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Cytokine 33 (2006) 36e40
Alteration pattern of tear cytokines during the course of a day:
Diurnal rhythm analyzed by multicytokine assay
Eisuke Uchino a, Shozo Sonoda a, Naoko Kinukawa b, Taiji Sakamoto a,*
a Department of Ophthalmology, Kagoshima University Graduate School of Medicine and Dental Sciences,
8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
b Department of Medical Information Science, Graduate School of Medical Sciences, Kyushu University,
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Received 26 July 2005; received in revised form 15 November 2005; accepted 18 November 2005
Abstract
Our aim was to study the alteration pattern and interaction of inflammatory tear cytokines during the course of a day. Using a prospective,
experimental design, tears were collected from 28 healthy volunteers with normal eyes during the period from April 2004 to March 2005. Tears
(10 ml) were collected by capillary outflow from each eye at 9:00, 12:00, 16:00, 21:00, and 24:00 h. The concentrations of inflammatory cyto-
kines, IL-1b, IL-6, IL-8, IL-10, IL-12p70, and TNF-a were measured using cytometric bead arrays. Although the concentration of tear cytokines
varied widely among eyes, the amount of cytokine had a specific alteration pattern in each eye during the course of a day. IL-1b, IL-6,
IL-10, IL-12p70, and TNF-a showed slight increases in the morning and the late evening. IL-8 remained low throughout the day. The alteration
pattern of IL-8 was significantly different from those of TNF-a and IL-12p70 (P < 0.01). The ratio of each pro-inflammatory cytokine to anti-
inflammatory cytokine IL-10 did not significantly change throughout the day. The amount of tear cytokines changed during daytime with a
specific pattern. This diurnal rhythm may influence symptoms of ocular surface diseases during the course of a day.
� 2005 Elsevier Ltd. All rights reserved.
Keywords: Dry eye; Conjunctivitis; ELISA; Protein standard; Cortisol
1. Introduction
Recently, the number of individuals suffering from ocular
surface problems such as dry eye and allergic conjunctivitis
has been increasing in many countries [1e3]. Although this
condition does not directly lead to severe visual loss, it is a se-
rious problem especially for urban workers because it might
be related to air pollution, air conditioning, or computer use
[4,5]. The symptoms of ocular discomfort vary widely, and
their severity also changes throughout the day [1,6,7]. So
far, changes of symptoms during a day have been explained
by tear evaporation, decreased tear production, and/or tired
eyes. However, the real mechanism has yet to be clarified
* Corresponding author. Tel.: þ81 99 275 5402; fax: þ81 99 265 4894.
E-mail address: tsakamot@m3.kufm.kagoshima-u.ac.jp (T. Sakamoto).
1043-4666/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cyto.2005.11.013
[2,6,8,9]. Because the tear film plays an essential role in main-
taining the ocular surface integrity by tightly regulating the
optimal extracellular environment that is critical to its func-
tions, it is highly probable that tears also play a putative role
in ocular surface discomfort.
Cytokines play major roles in various patho-physiological
conditions and accurate and sensitive methods for their mea-
surement in body fluids are absolute prerequisites for under-
standing their roles in clinical practice. Many factors
contribute to the complexity of cytokines quantitation: these
molecules are present at very low levels in body fluids (e.g.
pg/ml) under various molecular forms and the presence of
inhibitors such as binding proteins, soluble receptors, autoanti-
bodies, can potentially interfere with assays [10]. So far,
studies on tear cytokines have shown significant progress in
achieving an understanding of ocular surface diseases. But,
the amount of information that could be acquired from each
mailto:tsakamot@m3.kufm.kagoshima-u.ac.jp
http://www.elsevier.com/locate/issn/10434666
37E. Uchino et al. / Cytokine 33 (2006) 36e40
subject has, until recently, been severely hampered by limited
sample volume and assay sensitivity. More importantly, as our
understanding of cytokine networks has increased, it has
become apparent that the combination of different cytokines
present and the balance of their relative concentrations could
be more important than the absolute concentrations of any spe-
cific cytokine [11e13]. Therefore, sampling tears becomes
even more critical for understanding the role of cytokines in
ocular surface disorders.
