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C L I N I C A L A N D E X P E R I M E N TA L

RESEARCH PAPER

Longitudinal changes in Langerhans cell density of the cornea and


conjunctiva in contact lens-induced dry eye

Clin Exp Optom 2017; 100: 3340 DOI:10.1111/cxo.12399

Yahya Alzahrani MScOptom Background: The aim was to determine longitudinal changes in Langerhans cell density
Luisa H Colorado OD (LCD) in the human cornea and conjunctiva during asymptomatic and symptomatic contact
Nicola Pritchard BAppSc(Optom) PhD lens wear.
Nathan Efron PhD DSc Methods: Twenty-ve participants with contact lens-induced dry eye (CLIDE) and 35 without
Institute of Health and Biomedical Innovation, and School CLIDE (NO-CLIDE), diagnosed using a range of symptom questionnaires and objective tests
of Optometry and Vision Science, Queensland University (tear lm break up, cotton thread tear test and corneal staining) were enrolled. The central
of Technology, Kelvin Grove, Queensland, Australia
cornea and nasal bulbar conjunctiva were examined using a Heidelberg laser scanning
E-mail: n.efron@qut.edu.au
confocal microscope at baseline and following one, four and 24 weeks wear of daily disposable
hydrogel contact lenses. Twenty-three non-contact lens-wearing controls were also examined.
Langerhans cells were counted manually from randomly selected images.
Results: In the cornea, mean and standard error of the mean LCD was greater after one week
of lens wear in CLIDE (55 7 cells/mm2) versus NO-CLIDE (43 4 cells/mm2) (p = 0.041) and
controls (27 4 cells/mm2) (p < 0.001). LCD was also greater in NO-CLIDE versus controls
(p = 0.010). At week 4, LCD was greater in CLIDE (41 6 cells/mm2) versus controls (27 4
cells/mm2) (p = 0.004). There were no other signicant differences between groups at weeks
four or 24. In the conjunctiva, LCD was greater after one week of lens wear in CLIDE (17 1
cells/mm2) (p = 0.003) and NO-CLIDE (17 3 cells/mm2) (p = 0.001) versus controls (7 1
Submitted: 5 October 2015 cells/mm2). There were no signicant differences between groups at weeks four or 24.
Revised: 21 December 2015 Conclusions: The initial transient increase in corneal and conjunctival LCD in CLIDE (versus
Accepted for publication: 23 December 2015 NO-CLIDE) suggests an inammatory component in the aetiology of this condition.

Key words: conjunctiva, contact lens, cornea, dry eye, inammation, Langerhans cells

Contact lens discomfort is a major unsolved There are two general lines of enquiry that and extended wear of rigid and soft contact
problem in ophthalmic science. Between 28 could shed further light on the question of lenses and so serve as markers for contact
and 50 per cent of contact lens wearers expe- the degree to which contact lens wear and lens-induced ocular inammation.7,8
rience discomfort associated with contact especially CLIDE is inammatory.3 At a The laser scanning confocal microscope
lens wear, according to population-based clinical level, the primary sign of inamma- (LSCM) has found considerable value in
studies.1 It is a primary reason for dissatisfac- tion that has been used to study the impact assessing the response of the anterior ocular
tion with, and discontinuation from, lens of contact lens wear on the eye is limbal and structures to contact lens wear at a cellular
wear. Contact lens wearers use various terms bulbar conjunctival redness.4 In fact, the rst level.9 Various studies have used this instru-
to describe symptoms of discomfort, but the two clinical reports of contact lens wearing ment to observe inammatory cells, which
overwhelming majority categorise this trials on humans, conducted independently are presumed to be Langerhans cells in both
discomfort as dryness.1 in the late 1880s by Adolf Fick5 and August the central and peripheral cornea10 as well as
The cause of contact lens-induced dry eye Mller,6 used conjunctival redness as a mea- the bulbar11 and palpebral12 conjunctiva.
(CLIDE) is not well understood and is likely sure of the physiological impact of lens wear. They are about 15 m in diameter and
to be multifactorial. Factors such as eyelid Cytochemical analysis of ocular tissues appear in various forms. Immature
and ocular surface anatomy, blinking charac- provides a more direct measure of the inam- Langerhans cells either lack dendrites or
teristics, systemic factors, hormonal inu- matory status of the eye during contact lens have small dendritic processes. Mature
ences, lens-induced changes to the tear lm wear. Inammatory mediators, such as Langerhans cells develop long interdigitating
and ocular surface, lens design, lens material, histamine, granulocyte macrophage colony- dendrites and can reside alone or form a
lens surface characteristics, such as wettability stimulating factor, tumour necrosis factor al- wire net-like structure, the latter appearance
and lubricity, environmental factors, and pre- pha, interleukin-6, interleukin-8, leukotriene being especially prevalent in the conjunc-
existing ocular pathology, such as dry eye, are B4, matrix metallopeptidase-9 and epidermal tiva.10 Cross-sectional studies have shown an
all thought to have the potential to contribute growth factor can be assayed from the tear increase in Langerhans cell density (LCD)
to CLIDE.2 lm.7,8 These mediators are altered by daily in the cornea1315 and conjunctiva16 in

