Download Inflammatory Markers in the Tears of Patients with Ocular Surface

Original Paper
Ophthalmic Res 2008;40:315–321
DOI: 10.1159/000150445
Received: July 19, 2007
Accepted after revision: October 23, 2007
Published online: August 7, 2008
Inflammatory Markers in the Tears of
Patients with Ocular Surface Disease
Arantxa Acera a Germán Rocha d Elena Vecino a Isabel Lema b
Juan A. Durán a, c
a
Departamento de Oftalmología, Facultad de Medicina, Universidad del País Vasco, Leioa, b Instituto Galego de
Oftalmoloxía, Universidad de Santiago de Compostela, Santiago de Compostela, c Instituto Clínico-Quirúrgico de
Oftalmología, Bilbao, España; d Universidad Católica, Santiago, Chile
Key Words
Tear fluid Interleukins Matrix metalloproteinase Ocular
surface diseases
IL-6 were only found to be overexpressed in conjunctivochalasis. These findings illustrate the selective implication of different molecules in each disorder.
Copyright © 2008 S. Karger AG, Basel
Abstract
Purpose: To determine the concentration of interleukins (IL1 and -6) and matrix metalloproteinase 9 (pro-MMP-9) in
the tears of patients with different ocular surface diseases
and to examine the possible relationship between the disorders and molecular inflammation. Methods: 77 patients diagnosed as having different ocular surface disorders and 18
normal control subjects were studied. Patients were routinely examined and separated into 5 groups: (1) control, (2)
blepharitis, (3) ocular allergic disease, (4) dry eye and (5) conjunctivochalasis. Ten microliters of tears were collected by a
Weck cell sponge. The concentrations of IL-1, IL-6 and proMMP-9 were measured by enzyme-linked immunosorbent
assay, and the MMP-9 activity was evaluated with gelatin zymography. Results: Levels of IL-1 and IL-6 in tear fluid were
significantly higher in conjunctivochalasis (p = 0.0062 and
p = 0.0134) than in the control group. Pro-MMP-9 levels were
significantly elevated in blepharitis (p = 0.013), in allergic eye
disease, in dry eye and in conjunctivochalasis (all p ! 0.001),
in comparison to controls. Conclusions: Pro-MMP-9 levels in
tears are elevated in all of the studied pathologies especially
in ocular allergy and conjunctivochalasis. However, IL-1 and
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Introduction
The cornea, conjunctiva and intervening transition
area comprise the tissues at the ocular surface. All three
are covered by a stratified, squamous, nonkeratinizing
epithelium at the surface of the eye. Functionally, all three
regions of the epithelium support the tear film which
plays a pivotal role in providing a smooth refracting surface for optimal visual acuity and in maintaining a
healthy, well-differentiated ocular surface [1].
A number of chronic diseases of the ocular surface
give rise to symptoms known as chronic ocular discomfort. They account for a large amount of ophthalmic consultations and the specific diagnosis is not always straightforward. Moreover, symptoms are not always consistent
with clinical signs and the associated pathogenic mechanisms are not clearly understood. Blepharitis (lid margin
inflammation), allergic eye disease, hyposecretive dry
Presented at the annual meeting of the Association for Research in
Vision and Ophthalmology, Fort Lauderdale, Fla., USA, May 4, 2006.
Juan A. Durán, MD, PhD
Instituto Clínico-Quirúrgico de Oftalmología
Virgen de Begoña 34
ES–48006 Bilbao (Spain)
Tel. +34 94 473 3545, E-Mail [email protected]
eye and conjunctivochalasis are among the most frequent
ocular surface disorders. It has been demonstrated that
inflammation plays an essential role in the pathogenesis
of these conditions.
In recent years, many studies of the biochemical
changes occurring in tears have been published. Smith et
al. [2] observed a correlation between tear metalloproteinase (MMP) content and clinical evidence of disease
progression. Meller et al. [3] reported that inflammatory
cytokines such as interleukin (IL) 1 and tumor necrosis
factor (TNF) , which can be derived from the ocular
surface and tears, may be responsible for the increased
expression of MMPs in conjunctivochalasis fibroblasts.
This suggests that ocular inflammation might be one important denominator in the pathogenesis of conjunctivochalasis [3]. Solomon et al. [4] studied the expression of
the pro- and anti-inflammatory forms of IL-1 in the tear
and conjunctival epithelium of normal eyes and those
with dry-eye disease and concluded that dry-eye disease
is accompanied by an increase in the proinflammatory
forms of IL-1 (IL-1 and mature IL-1).