It is widely known that concentrations of cytokines in body
fluids have circadian or diurnal rhythms, which are considered
to influence diurnal symptoms [14e16]. To our knowledge,
however, there are no reports regarding circadian changes of
tear cytokines focusing especially on multi-cytokines. Cyto-
metric bead array is a microparticle-based flow cytometric
assay that allows us to quantify multiple molecules from
a very small sample [17e20]. Using this method, we found
for the first time that there is a specific diurnal alteration pat-
tern in inflammatory cytokines in tears. In this manuscript, we
present some of these data and discuss their potential role in
the symptomatic changes during daytime.
2. Materials and methods
2.1. Tear sampling
The study was carried out with the approval of the institu-
tional review board, and was performed in accordance with the
ethical standards laid down by the Declaration of Helsinki.
All participants gave their informed consent to participating
in this study. After checking that each eye was free from ocu-
lar diseases by slit-lamp microscopy a total of 28 healthy
adults (office workers and students, age ranging from 23 to
45 years old, 14 males and 14 females) were enrolled in the
study. Tear samples were obtained by capillary flow, with no
nasal stimulation or previous instillation of drugs or vital
dyes. No anesthetic drops were instilled. The samples were
collected non-traumatically from the inferior meniscus. Care
was taken to avoid touching the corneal and conjunctival sur-
faces. Tears were collected from the right eye of the partici-
pants at 9:00, 12:00, 16:00, 21:00, and 24:00 h. During this
period, the participants maintained a regular life style and
remained awake. The collected tears were frozen at �80 �C
within 30 min and stored until measurement.
2.2. Measuring cytokines
The amounts of six inflammatory molecules (IL-1b, IL-6,
IL-8, IL-10, IL-12p70, and TNF-a) were measured using
a cytometric bead array (BD Biosciences Pharmingen, San
Diego, CA), according to the manufacturer’s instructions.
This method allows us to quantify six different molecules
from a small sample volume [17,18]. Briefly, for the tear
sample and cytokine standard mixture, 5e10 ml of sample or
standard was added to a mixture of 50 ml each of capture
Ab-bead reagent and detector Ab-phycoerythrin (PE) reagent.
This mixture was subsequently incubated for 3 h at room
temperature, and washed to remove any unbound detector
Ab-PE reagent before data acquisition. A two-color flow cyto-
metric analysis was performed using a FACScan flow cytometer
(BD Biosciences Immunocytometry Systems, San Jose, CA).
Data were acquired and analyzed using BD cytometric bead
array software.
To confirm the ability of the assay to measure cytokines,
spike-recovery experiments were also performed by the previ-
ously described method [18]. The pooled tears of five samples
(95%) and spike volume (5%) were mixed. Cytokine spike
concentrations were 80, 625, and 2500 pg/ml. A mixture of
50 ml each of capture Ab-bead reagent and detector Ab-PE re-
agent was added to 10 ml of pooled spiked sample or calibrator,
and incubated for 3 h at room temperature.
2.3. Statistical analysis
Alteration patterns of cytokines and value of each cytokine
at each time point were compared using two-way repeated
measures ANOVA and paired t-test. In particular, differences
in alteration patterns among cytokines were analyzedduring
the interaction between cytokines and time points of two-
way repeated measures ANOVA. Statistical significance was
evaluated by multiple comparisons using Bonferroni’s method.
The null hypothesis was rejected for P values of less than 0.05.
The BMDP software package (BMDO Statistical Software,
Inc., Los Angeles, CA, USA) was used to perform the statis-
tical analyses.
3. Results
3.1. Cytokines compared to those at 9:00 h
Although there were no pathological signs or symptoms in
any eyes, the concentration of individual cytokines variedwidely
between individuals. The median of IL-1b was 227.4 pg/ml,
ranging from 20.9 to 1691.8 pg/ml; IL-6 was 31.7 pg/ml,
ranging from 1.4 to 263.9 pg/ml, IL-8 was 283.0 pg/ml ranging
from 8.3 to 1484.8 pg/ml, IL-10 was 28.8 pg/ml, ranging from
2.7 to 262.5 pg/ml; IL-12p70 was 49.9 pg/ml, ranging from 1.7
to 714.9 pg/ml, and, TNF-awas 18.8 pg/ml, ranging from 1.5 to
286.0 pg/ml, respectively (Table 1).
