma, iris, lens and retina (35). Notably, transmit- to UV have been identied in the cornea, including nuclear fac-
tance of UVR through various ocular structures is highly tor-jB (NF-jB), cytokines, MMPs and PAX6. NF-jB has been
dependent on age. identied as an initiator of cell death in the cornea and is also
Much remains to be learnt about genetic changes that occur in known to be a key driver of cytokine production in response to
the corneal epithelium as a result of overexposure to UV. Recent injury. Likewise, cytokines can upregulate MMP production
advances in understanding the effects of mutagenic changes to (43,44). Although the current literature describes these
6 Naomi C. Delic et al.

independently of each other, it is likely that these factors work in extracellular matrix components and other bioactive proteins
concert and in a cascade-type arrangement to exhibit these identi- such as cytokines (50). In normal circumstances, MMPs are
ed molecular changes. The relationship of PAX6 to the NF-jB/ expressed at low levels and their activity is tightly regulated by
cytokine/MMP pathway in the cornea remains to be explored. their natural inhibitors (TIMPs); therefore, an overexpression and
activation of MMPs are associated with excessive degradation of
NF-jB the ECM, which can be potentially devastating for the structure
and clarity of the cornea (51). The gelatinases, MMP-2 and
Although cell death does not appear to be the primary route for MMP-9, and two of the collagenases, MMP-1 and MMP-8, have
removal of UVR-exposed corneal epithelia (see the section on been studied in the cornea in depth (5256). MMP-2 and MMP-
cellular changes in response to UV irradiation), NF-jB may play 9 were originally identied as enzymes involved in the degrada-
a role in initiating cell death. NF-jB is a transcription factor that tion by epithelial cells of type IV collagen, a principal
remains inactive via ubiquitous expression of its inhibitor IjB or component of basal membranes (57,58).
contiguous latency peptide sequence (45). Targeted phosphoryla- MMP-1 has been demonstrated to be one of the most abun-
tion and subsequent degradation of IjB or latency peptide allow dant MMPs present in pterygia. Di Girolamo et al. (52) showed
activation of NF-jB followed by its translocation to the nucleus that UVB could signicantly increase MMP-1 production in both
where its dimers are free to bind to its target DNA elements. pterygium epithelial cell lines and ex vivo pterygia explants. It is
This activates transcription of genes involved with responses to regulated via the UVR-inducible ERK1/2 pathway. MMP-2,
inammation or cell growth. Using the SV40 immortalized cor- MMP-9, MMP-7, MMP-8 and to a lesser degree MMP-14 have all
neal epithelial cell line T-HCEC, Lee et al. (46) showed that cell been shown to be upregulated by UVB exposure in vivo
death followed nuclear translocation of NF-jB after UVR irradi- (51,59,60), but their expression remains unaltered when corneas
ation. Furthermore, cell death could be blocked by treating cell are exposed to UVA (51). MMP-14 is active in multiple roles such
lines with the potent NF-jB inhibitors sulfasalazine and SN-50 as the cleavage of ECM, activation of other MMPs and activating
prior to UVR exposure. Immunocytochemical analysis conrmed signaling pathways for cell migration during wound healing in the
that NF-jB was inhibited from translocating to the nucleus after cornea (6163), but interestingly, its expression is only mildly
irradiation, and downstream cell death was blocked. Elec- increased after UVB exposure in rabbit corneal epithelium (51).
trophoretic mobility shift assays (EMSA) in conjunction with
immunocytochemical staining showed that NF-jB translocated to
the nucleus prior to cell death being observed, and its binding Cytokines
activity remained increased for 2 h postirradiation, suggesting that UV-induced changes in the cornea can be heavily attributed to
NF-jB indeed plays an early role in the cellular death cascade. the upregulation of pro-inammatory cytokines including IL-1,
IL-6, IL-8 and TNF-a in the corneal epithelium and stroma (64
PAX6 67). Specically, it was found that IL-6 and IL-8 were expressed
in the supercial epithelium, with some IL-8 expression in the
PAX6 plays a major role in structural development and ocular vascular endothelium of pterygium tissue that was exposed to
cell differentiation in the eye of vertebrates and has therefore low-dose UVB (40 mJ cm 2) (66). In cultured pterygium epithe-
been coined a master oculogenic control gene (47,48). Its lial cells that were irradiated with UVB, expression of IL-6 and
expression is essential for development, maintenance and repair IL-8 increased in a time- and dose-dependent manner (66). Nor-
of the eye. PAX6 is transcriptionally regulated by CTCF, a zinc mal limbal broblasts and corneal epithelial cells upregulate
nger phosphoprotein that binds to the CCCTC motif, located pro-inammatory and macrophage recruiting cytokines, TNFa,
upstream of the PAX6 P0 promoter. Because overexpression of interferon gamma (IFNc) and MCP-1 after low-dose
the CTCF gene in corneal epithelial cells resulted in a downregu- (20 mJ cm 2) UVB (64). Conversely, Notara et al. (64) also
lation of PAX6 activity, Wu et al. (49) explored PAX6 expres- showed that UVB treatment of limbal broblasts downregulates
sion regulation by CTCF in cultured rabbit and human corneal their production of soluble factors usually involved in mediating
epithelial cells that were stimulated with UV. Within 8 hours of lymphatic and blood endothelial activity, most likely a regulatory
exposure to UV, PAX6 expression increased while CTCF mechanism to avoid neovascularization of the limbus and cornea.