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Clinical andOptometry 100.1
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Langerhans cells
cells in
in contact
contactlens
lenswear
wearAlzahrani,
Alzahrani,Colorado,
Colorado,Pritchard
Pritchard and
and Efron
Efron

response to contact lens wear; however, longi- The following exclusion criteria were NON-INVASIVE TEAR BREAK-UP TIME
tudinal studies of LCD in response to contact applied: recent ocular inammation, history (NITBUT) TEST
lens wear have not been undertaken. of ocular trauma or surgery, currently using This is a non-invasive measure of the capacity
ocular medication with the exception of of the tear lm to remain intact on the sur-
This study sought to determine: non-preservative articial tear supplements, face of the cornea.20 The right and left eyes
systemic disease that may affect the cornea were assessed and the average of the three
1. longitudinal changes in LCD during or conjunctiva, hypertension, diabetes, dry readings per eye was taken as the mean value.
asymptomatic contact lens wear and eye, pregnancy or breastfeeding. The participant was positioned behind a
2. whether there is an increased LCD associ- Additional exclusion criteria for the con- keratometer and asked to blink once and
ated with CLIDE. tact lens wearing group were as follows: astig- then refrain from blinking. The time elapsed
matism of more than 1.50 D, myopia more until the rst sign of any distortion of the im-
A higher LCD may suggest in vivo cytological than -7.00 D or hyperopia more than +2.00 D. age of the keratometer mires (NITBUT) was
evidence of an inammatory basis for CLIDE. Participants entered into the study who recorded using an electronic timing device.
were interested in wearing contact lenses A reading of less than nine seconds was con-
were tted with daily disposable lenses and sidered as indicating dry eye.21
METHODS given instructions in their use. After one week
Ametropic participants who were willing to of lens wear, participants were stratied into PHENOL RED THREAD TEST (PRTT)
wear contact lenses for six months were CLIDE and NO-CLIDE. Recruitment contin- This is a test of the volume of tears in the
enrolled and stratied into those with CLIDE ued until there were at least 25 participants lower cul de sac, based on the Hamano and
and those without CLIDE (NO-CLIDE) (see in each contact lens subgroup. Twenty-three colleagues22 cotton test. One end of a yellow
below). A control group of non-contact lens non-contact lens wearing control participants cotton thread impregnated with phenol red
wearers was also enrolled. LCD in the cornea were also recruited. (Pcot-test, Tianjin Jingming New Technology
and bulbar conjunctiva were monitored At each visit, participants were examined Development Ltd, Tianjin Hi-Tech Industrial
using a LSCM in all three groups at baseline using a variety of dry eye examination tech- Park, China) is draped over the temporal as-
(when contact lenses were dispensed) and niques. All examinations were performed in pect of the lower eyelid and into the lower
after one, four and 24 weeks. the morning between 7:00 hours to 12:00 conjunctival sac for 20 seconds. The greater
Sample size calculation (G*Power 3.1)17 to hours to avoid any potential confounding the length of the thread that becomes wet
detect a difference of 10 cells/mm2 at a signif- inuence of diurnal variation. with tears, indicated by that portion of the
icance level of p 0.05, assuming a measure- thread turning red, the greater is the tear vol-
ment standard deviation of 8 cells/mm2, ume. A wet length of 10 mm or less was con-
revealed that a minimum of 12 participants Dry eye assessment sidered to indicate dry eye.
per group were required to be able to reject,
DRY EYE QUESTIONNAIRE-5 (DEQ-5)
with 80 per cent power, the null hypothesis CORNEAL FLUORESCEIN STAINING (CFS)
that the population group means are equal. This is a self-administrated questionnaire
A drop of saline was instilled on a uorescein-
Allowing for participant drop-out over the consisting of ve questions about frequency
impregnated strip, which was then touched
24 weeks of the study, the minimum number and intensity of eye discomfort and eye
gently on the lower bulbar conjunctiva. The
of participants recruited per contact lens dryness and frequency of watery eyes.18
cobalt blue light on the slit-lamp bio-
subgroup was set at 25. Scores for each question are assigned, rang-
microscope and yellow observation lter were
Ethics approval for this study was obtained ing from Never (score of 0) to Constantly
used to evaluate the extent of corneal stain-
from the Queensland University of Technol- (4) or Very intense (5). A total score of more
ing with reference to the validated Efron
ogy Human Research Ethics Committee (ap- than six (out of 22) indicates dry eye.
grading scale.23 The extent of staining was
proval number 1300000117). The research graded from 0 (normal) to 4 (severe stain-
followed the tenets of the Declaration of Hel- ing). A grade of staining of two or more was
CONTACT LENS DRY EYE QUESTIONNAIRE-
sinki and informed consent was obtained considered as being abnormal.23
8 (CLDEQ-8)
from the subjects after explanation of the na-
ture and possible consequences of the study. The Contact Lens Dry Eye Questionnaire-8
was developed specically to assess dry eye Diagnosis of dry dye
symptoms associated with contact lens wear.19 The following tests were conducted as part of
Participants This questionnaire consists of eight questions the baseline examination protocol on all
Potential participants were recruited from which are then analysed to reveal a score out those recruited for potential participation in
the staff and student population of the of 37, where higher scores indicate greater the study: DEQ-5, NITBUT, PRTT and CFS.
Queensland University of Technology, discomfort. In order to stratify our cohort In accordance with the recommendation of
Brisbane, Australia. Inclusion criteria were into CLIDE and NO-CLIDE, we adopted a various authors24,25 that a combined battery
as follows: good general health (self-re- threshold score of more than 17 as indicating of subjective and objective tests should be
ported), no contact lens wear for the previous CLIDE (and up to 17 indicating NO-CLIDE), used to diagnose dry eye, we adopted the
six months, age ranging from 18 to 50 years based upon the validation study of Chalmers criteria that participants who passed DEQ-5
and willingness to be tted with, and if and colleagues,19 who demonstrated that a and at least one of the objective dry eye tests
suitable to wear, daily disposable lenses on a mean score of 17.4 or less was associated with (NITBUT, PRTT or CFS) were considered el-
regular basis for 24 weeks. an overall opinion rating of fair or better. igible for inclusion in the study.