IL-1 is an important mediator of inflammation and
immunity. Both proinflammatory forms of IL-1 (IL-1
and IL-1) are multifunctional cytokines which in general produce similar biological effects. IL-1 is a potent
inducer of other inflammatory cytokines such as IL-6,
IL-8 and TNF-, and it can stimulate the production of
MMP enzymes [4–6].
IL-6 is a pleiotropic proinflammatory cytokine synthesized by various cells, such as fibroblasts, endothelial
cells and keratinocytes, in response to numerous cytokines including TNF- and IL-1. IL-6 can also induce the
expression of MMPs [7, 8]. There is much evidence to suggest that MMPs play a vital role in several physiological
and pathological processes. They participate in extracellular matrix (ECM) remodeling after wounding of the
corneal surface and have been implicated in the pathogenesis of sterile corneal ulceration, dry eye and other
ocular diseases [9]. Gelatinase B (MMP-9) and gelatinase
A (MMP-2) are the primary matrix-degrading enzymes
produced by the corneal epithelium and fibroblasts.
MMP-9 has been found to be of central importance in
catalyzing the cleavage of epithelial basement membrane
components [10, 11].
The aim of this research was to determine the concentration of IL-1, IL-6 and pro-MMP-9 in the tears of patients with different ocular surface diseases employing
the enzyme-linked immunosorbent assay (ELISA) and to
analyze the possible relationship between the disorder
and molecular inflammation.
316
Ophthalmic Res 2008;40:315–321
Methods
Patients
We designed a prospective, case-controlled study, in which 77
patients with different ocular surface disorders and 18 normal
subjects (controls) were enrolled. Patients and normal subjects
were recruited from the Cornea and Ocular Surface Unit, Instituto Clínico Quirúrgico de Oftalmología, Bilbao, Vizcaya,
Spain.
The collection of tears was performed from 13 chronic blepharitis patients whose mean age was 44 8 9.8 years (10 women, 3
men). The second group comprised 19 patients diagnosed for allergic eye disease with a mean age of 43 8 12.7 years (13 women,
6 men). All patients were diagnosed as having chronic allergic
conjunctivitis. Diagnosis was based on symptoms and on their
medical history.
The third group comprised 20 hyposecretive dry eye patients,
whose mean age was 56 8 12.63 years (13 women, 7 men). Dry
eye patients included in the study had moderate dry eye with typical symptoms including Schirmer test (with anesthesia) values
below 5 mm, and low tear meniscus and fluorescein and/or rose
Bengal staining (between 1+ and 3+). Patients diagnosed as having Sjögren syndrome were not included in the study.
The last group comprised 25 patients with conjunctivochalasis
disease manifesting itself at the lower tear meniscus. Diagnosis
was based on fluorescein- and rose-Bengal-assisted slitlamp examination. The mean age was 62 8 7.1 years (11 women, 14 men).
We collected tear samples from 18 healthy individuals whose
mean age was 40 8 12.21 years (11 women, 7 men) and who were
not suffering from any ocular disease (no allergic or atopic history). The most symptomatic eye from each patient was analyzed.
The exclusion criteria were the presence or history of any systemic or ocular disorder or condition (including ocular surgery,
trauma and disease) that could possibly interfere with the interpretation of the results. Current or recent use of topical ophthalmic or systemic medications that could affect the pathology condition and patients wearing contact lenses were also excluded
from this study.
Tear Collection
This research was conducted by medically qualified personnel
after approval by the Instituto Clínico Quirúrgico de Oftalmología Ethical Committee and in strict accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from all patients after the nature and possible consequences of the study had been explained. For all experiments, tears were
collected from the inferior lateral tear meniscus, minimizing irritation of the ocular surface or lid margin. Anesthetic drops were
not instilled. We obtained the tear sample using a polyvinyl acetal
surgical sponge (Merocel, ref. 0525, Oasis, Glendora, Calif., USA).
After collection, the sponge was introduced into a 0.5-ml tube
(Eppendorf, Fremont, Calif., USA), and the tear fluid was subsequently recovered by centrifugation at 13,000 rpm at 4 ° C for 15
min. The tears used for protein quantification were immediately
placed on ice !1 h before freezing and storage at –80 ° C until use.
We obtained small volumes of tear samples (10 l) and all the
sample was used to quantify the three markers by ELISA.