Cytokine concentrations at 9:00 h were taken as baseline
measurements and the concentrations at later time points were
expressed as ratios to these initial values. When expressed
in this way, it was clear that all the cytokines measured,
Table 1
Absolute concentration of tear cytokines by cytometric bead array
Median Range
IL-1b 227.4 20.9e1691.8
IL-6 31.7 1.4e263.9
IL-8 283.0 8.3e1484.8
IL-10 28.8 2.7e262.5
IL-12p70 49.9 1.7e714.9
TNF-a 18.8 1.5e286.0
The concentrations are expressed as pg/ml.
38 E. Uchino et al. / Cytokine 33 (2006) 36e40
except for IL-8, showed a similar pattern of change over a day
(Fig. 1), with a moderate increase in concentration in the
morning, followed by a slight decrease in the afternoon and
early evening and then a significant increase between 21:00
and 24:00 h. In contrast, the concentration of IL-8 was not
significantly higher than the concentration at 9:00 h at any later
time point. Repeated ANOVA measurements showed that the
changing patterns of six cytokines were unequal overall
(interaction P value ¼ 0.0000, data not shown). Multiple com-
parisons showed that the changing pattern of IL-8 is different
from that of TNF-a and IL-12p70 (Table 2, P < 0.01 and
P < 0.05 respectively). To find the specific time when the
composition of tear cytokine is different, the value of each
cytokine at each time point was compared to that at 9:00 h
using a multivariate paired t-test (Table 3). The results showed
that the values of IL-12p70 and TNF-a at 12:00 were
significantly higher than those at 9:00 (P < 0.05 in both), and
that the value of IL-6 at 24:00 h was significantly higher
than that at 9:00 h (P < 0.05). Spike-recovery experiments
showed that themicro-bead array systemhad high reproducibility
for tear cytokine measurement (data not shown).
3.2. Cytokines compared to IL-10
Several studies have shown that measuring the relevant
concentrations of several different cytokines yields more infor-
mation about pathologies than measurement of the absolute
amounts of a single cytokine [11e13]. Because IL-10 was
the only anti-inflammatory cytokine examined in this study,
the ratio of IL-1b, IL-6, IL-8, IL-12p70, and TNF-a to IL-
10 was calculated. As a result, although there was a morning
increase or an evening rise of IL-1b, IL-6, IL-12p70, and
TNF-a in a single cytokine quantification, this increase was
not found in the cytokine ratio to IL-10 (Table 4).
4. Discussion
The absolute concentrations of cytokines in tears measured
in this study showed some differences from those reported in
Fig. 1. Chronological alteration pattern of cytokines expressed as a ratio to
those collected at 9:00 h.
previous studies [11,21e23]. For example, base line concen-
trations of IL-6 were reported to be 226.2 pg/ml by Nakamura
et al. [22] and 2.2 pg/ml by Lema et al. [21] using an enzyme-
linked immunosorbent assay (ELISAs). In this study we
obtained a median value of 31.7 pg/ml. Cytometric bead assay
for soluble cytokines was reported to correlate well with
ELISA in sensitivity, accuracy, and reproducibility, however,
the absolute concentrations obtained from each assay differed
with kits from different manufacturers [11,17,19,20]. This is
due to the different cytokine standards and/or antibody provided
in a kit; for example, 1 ng IL-6 cytokine standard ranged from
3.9 to 505 according to the international units of the National
Institute for Biological Standards and Control (IU NIBSC 88/
514) among the kits of the different manufacturers [19]. Con-
sequently, it is difficult to compare the absolute concentrations
of cytokines measured by kits from different manufacturers,
but Khan et al. also showed that kits from the same manufac-
turer gave accurate and reproducible results [19]. Indeed, the