Clinical and
and Experimental
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34
Langerhans
Langerhanscells
cellsin
incontact
contact lens
lens wear Alzahrani, Colorado,Pritchard
Alzahrani, Colorado, Pritchardand
andEfron
Efron

One week after the baseline examination, whereby those cells partially cut off at the su-
CLDEQ-8 was used to assess those in the perior and right hand borders of the eld
contact lens-wearing group. Those failing were included in the count. The operator un-
the CLDEQ-8 test (score greater than 17) dertaking image selection and analysis was
were assigned to the CLIDE subgroup and masked with respect to the assigned group
the remainder formed the NO-CLIDE of participants.
subgroup. The subjective (CLDEQ-8) and
objective (NITBUT, PRTT or CFS) dry eye
tests were repeated four and 24 weeks from Contact lenses
the baseline examination. All participants in the contact lens groups
were tted with Biomedics 1 day Extra daily
disposable soft contact lenses (CooperVision,
Laser scanning confocal Pleasanton, California, USA). A hydrogel lens
microscopy was used in this experiment (the choice of
Images of Langerhans cells were captured brand was arbitrary) as this lens category will
using a Heidelberg LSCM (HRT3) in impart a greater physiological stress, at least
combination with a Rostock Corneal Module Figure 1. View captured from side- in terms of hyopxia, than a silicone hydrogel
(Heidelberg Engineering GmbH, Heidel- mounted charge-coupled device camera of lens. The Biomedics 1 day Extra lenses are
berg, Germany). A new disposable Perspex Heidelberg laser scanning confocal micro- made from the hydrogel material oculclon
applanating cap (Tomocap) was used for each scope, showing the plano face of the D. These lenses had the following parame-
participant. Before tting the Tomocap to the Perspex Tomocap (A) gently contacting ters and characteristics: water content 55 per
Rostock Corneal Module, it was lled with a cent, diameter 14.2 mm, base curve 8.6 or
the cornea (B)
GenTeal Gel (Novartis Pharmaceuticals 8.8 mm, centre thickness (at -3.00 D)
Australia Pty. Limited, Sydney, NSW, 0.07 mm, power range -10.00 to +6.00 D, oxy-
Australia). The gel facilitates an optical conjunctiva, about two to four millimetres gen permeability (Dk) 19 10-11 cm2 mlO2/
coupling of the Rostock Corneal Module from the limbus. Again, accurate positioning s ml mmHg, oxygen transmissibility (Dk/t; at
objective lens with the back surface of the was facilitated by the side-mounted CCD cam- -3.00D) 27 10-9 cm mlO2/s ml mmHg and
Tomocap. An anaesthetic drop (0.4% era (Figure 2). The participant was advised to light blue handling tint. Lens power was
oxybuprocaine hydrochloride; Chauvin Phar- xate on a target located in the horizontal determined by subjective refraction and any
maceuticals Ltd., London, UK) was instilled plane but approximately 45 degrees to the necessary vertex correction was applied.
prior to examination. left or right of the midline of the contralateral Participants were trained in the use of daily
Participants presented to the laboratory eye, depending on the eye being scanned. disposable contact lenses and were provided
wearing their contact lenses and the follow- The Tomocap was moved slightly in a vertical with a leaet and video recording that
ing procedure was undertaken within ve mi- and a horizontal direction, while the LSCM explained contact lens insertion, removal
nutes of lens removal on one randomly was focused at various depths between 20 to and care.
selected eye. The cornea was scanned rst. 150 m from the epithelial surface, which is
With the head securely positioned in the chin the known location of Langerhans cells in
and brow rest, the face of the Tomocap was the conjunctiva.
brought into gentle contact with the central
