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Goblet Cell Numbers and Epithelial Proliferation in the Conjunctiva of Patients With Dry Eye Syndrome Treated With Cyclosporine
Kathleen S. Kunert;
Ann S. Tisdale;
Ilene K. Gipson
Arch Ophthalmol. 2002;120:330-337.
ABSTRACT
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Objectives To compare conjunctival goblet cell numbers as well as epithelial turnover
in patients with non–Sjögren syndrome–associated keratoconjunctivitis
sicca (NSS-KCS) and those with SS-KCS before and after 6 months of treatment
with topical cyclosporine A (CsA) ophthalmic emulsion.
Methods Conjunctival biopsy specimens from 16 patients with NSS-KCS and 12 with
SS-KCS were obtained at baseline and after 6 months' therapy with CsA or vehicle
alone. Conjunctival biopsy specimens were also obtained from 11 normal subjects.
Periodic acid–Schiff staining determined the number of goblet cells
present. Immunofluorescence microscopy for Ki-67 localization was used to
evaluate the number of actively cycling cells.
Results Periodic acid–Schiff staining showed fewer goblet cells at baseline
in both dry eye populations when compared with normal subjects (P<.001). After 6 months of CsA treatment, conjunctival biopsy specimens
of both NSS-KCS and SS-KCS groups revealed an increase in goblet cells compared
with baseline (P<.05). More Ki-67–positive
cells were observed in NSS-KCS conjunctiva at baseline than in normal conjunctiva
(P<.05) whereas numbers of these cells in SS-KCS
conjunctiva were similar to normal at baseline. After 6 months of CsA treatment,
conjunctival biopsy specimens of NSS-KCS revealed a decrease in Ki-67–labeled
cells compared with baseline (P<.001). In contrast,
no substantial change was observed for CsA treatment in patients with SS-KCS.
Conclusions Treatment of dry eye syndrome for 6 months with topical CsA resulted
in an increase in goblet cell numbers in patients with NSS-KCS and SS-KCS
and a decrease in epithelial turnover in those with NSS-KCS. Reducing ocular
surface inflammation might have an effect on the proliferative activity of
the epithelium.
INTRODUCTION
KERATOCONJUNCTIVITIS sicca (KCS), or dry eye syndrome, is a frequently
encountered problem in ophthalmologic practice. Keratoconjunctivitis sicca
is characterized by chronic dryness of the corneal and conjunctival surfaces.1 Patients with dry eye syndrome typically have symptoms
of ocular discomfort ranging from irritation to severe pain. Redness, burning,
itching, foreign body sensation, contact lens intolerance, photophobia, and
blurred vision can occur.2 The diagnosis of
dry eye is difficult since it has no single characteristic sign or symptom
and no single diagnostic measure. The recent committee of the National Eye
Institute/Industry Workshop on Clinical Trials in Dry Eyes reported a new
classification system for the various types of dry eye syndrome.3
The 2 major categories are aqueous tear production–deficient and evaporative
dry eye. The aqueous tear production–deficient category includes Sjögren-associated
KCS (SS-KCS) and non-Sjögren associated KCS (NSS-KCS).
In NSS-KCS and SS-KCS, T-cell infiltration of the conjunctiva has been
observed.4-5 Increased levels
of the cytokines tumor necrosis factor (TNF)  , interleukin (IL) 1,
IL-6, IL-8, and IL-10, as well as expression of immune-activation markers
such as HLA-DR, intracellular adhesion molecule 1 (ICAM-1), and CD11a, have
been described in these patients.5-8
This chronic inflammatory environment on the ocular surface is in part responsible
for the characteristic pathologic alterations of the conjunctival epithelium
known as squamous metaplasia,6, 9
which is accompanied by an increase in epithelial stratification, enlargement
of the superficial epithelial cells, and loss of goblet cells in patients
with both NSS-KCS and SS-KCS.4, 9-12
Another feature of SS-KCS is an increase in the epithelial mitotic rate.13 It is not known, however, whether patients with NSS-KCS
have an increased epithelial proliferative rate.
Currently, the only treatments available are palliative, consisting
primarily of lubricating eye drops to supplement a patient's natural tears
or punctal occlusion to improve the residence time of the tears that the patient
can produce. Less frequently, topical corticosteroids and oral doxycycline
are used, particularly for dry eye that results from meibomitis. Attempts
to develop therapeutic treatments for KCS have been difficult due to the limited
understanding of the underlying pathophysiologic mechanisms. Although the
pathology is still not completely understood, there is now sufficient evidence
to suggest that dry eye syndrome is in part the result of an underlying immune-mediated
inflammation affecting the lacrimal gland and the ocular surface.14-19
This hypothesis is further supported by results obtained in human studies
using topical treatment with the immunomodulatory agent cyclosporine A (CsA)
ophthalmic emulsion.5, 8, 20-22
This drug prevents synthesis and/or secretion of several proinflammatory cytokines,
such as TNF-23 and IL-6,24
and has beneficial effects on the underlying inflammatory pathology of dry
eye syndrome.5 Clinical trials using CsA have
shown improvement in objective measures of dry eye syndrome, such as Schirmer
test values and corneal staining.21, 25
Our study was designed to determine whether goblet cell numbers and
the epithelial mitotic rate are altered when comparing patients with NSS-KCS
and SS-KCS and to evaluate whether therapy with topical CsA influences goblet
cell numbers and epithelial turnover in these 2 types of dry eye syndrome.