decreased, both detectable by Western blot analysis. The upregu-
lation of PAX6 by decreased activity of CTCF was conrmed by
EMSA that showed that the binding activity of CTCF on PAX6
CELLULAR CHANGES IN RESPONSE TO UV
IRRADIATION
P0 upstream enhancers was signicantly decreased after UVR
exposure. Finally, they showed that CTCF had a direct effect on Corneas of mice that are chronically exposed to low doses
PAX6 using site-directed mutagenesis on the 80-bp PAX6 P0 (equivalent to two minimal erythemal doses) of UVR display
enhancer sequence to genetically alter the ve binding motifs, abnormalities from as early as 25 weeks with epithelial hyper-
resulting in decreased binding activity of CTCF. plasia, dysplasia, atrophy of the corneal epithelial center, signi-
cant hypocellularity of the stroma, brosis, vascularization and
inammation (37,68). These changes eventually culminate in the
Matrix metalloproteinases
formation of OSSNs.
MMPs are a family of proteolytic enzymes that are important for The acute histological changes to corneas exposed to UVR
tissue remodeling and wound healing. They are synthesized as occur in a dose-dependent manner (37,68,69). Pitts and col-
inactive zymogens and become activated once proteolytically leagues (69) showed that UVR enhanced the premature shed-
cleaved at their propeptide domain on the N-terminus. Once ding/sloughing of the outermost epithelial cell layers and
active, MMPs are capable of either degrading or processing increased cell death 24 hours after radiant exposure. Gradually
Photochemistry and Photobiology 7

increasing the radiant exposure caused loss of corneal epithelia; indeed elicit a systemic immune response (7880). Today, it is
keratocytes became fragmented and the endothelium became dis- well established that the cornea contains resident antigen-present-
organized, vacuolated and detached from the stroma (69). ing cells of dendritic and macrophage lineages that seem to be
Through the use of ex vivo organ cultivated corneas, Ren et al. entrenched in the limbal transition zone, perhaps playing a key
(14) showed that a single dose of 1.05J cm 2 of UVB increased role as rst-line defense cells able to sample antigen at a sight
the shedding rate of epithelial cells from the ocular surface. They prone to infection, inammation and tumorigenesis (81).
found that, after irradiation, there was a delay of approximately Although much is now known about the immune system in
3 h before shedding was increased and maintained for the ensu- the eye, little is known about the immune response following
ing 3 h (14). UVR irradiation. Immunosuppression by UVR has been well
Using computer modeling, Lobo and colleagues (5) studied in the skin, with these effects being attributed partially to
recently demonstrated that the well-documented spoke pattern that cis-urocanic acid (UCA) which is naturally abundant in the skin
corneal epithelial cell clones form as they move from the limbus as the trans isomer (82). UV irradiation causes its isomerization
(1,70) requires no external physiological cues. Rather, this pattern from trans to cis. Treatment with a topical form of cis-UCA on
can be simply explained by population pressure from the periph- a corneal allograft in BALB/c mice suppressed the immune sys-
ery. However, the spoke formation does not rule out the role of tem therefore prolonging the graft survival time compared to
UV-regulated factors, such as signaling molecules, neurons and untreated groups (83). This presence of cis-UCA inhibits the
biophysical cues in response to inammation and injury (5). expression of the cytokines, IL-6 and IL-8, and general cytotoxi-
Using the Confetti mosaic mouse model, it was shown that a city associated with inammation (84). In human corneal epithe-
single dose of 150 mJ cm 2 UVB increases the spoke growth lial cells, cis-UCA is able to inhibit UVB-induced apoptosis and
rate from the control value of approximately 10 lm day 1 to latent proliferation by blocking the binding of the transcription
approximately 50 lm day 1 after UVR exposure (1,5). Mathe- factors, c-Jun and c-Fos, but not NF-jB (84). It has also been
matical modeling (5), together with in vivo experiments proposed that UVR-induced upregulation of JunB is able to
(5,68,71,72), has shown that, although apoptosis peaks at around antagonize the pro-apoptotic activity of c-Jun (85). Additionally,
24-hour post-UV exposure, it is insufcient to account for the in cultured spleen cells, cis-UCA increases the production of IL-
signicant inux of cells in response to UVR exposure without 10, an important factor in immunosuppression because it inhibits
causing a substantial thickening of the corneal epithelium. On antigen presentation and production of T helper 1 (TH1) cytoki-
the contrary, the corneal epithelium thins following UVR expo- nes by epidermal antigen-presenting cells (83).