region of the cornea. When capturing the Image analysis
images, the participant was advised to xate Approximately 100 digital images were cap-
on a target located directly in front of the tured during each examination. Pilot studies
contralateral eye. Accurate positioning of revealed that the analysis of ve and six im-
the Tomocap on the cornea was facilitated ages from the cornea and conjunctiva, re-
by a side-mounted charge-coupled device spectively, provided repeatability for
(CCD) camera that transfers a magnied determining LCD with a standard deviation
and real-time image onto a computer display of 8 cells/mm2 and an intra-class correla-
screen (Figure 1). The Tomocap was moved tion coefcient of 0.95. Thus, ve and six ran-
slightly in a vertical and a horizontal direc- domly selected, high-quality images that were
tion, while the LSCM was focused at the level overlapping less than 20 per cent were
of the sub-basal nerve plexus, approximately analysed to determine corneal and conjuncti-
60 m from the epithelial surface, which is val LCDs, respectively. The number of
the known location of Langerhans cells in Langerhans cells was counted using the in-
Figure 2. View captured from side-
the cornea. built general-utility manual counting tool of
After scanning the central cornea, the par- the Heidelberg instrument and data were mounted charge-coupled device camera of
ticipant was instructed to sit back for a brief expressed as LCD, which is the mean of the Heidelberg laser scanning confocal micro-
time before scanning the conjunctiva. In all number of cells per square millimetre from scope, showing the plano face of the
participants, the face of the Tomocap was the images. The L-method was used to select Perspex Tomocap (A) gently contacting
brought into gentle contact with the nasal which cells were included in the count, the bulbar conjunctiva (B)

2016 Optometry Australia Clinical and Experimental


Clinical andOptometry 100.1
Experimental January 2017
Optometry 2016
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35
Langerhans cells
cells in
in contact
contactlens
lenswear
wearAlzahrani,
Alzahrani,Colorado,
Colorado,Pritchard
Pritchard and
and Efron
Efron

Statistical approach
Normality of data relating to demographic
and clinical characteristics was examined
using the ShapiroWilk test and the most
appropriate test was applied for each analy-
sis. To compare the demographic and clin-
ical characteristics between group pairs,
parametric data were analysed using an
independent sample t-test and non-para-
metric data were analysed using an 2 test
and MannWhitney U-test.
A linear mixed model was used to examine
changes over time in LCD and whether the
changes were different among the three test
groups (CLIDE, NO-CLIDE and controls).
Although our data set displayed relatively
high kurtosis, a linear mixed model was
deemed most suitable for our longitudinal
data set, bearing in mind that these models
are robust to normality violations.26 A re-
peated-measures multivariate analysis of vari-
ance (using Pillais trace and Wilks lambda)
was performed for the groups and include
the factors, age and sex. Separate analyses
were undertaken for data relating to the cor-
nea and conjuctiva.
Tests of association between LCD and level
of severity as indicated by any of the dry eye
assessment techniques (DEQ-5, CLDEQ-8,
NITBUT, PRTT and CFS) at the one week
(when there was a maximum inammatory
response) were conducted using Spearmans
rank correlation test.
Data were analysed using IBM SPSS Statis-
tics for Windows, Version 21.0 (IBM Corpora-
tion, Armonk, New York, USA). A p-value of
less than 0.05 was considered to be statistically Figure 3. Flow diagram of study. The control group was recruited separately from the con-
signicant.
tact lens-induced dry eye (CLIDE) and NO-CLIDE groups.