SUBJECTS AND METHODS
SUBJECTS
Conjunctival biopsy specimens from 16 patients with NSS-KCS and 12 with
SS-KCS were obtained at the baseline visit and after 6 months of twice-daily
therapy with 0.05% or 0.1% CsA or the vehicle alone.25
This subject group was randomly chosen from a double-masked, vehicle-controlled
clinical study designed by Allergan Inc (Irvine, Calif) to investigate the
efficacy and safety of topical CsA in the treatment of moderate to severe
dry eye syndrome.21 The study was conducted
in compliance with Good Clinical Practices, investigational site institutional
review board regulations, sponsor and investigator obligations, informed consent
regulations, and the Declaration of Helsinki. Potential patients signed a
prescreening informed consent and a second written informed consent prior
to actual enrollment.21 The protocol for this
study is described briefly below. Adult patients of either sex were eligible
for participation if they were diagnosed as having moderate to severe KCS
as defined by the following criteria: (1) Schirmer test reading without anesthesia
of less than or equal to 5 mm/5 min in at least 1 eye (if reading was 0 mm/5
min, then Schirmer reading with nasal stimulation had to be greater than 3
mm/5 min in the same eye); (2) sum of corneal and interpalpebral conjunctival
staining of greater than or equal to +5 in the same eye, where corneal staining
was greater than or equal to +2; (3) a baseline Ocular Surface Disease Index26 score of 0.1 with no more than 3 responses of "not
applicable"; and (4) a score of greater than or equal to 3 on the Subjective
Facial Expression Scale.21 Signs and symptoms
must have been present despite conventional management. Individually packaged
preservative-free artificial tears (REFRESH Lubricant Eye Drops; Allergan
Inc) were provided as an adjunctive treatment to be used as frequently as
needed.
Patients were excluded from the study if they had participated in an
earlier clinical trial with CsA ophthalmic emulsion or had used systemic or
topical ophthalmic CsA within 90 days prior to the study. Other exclusion
criteria were the presence or history of any systemic or ocular disorder or
condition (including ocular surgery, trauma, and disease); current or recent
use of topical ophthalmic or systemic medications that could affect a dry
eye condition; known hypersensitivity to any component of the drug or procedural
medications such as stains or anesthetics; required contact lens wear during
the study; recent (within 1 month) or anticipated use of temporary punctal
plugs during the study; permanent occlusion of lacrimal puncta within 3 months
of the study; or if the patients were pregnant, lactating, or planning a pregnancy.
Patients were also excluded if they appeared to have end-stage lacrimal gland
disease (Schirmer reading with nasal stimulation of <3 mm/5 min) or if
their dry eye syndrome was secondary to the destruction of conjunctival goblet
cells or scarring.
A retrospective diagnosis of SS was used with modified criteria reported
by Vitali et al27 to ensure that a consistent
definition of SS was assigned to the patients enrolled. Diagnosis included
presence of at least one of the following autoantibodies in sera: antinuclear
antibody, rheumatoid factor, and SS autoantibodies class SS-A (Ro) and class
SS-B (La). In addition, oral and ocular symptoms were used to classify patients
with SS.
Full-thickness conjunctival biopsy specimens of a standard size (2 x
3 mm) were removed from the "worse" eye by surgeons following standard procedure.
The "worse" eye was defined as the eye with the lowest Schirmer tear test
reading (without anesthesia) and the lowest sum of corneal and interpalpebral
conjunctival staining. If both eyes were comparable, then the right eye was
used. At the baseline visit, the conjunctival biopsy specimen was taken from
the inferonasal quadrant of the bulbar conjunctiva, close to the midline.
At the 6-month visit, the specimen was removed from the same eye but from
the inferotemporal quadrant of the bulbar conjunctiva, also close to the midline.
Conjunctival biopsy specimens from 11 control subjects (generously supplied
by C. Steven Foster, MD, Massachusetts Eye and Ear Infirmary, Boston, Mass)
were also examined. Controls were patients aged 47 to 89 years who were undergoing
ocular surgery for conditions unrelated to ocular surface disease. Exclusion
criteria for controls included evidence of ocular surface disease or of trauma
during the past 6 months, age younger than 18 years, the presence of dry eye
syndrome, or the intake of medications known to affect the ocular surface.