Acera /Rocha /Vecino /Lema /Durán
Gelatinolytic Activity of MMP-9 Assay (Zymography)
SDS-gelatin polyacrylamide gel electrophoresis (zymography)
affects in situ activation of gelatinolytic proteases and was used to
separate and visualize the gelatinase activities present in tear
samples [3, 12, 13]. All samples were analyzed without heating or
reduction. The samples contained the same volume of tears, but
not necessarily the same protein content. High-molecular-weight
marker proteins (Sigma Chemical Co.) were routinely included in
each gel (120 g/well), and electrophoresis was performed at 75 V
for approximately 2.5 h. The gels were then removed from their
casts, washed and incubated sequentially at 37 ° C in Triton-X100
(30 min), 0.05 M Tris-HCl and 5 mM CaCl2. After this incubation, the gel was briefly rinsed in distilled water and stained with
0.25 % Coomassie brilliant blue for 1 h. The gel was destained
with 7% acetic acid. Gelatinase activity in the gel was visible as a
clear area in the blue background, indicating an area where the
gelatin had been digested.
Statistical Analysis
The Mann-Whitney U test was used for statistical comparisons between groups. Data are expressed as means 8 SD and the
differences were considered statistically significant at p ! 0.05.
300
*
IL-1 (pg/ml)
250
200
150
100
50
0
C
BL
AD
DE
CCH
Fig. 1. The concentration of IL-1 in tear samples from control sub-
jects (C) and patients with chronic blepharitis (BL), allergic eye
disease (AD), hyposecretive dry eye (DE) and conjunctivochalasis
(CCH), measured by the ELISA technique. * p ! 0.05 compared
with control subjects. Conjunctivochalasis is the only studied ocular surface disease characterized by a massive increase in IL-1
levels.
*
140
120
IL-6 (pg/ml)
Enzyme-Linked Immunosorbent Assay
The tear samples collected to measure the levels of proinflammatory molecules were obtained from different patients with the
same pathologies, but we used one ELISA kit per molecule. Double-sandwich ELISAs for human IL-1 (17 kDa), IL-6 (28 kDa)
and pro-MMP-9 (92 kDa) were performed with commercial kits
(Calbiochem, Darmstadt, Germany), according to the manufacturer’s protocol. In brief, 50 l of assay buffer and 50 l of standard dilutions of recombinant human (IL-1, IL-6 and proMMP-9) proteins, and 10 l of tears from each patient were dispensed into a 96-well microtiter plate coated with the relevant
monoclonal antibodies. The plate was sealed and incubated at
room temperature for 2 h.
After that, the plate was washed 4 times. Then, 200 l of the
corresponding rabbit secondary antiserum conjugated with
horseradish peroxidase was added into each well and incubated
at room temperature for 2 h. Aliquots of 200 l of the color reagent 3,3,5,5-tetramethylbenzidine were then applied for 20–30
min to develop a blue color, and the reaction was stopped by adding 50 l of 1 M H2SO4. Absorbance was read at 450 nm by an
automatic plate reader. Accurate sample concentration was calculated from the linear correlation made with standard antigen versus absorbency.
100
80
60
40
*
20
0
C
BL
AD
DE
CCH
Fig. 2. The concentration of IL-6 in tear samples from control
subjects (C) and patients with chronic blepharitis (BL), allergic
eye disease (AD), hyposecretive dry eye (DE) and conjunctivochalasis (CCH), measured by the ELISA technique. Elevated levels
of this cytokine are specifically associated with both blepharitis
and conjunctivochalasis. * p ! 0.05 compared with control subjects. The results show increased levels of IL-6 in allergic disease,
but this increase was not statistically significant. This apparent
contradiction could be resolved by a large cohort study.
No statistically significant age- or gender-related differences were detected between patients with different
ocular disorders and control subjects.
The cytokines analyzed were directly related to the
MMP-9 enzyme and ocular surface inflammation.
The mean level of IL-1 in tear fluid was significantly
higher in conjunctivochalasis (4.2-fold; 211.45 8 58.5 pg/
ml; p = 0.0062) than in the control group (49.6 8 26.99
pg/ml). However, there was no significant difference in
the concentration of IL-1 in blepharitis (36.54 8 1.28
pg/ml; p = 0.6547), allergic eye disease (14.5 8 14.48 pg/
ml; p = 0.3272) or dry eye (32.57 8 2.38 pg/ml; p = 0.6242)
compared to controls (fig. 1).