RESULTS The mean and standard deviation (SD) lens basal epithelium and sub-basal nerve plexus
powers tted to participants were: R -1.75 of the cornea (Figure 4). There was no
A ow diagram showing the number of 1.99 D, L -1.64 1.80 D for CLIDE and R change in corneal LCD over time in the
participants recruited and enrolled into the -1.70 1.95 D, L -1.63 1.79 D for NO-CLIDE. control group (p = 0.272) throughout the 24-
study, discontinuing from the study (and the Demographic and clinical data of the three week observation period.
reasons for this) and nally examined, is study groups at baseline and week 24 are Figure 5 shows the mean and standard
presented in Figure 3. After screening 110 shown in Table 1, together with paired error of the mean (SEM) of corneal LCD at
potential study participants, 92 were enrolled differences among groups. The groups were baseline, one, four and 24 weeks for each of
into the study. Sixty participants were tted age-matched but were not effectively gender the three groups. At baseline, there were no
with contact lenses and 23 served as controls. balanced, with a signicantly higher propor- differences in corneal LCD overall or
When signs and symptoms of CLIDE were tion of males among controls versus CLIDE between any paired combinations of CLIDE
assessed among contact lens wearers at one and NO-CLIDE. The signicantly higher (24 3 cells/mm2), NO-CLIDE (24 3 cells/
week, the group assignment comprised 25 mean CLDEQ-8 scores for CLIDE versus mm2) and controls (26 4 cells/mm2). At
CLIDE and 35 NO-CLIDE. Sixteen partici- NO-CLIDE at baseline constituted the basis week 1, LCD was greater in CLIDE (55 7
pants (eight CLIDE, six NO-CLIDE and two for group assignment. cells/mm2) versus NO-CLIDE (43 4 cells/
controls) dropped out of the study over the mm2) (p = 0.041) and controls (27 4 cells/
24-week study period, leaving 17 CLIDE, 29 Changes in corneal LCD mm2) (p < 0.001). LCD was also greater in
NO-CLIDE and 21 controls who completed Both immature and mature Langerhans cell NO-CLIDE (p = 0.041) versus controls
the study. phenotypes were observed at the level of the (p = 0.01). At week 4, LCD was greater in

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Parameter Baseline 24 weeks p-value

CLIDE NO-CLIDE Control CLIDE NO-CLIDE Control AvB AvC BvC DvE DvF EvF AvD BvE CvF
(A) (B) (C) (D) (E) (F)

Male/female 7/18 10/25 19/4 15/12 8/21 17/4 0.96* <0.001* <0.001* 0.98* 0.001* <0.001* 0.63* 0.61* 0.88*

2016 Optometry Australia


(n)
Age (years) 28.5 6.4 32.1 9.8 30.0 8.0 28.1 5.9 31.2 9.3 30.3 8.0 0.10 0.54 0.34
DCLWT (h) 8.3 3.9 9.8 3.9 0.38
DEQ-5 (0-22) 4.5 1.8 3.8 2.1 1.9 2.1 2.2 2.3 0.18+ 0.002+ 0.01+ 0.64#
CLDEQ-8 21.4 4.2 11.6 3.6 19.6 9.0 10.6 6.2 <0.001 <0.001 0.12 0.43
(037)
NITBUT 13.4 5.7 11.9 5.6 12.7 6.1 9.7 5.9 11.0 4.7 10.0 4.3 0.31+ 0.67+ 0.58+ 0.12+ 0.87+ 0.52+ 0.24# 0.52# 0.10#
(seconds)
CFS (04) 0.4 0.5 0.3 0.5 0.5 0.6 1.0 0.8 0.7 0.6 0.3 0.4 0.51+ 0.73+ 0.30+ 0.17+ 0.01+ 0.13+ 0.006# 0.008# 0.58#