sure (14). Transplantation is often the only solution for treating patients
A hypothesis to explain this lack of apoptotic cell death in with limbal stem cell deciency, which is frequently caused by
response to UVB comes from Leerar et al. (73). Following chemical and thermal burns. Rama and colleagues showed that
UVB, human corneal epithelial cells lose potassium ions (K+) grafts generated from the patients own limbal stem cell popula-
that would normally activate caspases, triggering the apoptotic tion were a potential solution, although there was still a 10%
cascade (7375). However, tears are naturally high in K+, and failure rate and cited inammation as one of the contributing
therefore, it was argued that they would reduce the loss of intra- complications (86). Unlike the skin, an inux of immune cells
cellular K+, preventing apoptosis (73,74,76). Additionally, Ren and chronic inammation in the cornea can potentially cause
and Wilson (14) observed that cells which were shed in response irreparable damage, and so, understanding the immunogenicity
to UVR were smaller than a typically differentiated cell, suggest- of the cornea is imperative for positive advancements in thera-
ing that the epithelial wing cells were failing to fully terminally peutic applications.
differentiate before leaving the epithelium.
Thus, although the cornea is anatomically simple, the mecha- CONCLUSIONS AND FUTURE NEEDS
nisms which it uses to maintain its integrity are complex and
remain relatively unexplained. While apoptosis and increased cell It is clear from the available data that the level of UVR required
shedding/terminal differentiation appear to be means of stabiliz- to provoke a response in the cornea is low and, when provoked
ing the structure after a UV insult, the current evidence indicates chronically, will result in clinical disease. UVR-protective eye-
these mechanisms may not be wholly responsible, and other reg- wear should be worn whenever outdoors, with the need being
ulatory mechanisms await discovery. greater on days with cloud cover and in surroundings with highly
reective surfaces, due to increased reectance and scatter.
There is currently a need for deeper understanding of the
IMMUNOGENICITY AND THE CORNEA
genetic, cellular and immunological changes that occur in the
The eye is considered an immune privileged site. It was origi- cornea after UV exposure, compared to the greater amount of lit-
nally hypothesized that the anterior chamber of the eye was lack- erature on the skin. Galor and colleagues (42) have pioneered
ing in lymphatic vessels, protecting this organ from an the area of somatic genetic changes of the cornea by identifying
overwhelming immune response if an antigen was detected. This mutational changes in human patients with OSSNs. Although a
was proposed to explain the results of studies performed on very small number of samples were analyzed, they demonstrate
transplanted cells from genetically different hosts, which sur- the potential for identifying mutations and correlating them with
vived signicantly longer in the anterior chamber than in other efcient therapies. The mechanism for the nonapoptotic cell loss
sites of the body (77,78). However, this hypothesis was chal- that results in thinning of UVR-exposed corneas remains to be
lenged when Kaplan and Streilein (79) found the presence of elucidated. Identifying the molecular pathways involved might
hemagglutinating antibodies in serum from rats, 4 days after shed insights into novel means of treating degenerative condi-
being immunized intracamerally with allogeneic lymphoid cells, tions involving the corneal epithelium. Studies identifying the
concluding that antigens present in the anterior chamber can semi-immunosuppressive state of the eye and its correlation with
8 Naomi C. Delic et al.

OSSN demonstrate the complexity of the immune system. This 15. Johnson, G. J. (1981) Aetiology of spheroidal degeneration of the
requires considerably more analysis. There are many mouse cornea in Labrador. Br. J. Ophthalmol. 65, 270283.
16. Taylor, H. R., S. K. West, F. S. Rosenthal, B. Munoz, H. S. New-
models available in which immune cells are uorescently tagged land and E. A. Emmett (1989) Corneal changes associated with
and these would assist in teasing out mechanisms of the ocular chronic UV irradiation. Arch. Ophthalmol. 107, 14811484.
immune response. 17. Cullen, A. P. (2002) Photokeratitis and other phototoxic effects on
Mathematical models might prove to be useful in improving the cornea and conjunctiva. Int. J. Toxicol. 21, 455464.
therapies such as UVA cross-linking and limbal stem cell trans- 18. Holopainen, J. M., A. Robciuc, T. A. Cafaro, M. F. Suarez, Y. T.