Mean levels of IL-6 in tear fluid were significantly
higher in conjunctivochalasis (6.3-fold; 51.78 8 81.53 pg/
ml; p = 0.0134) and blepharitis (2.02-fold; 16.53 8 1.26
Inflammatory Markers in the Tears
Ophthalmic Res 2008;40:315–321
Results
317
Pro-MMP-9 (ng/ml)
250
*
*
200
*
150
*
100
50
0
C
BL
AD
DE
CCH
Fig. 3. The concentration of pro-MMP-9 in tear samples from
control subjects (C) and patients with chronic blepharitis (BL),
allergic eye disease (AD), hyposecretive dry eye (DE) and conjunctivochalasis (CCH), measured by ELISA. Elevated levels of
this enzyme are found in all 4 ocular pathologies. * p ! 0.05 compared with control subjects.
C
CCH DE
MMP-9 (92 kDa)
MMP-2 (72 kDa)
a
AD AD
BL
BL
DE
DE
C
Discussion
MMP-9 (92 kDa)
MMP-2 (72 kDa)
b
Fig. 4. a Gelatin zymogram of tear samples from a control subject
(C), a patient with conjunctivochalasis (CCH) and a patient with
hyposecretive dry eye (DE). The 92-kDa pro-MMP-9 and 72-kDa
pro-MMP-2 bands are indicated with arrows. b Gelatin zymogram of tears from a control, 2 patients with allergic eye disease
(AD), 2 patients with blepharitis (BL) and 2 patients with dry
eye.
pg/ml; p = 0.0323) than in the control group (8.15 8 2.73
pg/ml). However, there was no significant difference in
the concentration of IL-6 in dry eye (16.52 8 10.58; p =
0.0814) or allergic eye disease (33.11 8 8.66 pg/ml; p =
0.5508) compared to controls (fig. 2).
318
The concentration of pro-MMP-9 (92 kDa) was measured by ELISA in the samples of tear film from controls
and patients with blepharitis, dry eye, conjunctivochalasis and allergic eye disease. Pro-MMP-9 levels were significantly elevated in blepharitis (2.5-fold; 58.56 8 30.1
ng/ml; p = 0.013), in allergic eye disease (5.6-fold; 132.33
8 77.99 pg/ml; p ! 0.001), in dry eye (4.1-fold; 97.25 8
49.5 ng/ml; p ! 0.001) and in conjunctivochalasis (5.3fold; 126.40 8 101.97 ng/ml; p ! 0.001), in comparison to
controls (23.61 8 17.4 ng/ml). Thus, this proteolytic enzyme exhibited increased levels in all pathological groups
(fig. 3).
Minimal 92-kDa pro-MMP-9 activity was observed
by gelatin zymography of tear fluid samples taken from
2 normal control subjects (representative samples shown
in fig. 4a, b). Higher levels of pro-MMP-9 were found in
tear fluid samples taken from patients with blepharitis,
allergic eye disease, dry eye and conjunctivochalasis
(fig. 4a, b). The strongest bands were observed in conjunctivochalasis and allergic eye disease, in keeping with
the ELISA results of figure 3. In fact, a general correlation
between the quantitative data measured by ELISA and
the gelatinolytic activity revealed by zymography can be
observed (order of enzyme activities: conjunctivochalasis
1 allergic eye disease 1 dry eye 1 blepharitis).
Ophthalmic Res 2008;40:315–321
We characterized 4 inflammatory-type ocular surface
disorders in terms of the concentration of the proinflammatory molecules IL-1, IL-6 and pro-MMP-9 present in
tears, with the aim to analyze the implication of these
proteins in each disorder.
In addition to pro-MMP-9, that is the inactive form of
MMP-9, we also studied its active form by zymography.
We found that these different molecules are selectively
implicated in each type of inflammation, raising the possibility of a biochemical diagnosis of ocular surface disorders in the future.
In patients with blepharitis, the concentration of proMMP-9 with respect to control was significantly elevated; however, this increase was smaller than those differences found in the other studied pathologies. Blepharitis
is characterized by tear film instability, which leads to a
reduction of nonpolar lipids, fundamentally sphingomyelin [14, 15], in turn, provoking conjunctival inflammation and tear evaporation. Evaporative dry eye which is
associated with blepharitis may be the cause of the increase in the concentration of ECM degrading enzymes,
Acera /Rocha /Vecino /Lema /Durán
since in this disorder the other analyzed molecules do not
appear to play a relevant role. Nevertheless, lid margin
inflammation is associated with the liberation of large
quantities of irritating lipids by the meibomian glands
[16].