PRTT 18.0 7.6 19.9 8.0 22.7 8.4 9.4 4.9 16.8 8.4 18.8 7.7 0.35 0.36 0.17 0.003 <0.001 0.38 0.001 0.12 0.10
(mm/20s)
DCLWT: daily contact lens wear time, DEQ-5: dry eye questionaire-5, CLDEQ-8: contact lens dry eye questionaire-8, NITBUT: non-invasive tear break-up time, CFS: corneal uorescein staining,
PRTT: phenol red thread test, CLIDE: contact lens-induced dry eye, NO-CLIDE: no contact lens-induced dry eye.
Results are expressed as mean SD, or counts for categorical variables.
Signicant differences (p < 0.05) are shown in bold.
* test.
Langerhans


Independent sample t-test.
+
MannWhitney U-test.
Langerhanscells


CLDEQ-8 baseline data is that collected after 1 week of contact lens wear.
cellsin

Table 1. Demographic and clinical data at baseline and 24 week visits of three study groups and pairwise differences
incontact
contact lens
lens wear Alzahrani,

epithelial surface.

Clinical and Experimental


(28 4 cells/mm2).
Alzahrani, Colorado,

Clinical andOptometry
standard error of the mean.

Experimental
Colorado,Pritchard

100.1
Pritchardand

Optometry
andEfron

pants over 24 weeks. Error bars indicate

univariate, within-group analyses of corneal


(up to 30 years and more than 30 years),
NO-CLIDE (29 3 cells/mm2) and controls
combinations of CLIDE (35 8 cells/mm2),
CLIDE (41 6 cells/mm2) versus controls
the cornea of contact lens-induced dry eye
cate immature Langerhans cells with short

5
between time and sex or between the time
LCD showed no signicant interaction
cells with long dendrites. Thin arrows indi-
scopic image of Langerhans cells in cornea.

2016
When participants were grouped by age
24, there were no differences in corneal
between CLIDE versus NO-CLIDE. At week
(27 4 cells/mm2) (p = 0.004); however,
(CLIDE), NO-CLIDE and control partici-
tured at a depth of 63 m from the corneal

CLIDE (35 4 cells/mm2) versus controls or


there were no differences between NO-
Thick arrows indicate mature Langerhans

LCD overall or between any paired


Figure 5. Langerhans cell density (LCD) in
or absent dendrites. This image was cap-
Figure 4. Laser scanning confocal micro-

37
January 2017
Efron
Langerhans cells
cells in
in contact
contactlens
lenswear
wearAlzahrani,
Alzahrani,Colorado,
Colorado,Pritchard
Pritchard and
and Efron
Efron