Full-thickness conjunctival biopsy specimens were taken from the eye at the
time of surgery from the superotemporal bulbar region. This site was chosen
based on the article by Kessing28 demonstrating
that this region has goblet cell numbers comparable with superotemporal bulbar
and inferotemporal/nasal bulbar quadrants of the normal human conjunctiva.
Prior to biopsy, standard measures were followed to ensure sterility of the
operated eye, including preparation with a drop of 5% povidone-iodine solution
in the eye followed by the use of 10% povidone-iodine on the skin.
TISSUE PROCESSING FOR PERIODIC ACID–SCHIFF STAINING AND IMMUNOHISTOCHEMISTRY
After removal, biopsy specimens from patients with NSS-KCS and SS-KCS
taken at day 0 and after 6 months of treatment with either CsA or vehicle,
and from controls, were immediately frozen in OCT embedding compound (Tissue-Tek;
Miles Laboratories; Elkhart, Ind) and stored at -80°C until patient-matched
6-month biopsy specimens were obtained and similarly frozen. Six-micrometer
sections were taken from each block, mounted on gelatin-coated slides, and
processed for periodic acid–Schiff (PAS) and immunohistochemistry. To
minimize differences due to experimental conditions, sectioning of tissue
blocks and histological and immunohistochemical experiments were done as pairs
of biopsies, pretreatment and posttreatment.
PAS STAINING TO DETERMINE NUMBER OF GOBLET CELLS
Periodic acid–Schiff staining of conjunctival biopsy specimens
of patients with NSS-KCS, SS-KCS, and control patients was performed by conventional
techniques to determine the numbers of goblet cells present. Image analysis
was performed as previously described.5 Counting
was done in a masked fashion by 2 independent observers. Counts were recorded
for 3 images of conjunctival epithelium from each biopsy specimen (original
magnification x20), and numbers of goblet cells/0.1 mm2 of
epithelium in patients with NSS-KCS, SS-KCS, and normal patients were compared.
IMMUNOHISTOCHEMISTRY FOR Ki-67 NUCLEAR ANTIGEN
Immunohistochemical localization of Ki-67 nuclear antigen—a marker
of actively cycling cells—on conjunctival sections of biopsy specimens
of patients with NSS-KCS, SS-KCS, and control patients was done as previously
described.29 Secondary antibody controls that
omitted the primary antibody for all biopsy specimens were run. As described
previously, labeled cells were counted in all layers overlying 100 basal epithelial
cells.30 Counting was done in a masked fashion
by 2 independent observers. Counts were recorded for 3 images of conjunctival
epithelium from each biopsy specimen (original magnification x20), and
numbers of goblet cells per 0.1 mm2 of epithelium in patients with
NSS-KCS, SS-KCS, and normal patients were compared.
STATISTICAL METHODS
The same statistical methods were applied for goblet cell numbers and
Ki-67–positive cells, comparing NSS-KCS and SS-KCS subpopulations. Baseline
characteristics were tabulated and summarized by patient populations. Overall
differences among patient populations were tested using a 2-way analysis of
variance for continuous variables and the Fisher exact test for categorical
variables. Percent changes in the numbers of goblet cells and Ki-67–positive
cells were summarized using descriptive statistics (ie, sample size, mean,
SEM, minimum, maximum, and median). A 1-way analysis of variance with main
effect for treatment was used to test for differences in percent change from
baseline. If the test for among-group differences in main effect was significant,
then all pairwise comparisons were made. Within-group changes from baseline
were analyzed by the paired t test method.
RESULTS
QUANTITATION OF GOBLET CELLS
In general, PAS staining documented fewer goblet cells at baseline in
both KCS populations than in normal control subjects. After 6 months of treatment
with CsA, conjunctival biopsy specimens of both NSS-KCS and SS-KCS groups
revealed an increase in PAS-stained goblet cells compared with baseline. There
were no differences observed between the 0.05% and 0.01% treatment groups,
and thus, data for the 2 groups were combined. Vehicle treatment, on the other
hand, resulted in a further decrease in the numbers of goblet cells.
No difference in goblet cell patterns was detected between patients
with NSS-KCS and those with SS-KCS (Figure
1 and Figure 2). A decrease
in goblet cell numbers from normal at baseline and an increase from baseline
with CsA treatment was found for both KCS subtypes (Figure 1 and Figure 2). Figure 1 shows micrographs of PAS-stained
conjunctival sections in which the goblet cells are clearly delineated from
the surrounding stratified epithelial cells. Biopsy specimens are shown from
2 patients with NSS-KCS (Figure 1A-D)
and 2 patients with SS-KCS (Figure 1E-H)
taken at baseline and after 6 months of treatment with either CsA or vehicle.