We found a significant increase in pro-MMP-9 levels
in patients with allergic eye disease with respect to control subjects. This finding may be due to an increased
production of collagen in allergic processes. In fact, as
reported Leonardi et al. [17], histamine, as well as the majority of allergic mediators, can increase collagen production by conjunctival fibroblasts.
Although the IL-6 level in the tears of patients with
allergic eye disease was increased compared with the
controls (33.11 8 8.66 vs. 8.15 8 2.73 pg/ml), it was not
statistically significant.
In hyposecretory dry eye, which is characterized by
reduced tear production and a decreased tear clearance,
pro-MMP-9 enzyme is accumulated and activated subsequently in the tears. In addition, the accumulation of other substances, such as the zonula occludens protein 1 and
occludin binding protein, which are components of the
basal membrane, acts as a stimulus for MMP-9 overexpression [18, 19]. Moreover, tears from dry eyes exhibit an
increase in levels of inflammatory cytokines such as IL-1
and a reduction in anti-inflammatory factors such as lactoferrin [4]. Pflugfelder et al. [20] observed increased levels of IL-1 and MMP-9 in cultures of human corneal
epithelium, concluding that this cytokine was the most
potent inducer of MMP-9 mRNA synthesis, leading to an
increase in pro-MMP-9 synthesis by the corneal epithelium and activated MMP-9 in the tear. Our results show
a substantial increase in pro-MMP-9 levels in the tears of
patients with hyposecretory dry eye. However, in the
present study, the concentration of IL-1 was found to be
similar to that of the control group. This finding may be
due to the increase in the concentration of this enzyme at
an intracellular level, whereas we measured levels of this
interleukin in tears. Our results also corroborate those of
Narayanan et al. [21], who did not find significant differences in the tears of patients with moderate dry eye with
respect to control. We included only patients with hyposecretive dry eye not associated with Sjögren syndrome.
Consequently, IL-1 may not be a good marker of inflammation in the tears of individuals with hyposecretory dry
eye without Sjögren syndrome. Nevertheless, this conclusion should not be extrapolated to Sjögren-type dry eye,
since in this case, IL-1 has been claimed to play a role as
an inducer of inflammation [4].
It appears that in dry eye there is an increased production of proinflammatory cytokines such as IL-1 and
TNF- in the conjunctiva [4] and IL-6 in the tears [22].
Although our results show also an increase in IL-6, it was
not statistically significant. This apparent contradiction
can be explained by the high range in the results observed
in all the three studies. Besides, patients in our study did
not suffer from Sjögren syndrome as did some of the patients from the previous studies. The results presented
recently at the International Dry Eye Workshop (2007)
show that levels of IL-1, TNF-, IL-8 and IL-6 are increased in Sjögren syndrome and in rosacea dry eye, but
not in undifferentiated keratoconjunctivitis sicca or in
non-Sjögren dry eye, as measured in tears [23]. One intrinsic limitation of tear analysis is the potential variability of the results in samples obtained from disorders that
may fluctuate even during the day. In all of our patients
we took the tear samples in the morning (9.30 to 11.30
a.m.). Also, as discussed later, the method for the tear uptake may influence the quality of the sample. In any case,
we consider that the role of these cytokines as dry eye
markers has to be reevaluated.
Our most surprising result is the significant increase
in the concentration of IL-1, IL-6 and pro-MMP-9 in
tears from patients with conjunctivochalasis. An alteration in the ECM of the inferior bulbar conjunctiva has
been proposed to occur in conjunctivochalasis, which
would provoke the degeneration of the elastic fibers but
not their fragmentation [24]. The redundant conjunctiva
which appears in conjunctivochalasis suggests that ECM
degradation enzymes may contribute to the pathogenesis
of conjunctivochalasis. We found overexpression of proMMP-9 which could be involved in the degeneration of
gelatin, type I and IV collagen of the basal membrane and
of elastin. As mentioned previously, pro-MMP-9 synthesis is regulated by proinflammatory cytokines such as IL1, which in turn regulate the expression of other cytokines such as IL-6. The high concentrations which we
observed may be due to the accumulation of these proinflammatory markers in tears due to the delay in tear
clearing which occurs in conjunctivochalasis, which in
turn translates into a worsening of ocular irritation. Together, these results strongly corroborate the inflammatory theory of the pathogenesis of conjunctivochalasis [3,
25].