and age group. There were signicant multi- neutrophils, granulocytes and macrophages
variate effects with respect to LCD for time from the surrounding tissues and the blood
(p < 0.001) and an interaction between vascular system, resulting in an inammatory
group and time (p < 0.001); however, there response with typical symptoms.27 Therefore,
were no signicant multivariate effects for activation of Langerhans cells is an early and
the study group (CLIDE/NO-CLIDE), sex, sensitive measure of an impending inamma-
age group, interaction between sex and time, tory process.
or interaction between age group and time. Langerhans cells are a subset of a broad
There were no signicant correlations category of cells known as dendritic cells, so
between corneal LCD and level of severity as all dendritic cells observed on the ocular
indicated by any of the dry eye assessment surface with the LSCM may not necessarily
techniques (DEQ-5, CLDEQ-8, NITBUT, be Langerhans cells. Evidence in the litera-
PRTT and CFS) at one week. Figure 7. Langerhans cell density (LCD) in ture suggests that there is a direct correlation
the conjunctiva of contact lens-induced dry between LSCM and immunohistochemical
eye (CLIDE), NO-CLIDE and control par- observations of dendritic cells in the
Changes in conjunctival LCD ticipants over 24 weeks. Error bars indi- cornea.28 The expression of Langerhans
Both immature and mature Langerhans cell cate standard error of the mean. cell-specic surface markers by dendritic cells
phenotypes were observed in the stroma of in the corneal and limbal epithelium has also
the bulbar conjunctiva (Figure 6). There was been reported.29,30 In view of this strong pre-
between any paired combinations of CLIDE
no change in conjunctival LCD over time in sumption that dendritic cells observed in the
(14 3 cells/mm2), NO-CLIDE (12 2 cells/
the control group (p = 0.944) throughout cornea using LSCM are Langerhans cells, we
mm2) and controls (7 1 cells/mm2). At week
the 24-week observation period. have adopted this term in the present study.
24, there were no differences in conjunctival
Figure 7 shows the mean and standard Cross-sectional studies have found
LCD overall or between any paired
error of conjunctival LCDs at baseline, one, Langerhans cells to be up-regulated in the
combinations of CLIDE (10 3 cells/mm2),
four and 24 weeks for each of the three cornea during contact lens wear.13,15 Zhivov
NO-CLIDE (10 2 cells/mm2) and controls
groups. At baseline, there were no differ- and colleagues15 reported that Langerhans
(8 2 cells/mm2).
ences in conjunctival LCD overall or between cells could be observed in the cornea of 59
When participants were grouped by age
any paired combinations of CLIDE (7 1 per cent of contact lens wearers and 33 per
(up to 30 years and over 30 years), univariate,
cells/mm2), NO-CLIDE (8 2 cells/mm2) cent on non-lens wearing controls. In those
within-group analyses of conjunctival LCD
and controls (7 1 cells/mm2). At week 1, with Langerhans cells, LCD of the central
showed no signicant interaction between time
conjunctival LCD was greater in CLIDE (17 cornea was found to be 78 cells/mm2 in lens
and sex or between the time and age group.
1 cells/mm2) (p = 0.003) and NO-CLIDE wearers versus 34 cells/mm2 in non-lens wear-
There were no signicant correlations be-
(17 3 cells/mm2) (p = 0.001) versus controls ing control subjects. The generally higher
tween conjunctival LCD and level of severity
(7 1 cells/mm2). At week 4, there were no values of LCD reported by Zhivov and
as indicated by any of the dry eye assessment
differences in conjunctival LCD overall or colleagues15 compared with our study could
techniques (DEQ-5, CLDEQ-8, NITBUT,
be attributed to the inclusion of all eyes in
PRTT and CFS) at one week.
our analysis, including those with zero LCD.
Sindt and colleagues13 reported the mean
DISCUSSION LCD to be signicantly higher in 53 contact
lens wearers (64 cells/mm2) compared with
Of the 60 participants who were entered into 10 non-lens wearing controls (29 cells/
the contact lens arm of this study all of whom mm2). These reported values of LCD are
were pre-screened to ensure that they were generally commensurate with those reported
free of dry eye symptoms at baseline 25 (42 in our study.
per cent) developed CLIDE after one week. Efron, Al-Dossari and Pritchard11 have also
This observation is broadly consistent with observed Langerhans cells in the bulbar con-
previous studies, which report a prevalence junctiva but were unable to differentiate LCD
of CLIDE of between 28 and 50 per cent.1 between those who had been wearing contact
Langerhans cells are antigen-presenting lenses for 10 years versus non-lens wearers.16
cells. They are observed in the skin, mucous This observation is consistent with the
membranes including the ocular surface, present study, which noted an increase in
lymph nodes and spleen. Although knowl- conjunctival LCD after one week of lens wear
Figure 6. Laser scanning confocal micro-
edge of the precise role of Langerhans cells but no difference after one month of wear.
scopic image of Langerhans cells in con-
is evolving,27 they are generally believed to The observation in this study of an ini-
junctiva. Arrows indicate the typically be essential in the processing of antigens tial up-regulation in corneal LCD in
mature Langerhans cells with long den- presented through the epithelial surface CLIDE versus controls at one and four
drites found in the conjunctiva. This image and perform as a histocompatibility antigen weeks is consistent with previous reports
was captured at a depth of 20 m from the to stimulate T and B cells. These cells in turn of higher levels of corneal LCD in dry eye
conjunctival epithelial surface. recruit further inammatory cells, such as patients. Machetta and colleagues31

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Langerhans
Langerhanscells
cellsin
incontact
contact lens
lens wear Alzahrani, Colorado,Pritchard
Alzahrani, Colorado, Pritchardand
andEfron
Efron