These sections show only a few goblet cells at baseline and an increase in
goblet cells with CsA treatment in both NSS-KCS (Figure 1A and B) and SS-KCS (Figure
1E and F). In contrast, sections from a patient with NSS-KCS (Figure 1C and D) and one with SS-KCS (Figure 1G and H) treated with the vehicle
alone show fewer goblet cells when compared with baseline. Figure 1I shows the distribution of conjunctival goblet cells in
a normal patient. There was a high density of goblet cells observed in comparison
with the baseline biopsy specimens of patients with NSS-KCS and SS-KCS.
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Figure 1. Goblet cells in human conjunctival
biopsy specimens from 2 patients with non–Sjögren syndrome–associated
keratoconjunctivitis sicca (NSS-KCS) (A-D) and 2 patients with SS-KCS (E-H).
A, Patient 1 with NSS-KCS at baseline. B, Same patient after 6 months of treatment
with topical cyclosporine A (CsA) ophthalmic emulsion. C, Patient 2 with NSS-KCS
at baseline. D, Same patient after 6 months of treatment with the vehicle
alone. E, Patient 1 with SS-KCS at baseline. F, Same patient after 6 months
of treatment with CsA. G, Patient 2 with SS-KCS at baseline. H, Same patient
after 6 months of treatment with the vehicle alone. I, Normal goblet cells
in normal human conjunctiva. Periodic acid–Schiff; bar = 50 µm.
All micrographs are the same magnification.
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Figure 2. Goblet cell numbers per 0.1 mm2 of conjunctival epithelium at baseline (A) and percent change from
baseline after 6 months of treatment with either topical cyclosporine A (CsA)
ophthalmic emulsion or the vehicle (B). A, At baseline, goblet cell numbers
(± SEM) are shown for patients with non–Sjögren syndrome–associated
keratoconjunctivitis sicca (NSS-KCS) (n = 16) and SS-KCS (n = 12) in comparison
witha normal patient population (n = 11). B, Percent change from baseline
at month 6 after treatment with either CsA or the vehicle for the numbers
of goblet cells (± SEM) is shown for patients with NSS-KCS and SS-KCS.
Asterisk indicates P<.001; dagger, P<.05.
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At baseline, goblet cell numbers appear to be significantly decreased
in both KCS populations compared with those of normal control subjects (P<.001) (Figure 2A).
When numbers of goblet cells at baseline were compared with numbers after
treatment with either CsA or vehicle, an increase from baseline was found
in the CsA-treated groups. In contrast, there was a reduction from baseline
in the vehicle-treated groups (Figure 2B).
The numbers of goblet cells for the patients with NSS-KCS increased by 234%
from baseline with CsA treatment and decreased by 114% with vehicle treatment.
For the patients with SS-KCS, the numbers of goblet cells increased by 198%
from baseline with CsA treatment and decreased by 75% with vehicle treatment.
Pairwise comparisons favored CsA over the vehicle (P<.001).
The increase from baseline in the numbers of goblet cells within the CsA group
was statistically significant (P<.05) for both
KCS populations.
QUANTITATION OF Ki-67–LABELED CELLS
Results of immunohistochemical analysis documented numerous Ki-67–positive
cells in NSS-KCS conjunctiva at baseline compared with low numbers of Ki-67–positive
cells in normal conjunctiva. In SS-KCS conjunctiva, however, numbers appeared
to be similar to those in normal conjunctiva. After 6 months of treatment
with either concentration of CsA, conjunctival biopsy specimens of patients
with NSS-KCS revealed a decrease in Ki-67–labeled cells compared with
baseline whereas vehicle treatment resulted in an increase in Ki-67–labeled
cells. In contrast, no substantial change was observed for either treatment
in the SS-KCS subset.
There was a difference in the Ki-67 binding pattern between patients
with NSS-KCS and those with SS-KCS (Figure
3 and Figure 4). Figure 3 shows representative sets of immunofluorescence
micrographs for Ki-67–positive cells in conjunctival specimens of 2
patients with NSS-KCS (Figure 3A-D)
and 2 patients with SS-KCS (Figure 3E-H)
taken at baseline and after 6 months of treatment with either CsA or vehicle.
Specific binding was mainly seen in the basal layers of the epithelium. At
the baseline visit, both NSS-KCS patient biopsy specimens (Figure 3A and C) show numerous Ki-67–positive cells whereas
both SS-KCS patient biopsy specimens show only a few Ki-67–labeled cells.