absolute concentrations of tear cytokines in the present results
were almost the same as those reported by Cook et al., who
used the same kit as ours [18]. Importantly, it is noteworthy
that we did not primarily analyze the absolute concentration
of cytokines, but analyzed the alteration pattern and the ratio
to other cytokines using the same assay kit, and that reproduc-
ibility was confirmed by spike-recovery experiments. As is
known well, the absolute concentrations of tear cytokines
Table 2
Results of multiple comparisons of cytokine change with Bonferroni’s method
Factor F-value DF P-value Bonferroni
IL12-TNFa 4.50 1, 27 0.0433
Interaction 3.74 4, 108 0.0069
IL12-IL10 4.74 1, 27 0.0384
Interaction 3.38 4, 108 0.0120
IL12-IL6 2.74 1, 27 0.1093
Interaction 3.38 4, 108 0.0120
IL12-IL1b 0.01 1, 27 0.9072
Interaction 0.48 4, 108 0.7519
IL12-IL8 7.67 1, 27 0.0100
Interaction 6.09 4, 108 0.0002 **
TNFa-IL10 0.13 1, 27 0.7195
Interaction 0.14 4, 108 0.9691
TNFa-IL6 1.02 1, 27 0.3218
Interaction 1.74 4, 108 0.1462
TNFa-IL1b 2.63 1, 27 0.1163
Interaction 2.65 4, 108 0.0373
TNFa-IL8 5.05 1, 27 0.0330
Interaction 4.30 4, 108 0.0029 *
IL10-IL6 0.74 1, 27 0.3973
Interaction 2.09 4, 108 0.0874
IL10-IL1b 3.07 1, 27 0.0913
Interaction 3.42 4, 108 0.0112
IL10-IL8 3.65 1, 27 0.0668
Interaction 3.65 4, 108 0.0080
IL6-IL1b 0.95 1, 27 0.3375
Interaction 1.61 4, 108 0.1779
IL6-IL8 9.15 1, 27 0.0054 y
Interaction 4.19 4, 108 0.0034 y
IL1b-IL8 6.56 1, 27 0.0163
Interaction 4.05 4, 108 0.0042 y
DF, degree of freedom. Bonferroni: **P < 0.01, *P < 0.05, yP < 0.1.
39E. Uchino et al. / Cytokine 33 (2006) 36e40
Table 3
Results of multiple comparison paired t-tests for each cytokine compared to the value at 9:00 h
Time To 12:00 h To 16:00 h To 21:00 h To 24:00 h
Cytokine t-Value P-value B t-Value P-value B t-Value P-value B t-Value P-value B
IL-1b �2.16 0.0395 �1.33 0.1961 �2.01 0.0541 �2.58 0.0156
IL6 �2.26 0.0323 �1.88 0.0715 �1.82 0.0801 �2.68 0.0124 *
IL8 0.38 0.7451 �0.41 0.6885 �0.65 0.5217 �0.2 0.8404
IL10 �2.34 0.0271 �1.18 0.2482 �1.05 0.3025 �2.36 0.0256
IL12 �2.85 0.0082 * �1.64 0.1133 �1.66 0.1081 �2.58 0.0157
TNF-a �2.83 0.0086 * �1.6 0.1213 �1.56 0.1309 �2.38 0.0245
B, P-value with Bonferroni’s method, *P < 0.05.
are sensitive to the collection technique. In view of the assay
method used in this study, our results better reflect the relative
concentration of cytokines, and therefore the balance on
pro- and anti-inflammatory effects they may have, than the
absolute, individual concentrations.
Although the concentrations of cytokines widely differed
between subjects, each cytokine showed a specific alteration
pattern, which was expressed as a ratio to its concentration
at 9:00. The increases in concentration seen in the morning,
and particularly in the evening, for IL-1b, IL-6, IL-10, IL-
12p70, and TNF-a, did not seem to simply reflect the effects
of tear evaporation, as the concentration of IL-8 remained con-
stant throughout the day, with the pattern of its concentration
at various time-points showing a significant difference from
that of IL-12p70 and TNF-a. Many cells can secrete these
cytokines into tears (e.g. conjunctival epithelial cells, corneal
epithelial cells, endothelial cells, mast cells, monocytes, eosi-
nophils,lacrimal gland cells). Although they are controlled in
order to maintain the homeostasis at the ocular surface, the ex-
act mechanisms are still uncertain. This study shows that
changes in the concentrations of the five cytokines, IL-1b,
IL-6, IL-10, IL-12p70, and TNF-a, follow similar patterns in
the course of a day and that the pattern for IL-8 is quite differ-
ent, suggesting that IL-8 and the other five cytokines may have
different sources or that different systems control their
secretion.