Tear sampling, which is crucial in the study methodology, has its difficulties and limitations. We have used for
tear sampling a Weck cell sponge (Merocel sponge, polyvinyl acetal, ref. 0525, Oasis) which enables optimal tear
volumes to be obtained in a rapid and simple manner. In
Inflammatory Markers in the Tears
Ophthalmic Res 2008;40:315–321
319
contrast to the method of tear extraction using capillaries
(which requires 1 min/l of sample), this method is less
traumatic for the patient and requires less time for the
same amount of tear fluid. Consequently, the method involves less alteration to the relative viscosity of the tear
[13]. A theoretical advantage of the capillaries compared
to the other methods is that it can collect the pure tear.
Nevertheless, López-Cisternas et al. [26] have demonstrated that the results of the electrophoretic protein profiles of tear samples were indistinguishable between polyurethane foam cylinders (similar material to polyvinyl
acetal) and to the one obtained with capillaries.
Other available methods include Schirmer strips [27]
or polyester rods [13], but the former requires the addition of external agents such as buffer solutions. Thus, the
method we report here does not alter the sample either
quantitatively or qualitatively.
We have made previous studies comparing the absorption and release rate of different materials (i.e. polyvinyl acetal and cellulose). The selected material was the
one that allowed us taking a relatively large quantity of
tears and releasing it practically in its totality. We cannot
exclude the adherence of certain molecules to the Merocel sponge. However, this would affect in a similar way all
the tear samples so that it would not cause any alterations
to the measured concentrations.
In conclusion, elevated pro-MMP-9 levels in tears appear to be a pan-marker for the 4 studied ocular surface
disorders, especially allergic eye disease and conjunctivochalasis. Increased IL-6 levels in the absence of IL-1
were found to be associated with blepharitis, whereas elevated IL-6 and IL-1 were characteristic of conjunctivochalasis. These findings illustrate the selective implication of different molecules in each type of inflammation,
bringing us closer to a biochemical diagnosis of ocular
surface disorders in the future. Such an application would
be of tremendous clinical benefit in light of the clinical
difficulties associated with the differential diagnoses of
these pathologies [23].
We consider that there is a high controversy and it is
necessary to obtain a basic knowledge on the mechanism
of inflammation on the ocular surface to develop major
hypotheses on the mechanism that can be tested.
References
1 Tseng SCG, Tsubota K: Important concepts
for treating ocular surface and tear disorders. Am J Ophthalmol 1997;124:825–835.
2 Smith VA, Rishmawi H, Hussein H, Easty
DL: Tear film MMP accumulation and corneal disease. Br J Ophthalmol 2001; 85: 147–
153.
3 Meller D, Li DQ, Tseng SCG: Regulation of
collagenase, stromelysin and gelatinase B in
human conjunctival and conjunctivochalasis fibroblasts by interleukin-1 and tumor
necrosis factor- . Invest Ophthalmol Vis Sci
2000;41:2922–2929.
4 Solomon A, Dursun D, Liu Z, Xie Y, Macri A,
Pflugfelder SC: Pro- and anti-inflammatory
forms of interleukin-1 in the tear fluid and
conjunctiva of patients with dry eye disease.
Invest Ophthalmol Vis Sci 2001; 42: 2283–
2292.
5 Matsumoto K, Ikema K, Tanihara H: Role of
cytokines in pseudomonal keratitis. Cornea
2005;24:43–49.
6 Cayphas S, Van Damme J, Vink A, Simpson
RJ, Billiau A, Van Snick J: Identification of an
interleukin HPI-like plasmacytoma growth
factor produced by L cells in response to viral
infection. J Immunol 1987;139:2965–2969.
320
7 Girolomo ND, Kumar RK, Coroneo MT,
Wakefield D: UVB-mediated induction of
interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells. Invest Ophthalmol Vis Sci 2002; 43: 3430–
3437.
8 Ray A, Tatter SB, Santhanam DC, Helfgott
DC, May LT, Sehgel PB: Regulation of expression of interleukin-6: molecular and
clinic studies. Ann NY Acad Sci 1989; 557:
353–362.