The specimen of the patient with NSS-KCS treated with CsA reveals a reduction
in positive cells at month 6 (Figure 3B),
whereas the biopsy specimen of the patient with NSS-KCS treated with the vehicle
alone (Figure 3D) reveals an even
higher number of Ki-67–positive cells compared with baseline (Figure 3A and C, respectively). Specimens
of the 2 patients with SS-KCS, 1 treated with CsA (Figure 3F) and 1 treated with the vehicle alone (Figure 3H), indicate little difference between Ki-67–positive
cells after 6 months of treatment and at baseline (Figure 3E and G, respectively). Very few Ki-67–labeled cells
are seen in normal conjunctival epithelium (Figure 3I).
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Figure 3. Immunohistochemical localization
of Ki-67 antibody binding on cryosections of human conjunctiva. The antibody
recognizes a nuclear antigen found only in proliferating cells. Representative
biopsy specimens are shown from 2 patients with non–Sjögren syndrome–associated
keratoconjunctivitis sicca (NSS-KCS) (A-D) and 2 patients with SS-KCS (E-H).
A, Patient 1 with NSS-KCS at baseline. B, Same patient after 6 months of treatment
with topical cyclosporine A (CsA) ophthalmic emulsion. C, Patient 2 with NSS-KCS
at baseline. D, Same patient after 6 months of treatment with the vehicle
alone. E, Patient 1 with SS-KCS at baseline. F, Same patient after 6 months
of treatment with CsA. G, Patient 2 with SS-KCS at baseline. H, Same patient
after 6 months of treatment with the vehicle alone. I, Normal Ki-67–labeled
cells in normal human conjunctiva. J, Normal, negative biopsy result in which
the primary antibody was omitted. Periodic acid–Schiff; bar = 50 µm.
All micrographs are the same magnification.
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Figure 4. Ki-67–labeled cells of conjunctival
epithelium at baseline (A) and percent change from baseline after 6 months
of treatment with either topical cyclosporine A (CsA) ophthalmic emulsion
or the vehicle (B). Data are expressed as the number of Ki-67–labeled
cells per 100 basal propidium iodide–labeled cells. A, At baseline,
Ki-67–labeled cells (± SEM) are shown for patients with non–Sjögren
syndrome–associated keratoconjunctivitis sicca (NSS-KCS) (n = 16) and
SS-KCS (n = 12) in comparison with a normal patient population (n = 11). B,
Percent change from baseline at month 6 after treatment with either CsA or
the vehicle for the number of Ki-67–labeled cells (± SEM) is
shown for patients with NSS-KCS and SS-KCS. Asterisk indicates P<.05;
dagger, P<.001.
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At baseline, the number of Ki-67–labeled cells per 100 basal epithelial
cells appeared to be significantly greater in conjunctival biopsy specimens
of patients with NSS-KCS when compared with normal controls (P<.05). In contrast, no significant difference from normal was observed
for the SS-KCS population (Figure 4A).
The number of Ki-67–labeled cells in patients with NSS-KCS decreased
by 73% from baseline with CsA treatment and increased by 178% with vehicle
treatment (Figure 4B). In the patients
with SS-KCS, the number of Ki-67–labeled cells decreased by only 29%
from baseline with CsA treatment and increased by 25% with vehicle treatment.
Pairwise comparisons show a significant difference between CsA and vehicle
treatment for the NSS-KCS (P<.05) but not for
the SS-KCS population. Changes from baseline were significant for CsA (P<.001) and vehicle (P<.05)
of the NSS-KCS but not for the SS-KCS population.
COMMENT
In this study, immunohistochemical analysis was used to evaluate goblet
cell numbers and epithelial mitotic rates in patients with NSS-KCS and those
with SS-KCS at baseline and following treatment with topical CsA or the vehicle.
We demonstrated that treatment of patients with KCS using topical CsA for
6 months resulted in an increase in goblet cell numbers for both patients
with NSS-KCS and SS-KCS, and a decrease in epithelial turnover in patients
with NSS-KCS.
We observed significantly fewer goblet cells at baseline compared with
normal specimens and an increase in goblet cell numbers after 6 months of
treatment with CsA in both KCS subsets. In concordance with our data, several
groups have found a decrease in the numbers of goblet cells in both forms
of aqueous-deficient dry eye.4, 9-12
Goblet cell densities are thought to be very sensitive indicators of ocular
surface disease.31 In eyes with KCS, the first
evidence of ocular surface injury is a decrease in conjunctival goblet cells.
As the disease progresses in severity, goblet cell numbers decrease further,
resulting in squamous metaplasia, enlargement of the epithelial area, and
occasional keratinization of the ocular surface.11
There have also been some attempts to look at changes in goblet cell
numbers in dry eye syndrome following several treatment modalities. These
include therapy with retinol palmitate ophthalmic solution and hypotonic electrolyte
solutions, both demonstrating an increase in goblet cell numbers in patients
with KCS following therapy.32-34
However, comprehensive studies of goblet cell density in patients with dry
eye syndrome treated with topical CsA are lacking. In this study, we demonstrate
a significant increase in goblet cell numbers following topical treatment
with CsA. Treatment with the vehicle alone, on the other hand, leads to an
even further decrease in goblet cells during the 6-month treatment course,
suggesting that the inflammatory process is still ongoing. This implies that
CsA, in reducing ocular surface inflammation, might help to restore conjunctival
goblet cells, which secrete mucins that prevent the formation of dry spots
associated with KCS.
