(Investigative Ophthalmology and Visual Science. 1999;40:3138-3146.)
© 1999
by The Association for Research in Vision and Ophthalmology, Inc.
Mucocutaneous Junction As the Major Source of Replacement Palpebral Conjunctival Epithelial Cells
Jonathan D. Wirtschafter1,2,3,
Jeffrey M. Ketcham1,
Robert J. Weinstock1,
Tara Tabesh1 and
Linda K. McLoon1,4
From the Departments of
1 Ophthalmology,
2 Neurology,
3 Neurosurgery, and
4 Neuroscience, University of Minnesota, Minneapolis.
 |
Abstract
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PURPOSE. The conjunctival epithelium performs an important role in the
homeostasis and integrity of the eye. To protect the integrity of the
ocular surface, these cells must be replaced from locally concentrated
or randomly distributed foci of stem cells. These slow-cycling stem
cells produce transient amplifying cells that undergo further divisions
before becoming mature conjunctival epithelial cells. In the current
study, the source of palpebral conjunctival cells was determined.
METHODS. Adult rabbits were injected intraperitoneally with bromodeoxyuridine
(BrdU) at a dose of 50 mg/kg body weight and killed after 1, 3, 5, and
7 days and 2 months. The orbital contents and eyelids were exenterated
en bloc, frozen to maintain the orientation between the eyelids and
globe, and sectioned in a parasagittal plane. Random midglobe sections
were stained for the presence of proliferating cell nuclear antigen
(PCNA). Additional sections were immunostained to detect BrdU-labeled
conjunctival epithelial cells. BrdU-positive cells were counted in a
series of 0.4-mm zones from the mucocutaneous junction of the eyelid,
through the fornix and bulbar conjunctiva. A second set of rabbits
received daily injections of BrdU for 2 or 4 weeks followed by a
2-month BrdU-free period before death and processing.
RESULTS. In all eyelid sections examined, there was a focus of
PCNA-positive cells in the mucocutaneous junction and a few scattered
PCNA-positive cells along the length of the palpebral conjunctiva
toward the fornix. In both the upper and lower eyelids, the peak
concentration of BrdU-labeled cells/0.4-mm zone was located at
progressively greater distances from the mucocutaneous junction in the
animals killed at 1, 3, and 5 days respectively and was unidentifiable
by 7 days. A focus of BrdU-labeled conjunctival cells remained within 1
to 2 mm of the mucocutaneous junction at all postinjection intervals.
These were always found within one cell height of the basement membrane
in the basal layer of the epithelium. In the long-term studies,
BrdU-labeled nuclei were retained at the mucocutaneous junction.
CONCLUSIONS. The mucocutaneous junction of the conjunctival epithelium is a
source of actively dividing transient amplifying cells that migrate
toward the fornix at a rate of approximately 1.7 mm/d with a transit
time of approximately 6 days. Long-term retention of label at the
mucocutaneous junction indicates that slow-cycling stem cells are
present at this location. It appears that most palpebral conjunctival
epithelial stem cells are located near the mucocutaneous junction.
These results are not necessarily at variance with previous studies,
but they diminish the relative importance of the forniceal region in
palpebral conjunctival homeostasis. The mucocutaneous junction may
provide a therapeutically significant source of replacement
conjunctival cells.
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Introduction
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The conjunctival epithelium is critical for maintaining
homeostasis and integrity of the ocular surface. To protect the
integrity of the ocular surface, these cells must be replaced from
locally concentrated or randomly distributed foci of stem cells. A
focal source of replacement cells for the conjunctiva has been ascribed
to the fornix.1
2
However, it is possible that there are
alternative focal concentrations or randomly distributed loci of stem
cells for specific regions of the conjunctiva. The specific source of
palpebral conjunctival cells has not been clearly demonstrated.
Epithelium is a constantly renewing tissue. In the skin, stem cells are
generally diffusely positioned and not focally concentrated. These stem
cells are slow-cycling cells that serve as progenitor cells for the
tissue. In epithelium, a two-stage system has been described for
proliferation.3
Epithelium contains a small population of
stem cells that divide infrequently, and their daughter cells produce
transient amplifying cells, which are a more actively proliferating
population of cells.4
Two compartments, one slow-cycling
and one proliferative, are present in the corneal limbus,5
the region responsible for corneal re-epithelialization after a corneal
injury.6
In the present study, we identified a focus of proliferating cell
nuclear antigen (PCNA)–positive cells at the mucocutaneous junction
region of normal rabbit eyelid. We investigated the location and
migration of transient amplifying cells in the palpebral conjunctiva in
rabbits. Cohorts of palpebral conjunctival transient amplifying cells
were identified and followed by using bromodeoxyuridine (BrdU), the
thymidine analogue, by pulse-labeling of DNA during the S phase of the
cell cycle. A preliminary report of the short-term labeling study has
previously appeared.7
Long-term retention of BrdU label
was examined along the length of the palpebral conjunctiva.