Konttinen, H. M. Alkatan, K. F. Tabbara, T. Tervahartiala, T. Sorsa,
plants to optimize locations of treatment and avoiding more dam- J. A. Urrets-Zavalia and H. M. Serra (2012) Pro-inammatory
age due to the treatment (87). Lobo et al. (5) demonstrated that cytokines and gelatinases in climatic droplet keratopathy. Invest.
their model was efcient in replicating the biology of the epithe- Ophthalmol. Vis. Sci. 53, 35273535.
lial cells under homeostatic and experimental conditions. Mathe- 19. Serra, H. M., J. M. Holopainen, R. Beuerman, K. Kaarniranta, M. F.
Suarez and J. A. Urrets-Zavalia (2015) Climatic droplet keratopathy:
matical simulations could potentially be programmed to an old disease in new clothes. Acta Ophthalmol. 93, 496504.
incorporate a vast number of experimental parameters to identify 20. Grossniklaus, H. E., W. R. Green, M. Luckenbach and C. C. Chan
appropriate targets. This could be used to help design experi- (1987) Conjunctival lesions in adults. A clinical and histopathologic
ments, reducing costs associated with large-scale experimental review. Cornea 6, 78116.
testing and optimizing therapeutic strategies. Therefore, not only 21. Chui, J., M. T. Coroneo, L. T. Tat, R. Crouch, D. Wakeeld and N.
Di Girolamo (2011) Ophthalmic pterygium: a stem cell disorder with
would mathematical modeling be benecial for developing treat- premalignant features. Am. J. Pathol. 178, 817827.
ment strategies, but also for planning research programs. 22. Young, R. W. (1994) The family of sunlight-related eye diseases.
Optom. Vis. Sci. 71, 125144.
AcknowledgementsN.C. Delic was supported by Australian 23. Mackenzie, F. D., L. W. Hirst, D. Battistutta and A. Green (1992)
Postgraduate Award and Sydney Catalyst Top-Up Scholarship from Risk analysis in the development of pterygia. Ophthalmology 99,
Cancer Institute NSW. This work was supported by Human Frontiers 10561061.
24. Yam, J. C. and A. K. Kwok (2014) Ultraviolet light and ocular dis-
Science Program Grant RGP0041. The authors thank Professor Minas
eases. Int. Ophthalmol. 34, 383400.
Coroneo (Department of Ophthalmology, Prince of Wales Hospital, 25. Austin, P., F. A. Jakobiec and T. Iwamoto (1983) Elastodysplasia
Randwick, Sydney, Australia) for providing the clinical image of a and elastodystrophy as the pathologic bases of ocular pterygia and
pterygium. pinguecula. Ophthalmology 90, 96109.
26. Kheir, W. J., M. T. Tetzlaff, M. L. Pfeiffer, K. Mulay, O. Ozgur, G.
Morrell and B. Esmaeli (2016) Epithelial, non-melanocytic and mela-
nocytic proliferations of the ocular surface. Semin. Diagn. Pathol.
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the rat eye. Br. J. Ophthalmol. 83, 225230. genic mouse model to
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AUTHOR BIOGRAPHIES Gary M. Halliday is a


Professor of Dermatology
at the University of Syd-
ney. He obtained his PhD
Naomi C. Delic is a PhD from Monash University
student at the University in 1981 and his Doctor of
of Sydney. She received Science from the Univer-
her Bachelor of Science sity of Sydney in 2001.
with rst class honors His research is directed
from the University of toward understanding the
New South Wales in role of sunlight in skin
2006. Her research inter- carcinogenesis, particu-
ests include cancer biol- larly ultraviolet radiation
ogy of the skin; however, suppression of immunity,
her current research is induction of gene muta-
focused on understanding tions, skin cancer cell
the response of the cornea biology and development
to ultraviolet radiation of effective prevention.
exposure, using intravital His research group is part
lineage tracing techniques. of the Sydney Cancer
Centre, the Bosch Insti-
tute, the department of
Dermatology of Royal
Prince Alfred Hospital
and the Central Clinical
School of the University
J. Guy Lyons received of Sydney.
his PhD from the Univer-
sity of Sydney in 1988.
He is a cancer biologist
with a long-term interest
in epithelial carcinogene-
sis. Squamous cell carci-
nomas of the skin, oral
mucosa and ocular surface
are of particular interest.
He is currently residing at
the Centenary Institute,
where he is using intravi-
tal lineage tracing and
genetic manipulation tech-
niques to study clonal
evolution during develop-
ment and carcinogenesis.