Data from studies of the gastrointestinal tract support this interpretation.
Treatment with keratinocyte growth factor or TNF- antibodies increases
mucus production and restores goblet cells in cases of intestinal inflammation.35-36 Alternatively, CsA may have a direct
effect on goblet cell differentiation. In a human colon adenocarcinoma cell
line, CsA induced a 94% increase in the volume of mucin within goblet cells.37 Our data furthermore suggest that there is no difference
in goblet cell numbers between NSS-KCS and SS-KCS, either at baseline or after
CsA treatment, indicating that both forms of aqueous-deficient dry eye syndrome
may benefit from CsA treatment.
A second finding of this study is that there were differences in the
epithelial mitotic rate between patients with NSS-KCS and SS-KCS at baseline
and after 6 months of treatment with CsA. More Ki-67–positive cells
were observed in NSS-KCS conjunctiva at baseline compared with normal conjunctiva
whereas in SS-KCS conjunctiva, numbers were similar to normal. After 6 months
of CsA treatment, conjunctival biopsy specimens of NSS-KCS revealed a decrease
in Ki-67–labeled cells when compared with baseline. In contrast, no
substantial change was observed for CsA treatment in SS-KCS. It should be
noted, however, that sample sizes for each group were relatively small.
Jones and colleagues3 found an increase
in mitotic rate as shown by bromodeoxyuridine-labeling in SS-KCS. Even though
we also found an increase in the number of proliferating cells in SS-KCS,
this increase was not significant when compared with normal controls. Possible
explanations for this observed difference might be that our study included
a larger sample size and that our control subjects were mostly elderly patients
undergoing cataract surgery as opposed to young human control subjects as
enrolled by Jones and colleagues.
Altered lubrication and drying of the ocular surface in KCS result in
ocular surface damage or epithelial wounding. Chung and colleagues38 have shown that the conjunctival epithelium responds
to corneal wounding by an increase in bromdeoxyuridine-labeled cells in the
bulbar conjunctival epithelium of rats. In addition to the mechanical surface
abrasion secondary to aqueous tear deficiency, local inflammatory processes
contribute to the ocular surface disease associated with KCS. In NSS-KCS as
well as in SS-KCS, conjunctival epithelial and stromal T-cell infiltration
(predominantly CD3+ and CD4+ T lymphocytes) have been shown.4, 6
Furthermore, several studies that focused on conjunctival cytokine expression
in patients with SS-KCS4, 7, 18
demonstrated increased levels of inflammatory cytokines, such as IL-1,
TNF-, IL-6, and IL-8, in the conjunctival epithelium of patients with
SS-KCS compared with that of controls.4, 7, 18
There are data suggesting the involvement of such inflammatory cytokines in
epithelial hyperproliferation. Interleukin 6 and IL-8, for example, have been
reported to influence growth and differentiation of epithelial cells and promote
hyperproliferation of the epidermis in psoriasis.39-40
Furthermore, the concentration of EGF, a cytokine that is capable of inducing
proliferation or differentiation of epithelium, has been reported to be lower
in the tear fluid of patients with KCS.41 Pflugfelder
and colleagues18, 22 also found
a decrease in tear EGF and an increase in the expression of EGF receptors
in the conjunctiva of patients with SS-KCS. Perhaps inflammation increases
the receptor density, making the conjunctival epithelial cells hypersensitive
to mitogens. The exact role of cytokines in KCS, however, and the possible
difference in cytokine profiles between the 2 types of KCS, NSS-KCS and SS-KCS,
remains to be elucidated. Perhaps there are different degrees of conjunctival
inflammation or a different cytokine profile in the 2 types of KCS, which
might in part explain the observed differences in the epithelial mitotic rate
in the NSS-KCS and SS-KCS groups.
In reducing ocular surface abrasion as well as inflammation by decreasing
the production and release of inflammatory cytokines, CsA may help to reconstitute
homeostasis of the conjunctival epithelium, resulting in an increase in goblet
cells as seen in both KCS populations and a decrease in epithelial turnover
as seen in our patients with NSS-KCS. The differential response to CsA treatment
of the 2 types of KCS in terms of epithelial mitotic rate remains unclear,
but different cytokine or growth factor profiles may be responsible for these
differences.
AUTHOR INFORMATION
Submitted for publication May 31, 2001; final revision received November
27, 2001; accepted December 13, 2001.