9 Li DQ, Shang TY, Kim H, Solomon A,
Lokeshwar BL, Pflugfelder SC: Regulated expression of collagenases MMP-1, -8, and -13
and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells. Invest Ophthalmol Vis Sci 2003;44:2928–2935.
10 Matsubara M, Girard MT, Kublin CL, Cintron C, Fini ME: Differential role for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodeling cornea.
Dev Biol 1991; 147:425–439.
11 Fukuda K, Fujitsu Y, Kumagai N, Nishida T:
Inhibition of matrix metalloproteinase-3 in
human conjunctival fibroblasts by interleukin-4 or interleukin-13. Invest Ophthalmol
Vis Sci 2006;47:2857–2864.
Ophthalmic Res 2008;40:315–321
12 Luo L, Li DQ, Doshi A, Farley W, Corrales
RM, Pflugfelder SC: Experimental dry eye
stimulates production of inflammatory cytokines and MMP-9 and activates MAPK
signaling pathways on the ocular surface. Invest Ophthalmol Vis Sci 2004; 45: 4293–
4301.
13 Jones D, Monroy D, Pflugfelder SC: A novel
method of tear collection: comparison of
glass capillary micropipettes with porous
polyester rods. Cornea 1997;16:450–458.
14 McCulley JP, Shine W: A compositional
based model for the tear film lipid layer.
Trans Am Ophthalmol Soc 1997;95: 79–88.
15 McCulley JP, Shine W: Meibomian gland
and tear film lipids: structure, function and
control. Lacrimal gland, tear film, and dry
eye syndromes. Adv Exp Med Biol 2002;506:
373–378.
16 Mathers WD: Ocular evaporation in meibomian gland dysfunction and dry eye. Ophthalmology 1993; 100:347–351.
17 Leonardi A, Cortivo R, Fregona I, Plebani M,
Secchi AG, Abantangelo G: Effects of Th2
cytokines on expression of collagen, MMP-1,
and TIMP-1 in conjunctival fibroblasts. Invest Ophthalmol Vis Sci 2003;44:183–189.
18 Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell behavior. Annu
Rev Cell Dev Biol 2001; 17:463–516.
Acera /Rocha /Vecino /Lema /Durán
19 Behzadian MA, Wang XL, Windsor LJ,
Ghaly N, Caldwell RB: TGF-beta increases
retinal endothelial cell permeability by increasing MMP-9: possible role of glial cells in
endothelial barrier function. Invest Ophthalmol Vis 2001;42:853–859.
20 Pflugfelder SC, Solomon A, Dursun D, Li
DQ: Dry eye and delayed tear clearance: ‘a
call to arms’. Lacrimal gland, tear film and
dry eye syndromes. Adv Exp Med Biol 2002;
506:739–743.
21 Narayanan S, Millar WL, McDermott M:
Conjunctival cytokine expression in symptomatic moderate dry eye subjects. Invest
Ophthalmol Vis Sci 2006;47:2445–2450.
Inflammatory Markers in the Tears
22 Yoon KC, Jeong IY, Park YG, Yang SY: Interleukin-6 and tumor necrosis factor- levels
in tears of patients with dry eye syndrome.
Cornea 2007;26:431–437.
23 Gipson IK, Argüeso P, Beuerman R, Bonini
S, Butovich I, Dana R, Dartt D, Gamache D,
Ham B, Jumblatt M, Korb D, Kruse F, Ogawa
Y, Paulsen F, Stern M, Sweeney DF, Tiffany J,
Ubels J, Willcox M: Research in dry eye: report of the research subcommittee of the International Dry Eye Workshop (2007). Ocul
Surf 2007;5:179–193.
24 Meller D, Tseng SCG: Conjunctivochalasis:
literature review and possible pathophysiology. Surv Ophthalmol 1998; 43:225–232.
25 Li DQ, Meller D, Liu Y, Tseng SCG: Overexpression of MMP-3 and MMP-1 by cultured
conjunctivochalasis fibroblast. Invest Ophthalmol Vis Sci 2000;41:404–410.
26 López-Cisternas J, Castillo-Díaz J, TraipeAlonso L, López-Solis RO: Use of minisponges to collect human tear fluid. Cornea
2006;25:312–318.
27 Tomosugi N, Kitagawa K, Takahashi N,
Sugai S, Ishikawa I: Diagnostic potential of
tear proteomic patterns in Sjögren’s syndrome. J Proteom Res 2005;4:820–825.
Ophthalmic Res 2008;40:315–321
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