There were two rises in the concentrations of the pro-
inflammatory cytokines, TNF-a and IL-12p70, in the morning
and late evening. However, the concentration of the anti-
inflammatory cytokine IL-10 also increased, which was appar-
ent from the changes in its ratio to the other cytokines.
Because the inflammatory status was determined from the
balance of pro- and anti-inflammatory factors, the increase
of pro-inflammatory cytokines does not, necessarily, mean
the ocular surface is pro-inflammatory, rather, the simulta-
neous increase of both pro- and anti-inflammatory cytokines
indicate that the inflammatory status might be well controlled.
This is also supported by the fact that none of the participants
of the present study complained of ocular surface discomfort
or pathologic signs, even in the morning and the late evening.
Because there are many other factors that affect inflammatory
status in tears, further study is necessary to determine if a tear
is pro- or anti-inflammatory.
In previous reports, the symptoms of ocular surface dis-
eases are deteriorated as the day progressed or during the night
[6,8,9]. Reduced tear production, tear evaporation, effects of
inflammation in the eyelid, osmolarity changes, and the effect
of eye closure have been proposed as explanations for this
[6,8,9,24,25]. In the present study, there was an increase in
the pro-inflammatory cytokine IL-6 during the evening. There-
fore, it is possible to suggest that the ocular surface becomes
pro-inflammatory during the evening, although the IL-6:IL-10
ratio is not changed. The quantitative change of tear cytokines
might play a role in the nocturnal worsening of ocular symp-
toms to some extent.
At present, we do not have any evidence to elucidate the
mechanism underlying these changes. It is widely known
that diurnal rhythms of immunological reactions exist in the
human body, which includes diurnal rhythmic changes of
cytokine levels in blood. The levels of pro-inflammatory cyto-
kines such as IL-1, IL-10, TNF-a, and IL-12 tend to be highest
during the evening and low during the afternoon, and this is
caused by the diurnal rhythm of cortisol [15]. Because these
alteration patterns are similar to those of tear cytokines in
this study, they might also be affected by plasma cortisol in
a similar way.
Table 4
Results of multiple comparison of cytokine ratio to IL-10 compared to that at 9:00 h
Time 12:00 h 16:00 h 21:00 h 24:00 h
Cytokine t-Value P-value B t-Value P-value B t-Value P-value B t-Value P-value B
IL-1b 0.16 0.8777 0.38 0.7047 �2.43 0.022 �2.13 0.0429
IL-6 �0.02 0.9862 �1.11 0.2762 �1.80 0.0838 �1.29 0.2086
IL-8 2.01 0.0546 �0.29 0.7753 0.24 0.8113 1.00 0.3262
IL-12 �2.09 0.0459 �1.02 0.3153 �0.95 0.3508 �1.47 0.1543
TNF-a �1.51 0.1424 �1.55 0.1324 �1.45 0.1596 �1.60 0.1222
B, P-value with Bonferroni’s method.
40 E. Uchino et al. / Cytokine 33 (2006) 36e40
In summary, we show the diurnal rhythm of tear cytokines
using cytometric bead assay, which enabled us to analyze
multi-cytokines from a single tear sample. The alterations of
pro-inflammatory cytokines might well explain symptomatic
changes of the ocular surface during a day. The present infor-
mation is important for studying ocular surface diseases, and
for understanding them correctly.
Acknowledgments
This work was supported by a Grant-in-aid for Young
Scientists (B) from the Ministry of Education, Culture, Sports,
Science and Technology.
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	Alteration pattern of tear cytokines during the course of a day: Diurnal rhythm analyzed by multicytokine assay
	Introduction
	Materials and methods
	Tear sampling
	Measuring cytokines
	Statistical analysis
	Results
	Cytokines compared to those at 9:00h
	Cytokines compared to IL-10
	Discussion
	Acknowledgments
	References