This project was funded in part by a Sponsored Research Agreement from
Allergan Inc, Irvine, Calif, and by grant R01-EY03306 from the National Eye
Institute of the National Institutes of Health, Bethesda, Md (Dr Gipson).
Corresponding author and reprints: Ilene K. Gipson, Schepens Eye
Research Institute, Boston, MA 02114 (e-mail: gipson{at}vision.eri.harvard.edu).
From the Schepens Eye Research Institute and Department of Ophthalmology,
Harvard Medical School, Boston, Mass.
REFERENCES
| |
1. Gilbard JP. Dry eye disorders. In: A ADMaJF, ed. Principles and Practice of Ophthalmology-Clinical
Practice. Philadelphia, Pa: WB Saunders Co; 1994:257-276.
2. Lubniewski AJ, Nelson JD. Diagnosis and management of dry eye and ocular surface disorders. Ophthalmol Clin North Am. 1990;3:575-594.
3. Lemp MA. Report of the National Eye Institute/Industry Workshop on clinical
trials in dry eyes. CLAO J. 1995;21:221-232.
PUBMED
4. Raphael M, Bellefqih S, Piette JCH, Le Hoang PH, Debre P, Chomette G. Conjunctival biopsy in Sjogren's syndrome: correlations between histological
and immunohistochemical features. Histopathology. 1988;13:191-202.
ISI
| PUBMED
5. Kunert KS, Tisdale AS, Stern ME, Smith JA, Gipson IK. Analysis of tropical cyclosporine treatment of patients with dry eye
syndrome: effect on conjunctival lymphocytes. Arch Ophthalmol. 2000;118:1489-1496.
FREE FULL TEXT
6. Pflugfelder SC, Huang AJ, Feuer W, Chuchovski PT, Pereira IC, Tseng SC. Conjunctival cytologic features of primary Sjögren's syndrome. Ophthalmology. 1990;97:985-991.
ISI
| PUBMED
7. Jones DT, Monroy D, Ji Z, Atherton SS, Pflugfelder SC. Sjogren's syndrome: cytokine and Epstein-Barr viral gene expression
within the conjunctival epithelium. Invest Ophthalmol Vis Sci. 1994;35:3493-3504.
FREE FULL TEXT
8. Turner K, Pflugfelder SC, Ji Z, Feuer WJ, Stern M, Reis BL. Interleukin-6 levels in the conjunctival epithelium of patients with
dry eye disease treated with cyclosporine ophthalmic emulsion. Cornea. 2000;19:492-496.
FULL TEXT
|
ISI
| PUBMED
9. Pflugfelder SC, Tseng SC, Yoshino K, Monroy D, Felix C, Reis BL. Correlation of goblet cell density and mucosal epithelial membrane
mucin expression with rose bengal staining in patients with ocular irritation. Ophthalmology. 1997;104:223-235.
ISI
| PUBMED
10. Ralph RA. Conjunctival goblet cell density in normal subjects and in dry eye
syndrome. Invest Ophthalmol. 1975;14:299-302.
FREE FULL TEXT
11. Nelson JD, Wright JC. Impression cytology of the ocular surface in keratoconjunctivitis sicca. In: Holly FJ, ed. The Preocular Tear Film in Health,
Disease, and Contact Lens Wear. Lubbock: Texas Dry Eye Institute; 1986:140-156.
12. Rivas L, Oroza MA, Murube-del-Castillo AP-E. Morphological changes in ocular surface in dry eyes and other disorders
by impression cytology. Graefes Arch Clin Exp Ophthalmol. 1992;230:329-334.
FULL TEXT
|
ISI
| PUBMED
13. Jones DT, Monroy D, Ji Z, Pflugfelder SC. Alterations of ocular surface gene expression in Sjögren's syndrome. Adv Exp Med Biol. 1998;438:533-536.
ISI
| PUBMED
14. Williamson J, Gibson AA, Wilson T, Forrester JV, Whaley K, Dick WC. Histology of the lacrimal gland in keratoconjunctivitis sicca. Br J Ophthalmol. 1973;57:852-858.
FREE FULL TEXT
15. Damato BE, Allan D, Murray SB, Lee WR. Senile atrophy of the human lacrimal gland: the contribution of chronic
inflammatory disease. Br J Ophthalmol. 1984;68:674-680.
FREE FULL TEXT
16. Pflugfelder SC, Wilhelmus KR, Osato MS, Matoba AY, Font RL. The autoimmune nature of aqueous tear deficiency. Ophthalmology. 1986;93:1513-1517.
ISI
| PUBMED
17. Stern ME, Beuerman RW, Fox RI, Gao J, Mircheff AK, Pflugfelder SC. A unified theory of the role of the ocular surface in dry eye. Adv Exp Med Biol. 1998;438:643-651.