reported a higher LCD in the central cor- followed by an apparent progressive reduc- cells, which have short or absent dendrites,
nea of patients with Sjgren syndrome tion in LCD over the next 23 weeks. This nd- could be misidentied for other inamma-
dry eye (SSDE) (79 cells/mm2) compared ing provides further physiological evidence of tory cell types known to reside in ocular
to those without dry eye (22 cells/mm2) an adaptation to contact lens wear. This surface tissues, such a monocytes and
(p = 0.0031). In a similar study, Lin and hypothesis must be tempered by the fact that, polymorphs. Even if these bright features,
colleagues32 reported a higher LCD in in our study, ve participants discontinued which we have identied as Langerhans cells,
the central cornea of patients with both due to contact lens discomfort from the are some other cell type involved in the
SSDE (128 cells/mm2) and non-SSDE (90 CLIDE group versus only one from the NO- immunologic cascade of events, our conclu-
cells/mm2) compared to those without CLIDE group; had there been no discontinu- sions are essentially unaltered because our
dry eye (35 cells/mm2). We failed to nd ations, higher LCD values may have been observations support the overriding principle
evidence of an up-regulation of conjuncti- recorded in the CLIDE group at week 24. of inammatory cell up-regulation in CLIDE.
val LCD in CLIDE (versus NO-CLIDE) at Our failure to demonstrate signicant Misidentication of tissue features as
any stage in this longitudinal study, correlations between corneal and conjuncti- Langerhans cells would constitute a random
suggesting that the cornea is a more val LCD and any of the ve tests for dry eye error that would manifest without bias across
sensitive barometer of ocular inamma- (DEQ-5, CLDEQ-8, NITBUT, PRTT and all groups.
tion associated with CLIDE. CFS) precludes us from developing predictive Although the use of only one lens type lim-
The concept of adaptation has long been models for LCD. This lack of correlation ited data variability, this approach precluded
discussed in the contact lens eld. It is an ill- perhaps also supports the notion that the examination of the impact of different
dened term and refers to various phenom- sensation of discomfort, which is subjectively surface characteristics (for example, lubricity
ena, such as physical, visual and psychological reported as dryness, might not actually and wettability) that may have impacted com-
processes. Short-term adaptation describes represent a true aqueous deciency but fort and thus LCD. The study was limited to
the subjective perception of lenses becoming rather may arise from complex neural coding six months, so the impact of longer periods
more comfortable in the brief period of of other adverse sensory inputs. of lens wear is unknown.
seconds or minutes following the initial In conclusion, CLIDE is associated with an
instantaneous discomfort experienced The strengths of this study are as follows. initial up-regulation of Langerhans cells in
during insertion.32 Long-term adaptation the cornea, which suggests an inammatory
describes the subjective perception of lenses 1. Our cohort was carefully characterised in basis for this condition during the initial
becoming more comfortable in the days, respect of those with and without CLIDE, phases of contact lens wear. The apparent re-
weeks or months following the commence- as well as a contemporaneous control duction in LCD following this initial increase
ment of lens wear.33 Adaptation is more evi- group of non-lens wearers. suggests a form of physiological adaptation to
dent in respect of the wearing of rigid 2. Experimental bias was minimised by lens wear. Assessment of LCD using laser
contact lenses, which have a greater physical masking the operator assessing LCD from scanning confocal microscopy offers new in-
impact on the anterior ocular structures than the clinical status of the participants. sights into the pathophysiology of the ocular
soft lenses.34 3. All participants wore the same lens type tissues in response to contact lens wear. As
The basis for adaptation may be, in part, for the same length of time, thus exclud- well, this approach introduces the potential
psychological (that is, getting used to the ing these factors as possible confounding to non-invasively and reiteratively monitor
persistent discomfort).35 The physiological variables. the inammatory status of the cornea and
basis of adaptation is poorly understood,35 4. The experiment was sufciently powered conjunctiva in vivo in response to various lens
although recent evidence from studies of to detect differences in LCD between the materials and designs and different modali-
up-regulation of tear components and cor- assigned groups. ties of contact lens wear. In this way, clinicians
neal inammatory cells during contact lens who have access to this technology may be
wear are providing possible answers. This study has a limitation related to the assisted in clinical decision making, as well
Markoulli and colleagues36 noticed a signi- gender imbalance of CLIDE and NO-CLIDE as the contact lens industry in developing
cant increase in matrix metalloproteinase-9 versus the control group, which may have contact lenses with material characteristics
in the tear lm on awakening in people wear- confounded comparisons between those that are less physiologically challenging to
ing extended wear contact lenses. They sug- groups, if signicant gender differences exist the eye and afford greater levels of comfort.
gested that return of this inammatory in the parameters assessed in this work. An-
mediator to baseline by one month indicates other study limitation is that the non-lens
an adaptive process. wearing control group was recruited sepa- ACKNOWLEDGEMENTS
Using LSCM, Zhivov and colleagues15 rately, rather than having been randomly Yahya Alzahrani was supported by a Saudi
observed that central corneal LCD values of assigned from a single combined recruitment Arabian Government Scholarship. The con-
144, 122 and 15 cells/mm2 in people who cohort of potential study participants. tact lenses used in this study were kindly
had been wearing contact lenses for less than The limitation of LSCM for unambiguously supplied by CooperVision Australia.
ve years, ve to 10 years and more than identifying the observed bright features in
10 years, respectively. They suggested that this LSCM images as Langerhans cells is another
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