ISI
| PUBMED
18. Pflugfelder SC, Jones D, Ji Z, Afonso A, Monroy D. Altered cytokine balance in the tear fluid and conjunctiva of patients
with Sjogren's syndrome keratoconjunctivitis sicca. Curr Eye Res. 1999;19:201-211.
FULL TEXT
|
ISI
| PUBMED
19. Mircheff AK, Gierow JP, Wood RL. Autoimmunity of the lacrimal gland. Int Ophthalmol Clin. 1994;34:1-18.
20. Laibovitz RA, Solch S, Andriano K, O'Connell M, Silverman MH. Pilot trial of cyclosporine 1% ophthalmic ointment in the treatment
of keratoconjunctivitis sicca. Cornea. 1993;12:315-323.
FULL TEXT
|
ISI
| PUBMED
21. Sall K, Stevenson OD, Mundorf TK, Reis BL CsA Phase 3 Study Group. Two multicenter, randomized studies of the efficacy and safety of cyclosporine
ophthalmic emulsion in moderate to severe dry eye disease. Ophthalmology. 2000;107:631-639. [published correction appears in Ophthalmology. 2000;107:1220].
ISI
| PUBMED
22. Liu Z, Carvajal M, Carothers Carraway CA, Carraway KL, Pflugfelder SC. Increased expression of the type 1 growth factor receptor family in
the conjunctival epithelium of patients with keratoconjunctivitis sicca. Am J Ophthalmol. 2000;129:472-480.
FULL TEXT
|
ISI
| PUBMED
23. Kim WU, Cho ML, Kim SI, et al. Divergent effect of cyclosporine on Th1/Th2 type cytokines in patients
with severe, refractory rheumatoid arthritis. J Rheumatol. 2000;27:324-331.
ISI
| PUBMED
24. Mitruka SN, Pham SM, Zeevi A, et al. Aerosol cyclosporine prevents acute allograft rejection in experimental
lung transplantation. J Thorac Cardiovasc Surg. 1998;115:28-37.
FREE FULL TEXT
25. Stevenson D, Tauber J, Reis BL the Cyclosporin A Phase 2 Study Group. Efficacy and safety of cyclosporin A ophthalmic emulsion in the treatment
of moderate-to-severe dry eye disease: a dose-ranging, randomized trial. Ophthalmology. 2000;107:967-974.
FULL TEXT
|
ISI
| PUBMED
26. Schiffman RM, Christianson MD, Jacobsen G, Hirsch JD, Reis BL. Reliability and validity of the Ocular Surface Disease Index. Arch Ophthalmol. 2000;118:615-621.
FREE FULL TEXT
27. Vitali C, Bombardieri S, Moutsopoulos HM, et al. Preliminary criteria for the classification of Sjogren's syndrome:
results of a prospective concerted action supported by the European Community. Arthritis Rheum. 1993;36:340-347.
ISI
| PUBMED
28. Kessing S. Mucous gland system of the conjunctiva: a quantitative normal anatomical
study. Acta Ophthalmol (Copenh). 1968;:Suppl 95:1+.
29. Francesconi CM, Hutcheon AE, Chung EH, Dalbone AC, Joyce NC, Zieske JD. Expression patterns of retinoblastoma and E2F family proteins during
corneal development. Invest Ophthalmol Vis Sci. 2000;41:1054-1062.
FREE FULL TEXT
30. Thoft RA, Friend J, Kinoshita S, Nikolic L, Foster CS. Ocular cicatricial pemphigoid associated with hyperproliferation of
the conjunctival epithelium. Am J Ophthalmol. 1984;98:37-42.
ISI
| PUBMED
31. Kinoshita S, Kiorpes TC, Friend J, Thoft RA. Goblet cell density in ocular surface disease: a better indicator than
tear mucin. Arch Ophthalmol. 1983;101:1284-1287.
ABSTRACT
32. Kobayashi TK, Tsubota K, Takamura E, Sawa M, Ohashi Y, Usui M. Effect of retinol palmitate as a treatment for dry eye: a cytological
evaluation. Ophthalmologica. 1997;211:358-361.
ISI
| PUBMED
33. Gilbard JP. Dry eye: pharmacological approaches, effects, and progress. CLAO J. 1996;22:141-145.
PUBMED
34. Gilbard JP. Dry eye, blepharitis and chronic eye irritation: divide and conquer. J Ophthalmic Nurs Technol. 1999;18:109-115.
PUBMED
35. Egger B, Tolmos J, Procaccino F, et al. Keratinocyte growth factor promotes healing of left-sided colon anastomoses. Am J Surg. 1998;176:18-24.
FULL TEXT
|
ISI
| PUBMED
36. Arnold JW, Klimpel GR, Niesel DW. Tumor necrosis factor (TNF alpha) regulates intestinal mucus production
during salmonellosis. Cell Immunol. 1993;151:336-344.
FULL TEXT
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