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Materials and Methods
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All experimental procedures conformed to the National Institutes
of Health guidelines for use of animals in research and the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
Animals were purchased from Birchwood Farms (Red Wing, MN) and
housed at the University of Minnesota.
A single injection of BrdU in sterile isotonic saline was administered
intraperitoneally to adult rabbits at a dose of 50 mg/kg body weight.
The rabbits were euthanized 1, 3, 5, and 7 days after the injections.
Three to four eyelids were prepared at each experimental postinjection
interval. The orbital contents including the lids were exenterated en
bloc, and the orbital specimens were cut vertically in the center
(midglobe) of the specimen so that each cross-section would then
include upper lid, globe, and lower lid, all in as close to normal
anatomic configuration as possible. Each half of the tissue specimen
was frozen in a manner that maintained the orientation and continuity
between the globe and the eyelids, and each specimen was sectioned in a
parasagittal plane at 12 µm. The sections were identified according
to their relationship to the midsagittal plane and whether each was in
the lateral or medial half of each eye.
A second series of rabbits was injected daily with BrdU for 2 weeks or
4 weeks, and this was followed by a 2-month BrdU-free period.
The sections were processed for immunohistochemical localization of
PCNA and BrdU. For PCNA immunostaining, after a 15-minute blocking
incubation in normal serum, the sections were incubated with an
antibody to PCNA (Chemicon, Temecula, CA) at a dilution of 1:40 for 1
hour. Sections were reacted using an alkaline phosphatase staining kit
(Vectastain ABC; Vector, Burlingame, CA). For visualization of
BrdU-positive nuclei, sections were incubated in 2 N HCl at 37°C for
1 hour, followed by several rinses in 0.1 M borate buffer. The sections
were incubated in normal horse serum, followed by a 1-hour incubation
with an antibody to BrdU (Boehringer–Mannheim, Indianapolis, IN) at a
dilution of 1:100. The sections were processed using the ABC
kit with avidin-biotin-peroxidase labeling. The sections were reacted
with the heavy-metal–intensified diaminobenzidine procedure.
Representative cross-sections were double immunostained for the
presence of heparan sulfate proteoglycan (HSPG) with an antibody
against HSPG (Chemicon), using the alkaline phosphatase kit and a
substrate kit (Vector Red). All nuclei that incorporated BrdU were
stained black. The HSPG was stained red. Additionally, some
BrdU-immunostained sections were lightly counterstained with
hematoxylin and eosin.
The mucocutaneous junction was defined as the point along the
superficial edge of the lid margin at the transition point from
keratinized stratified squamous epithelial cells of the eyelid skin to
cuboidal epithelial cells of the palpebral conjunctiva. Often the
openings of the tarsal glands could be seen at this junction.
The sections were examined using a light microscope interfaced with a
computer. Morphometric analysis was done with the aid of image analysis
software (Bioquant; R & M Biometrics, Nashville, TN). BrdU-labeled
conjunctival epithelial cells were counted sequentially starting at the
mucocutaneous junction of the eyelid continuously along the
conjunctival surface to the fornix and onto the bulbar surface. Counts
of BrdU-positive conjunctival epithelial cells were totaled for each
full-thickness 0.4-mm zone along the eyelid cross-section. Counts were
also made of BrdU-labeled cells that were within one cell of the
basement membrane of the conjunctival epithelium for each of the 0.4-mm
zones. Results were analyzed with computer software for statistical
significance (Prism and Instat; Graphpad, San Diego, CA).
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Results
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PCNA-positive cells were almost exclusively found in basal
conjunctival epithelial cells within 0.4 mm of the mucocutaneous
junction in rabbit eyelids (Fig. 1A
). A few PCNA-positive cells were found randomly distributed along the
length of the palpebral conjunctiva toward the fornix (Fig. 1B)
. This
was true in both the upper and lower eyelids.
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Figure 1. Photomicrograph of a cross-section through the mucocutaneous junction
region (A) and along the length of the palpebral conjunctiva
(B) of rabbit eyelid stained for the localization of PCNA.
Arrows indicate positive cells in the mucocutaneous
region of the eyelid. These labeled cells were always within 0.3 mm of
the mucocutaneous junction (A). There are no PCNA-positive
cells in this palpebral region approximately midway between the
mucocutaneous junction and the fornix of this eyelid cross-section
(B). This was typical of all eyelids examined for PCNA
immunostaining. Bar, 100 µm.
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After pulse labeling with BrdU, BrdU-labeled nuclei were observed in
almost all the 0.4-mm zones of the conjunctival epithelium including
the palpebral, forniceal, and bulbar regions. However, in the
full-thickness counts including all cells from the basement membrane to
the outermost cells, the position of heavily labeled areas varied as
the distance from the eyelid margin increased after each of the
postinjection intervals examined, that is 1, 3, 5, and 7 days after
BrdU injection. One day after BrdU injection, there were many
BrdU-positive cells found within the first 0.8 mm from the
mucocutaneous junction (Figs. 2
4)
. At 3 days after BrdU injection, the region with the highest
concentration of labeled cells was located within approximately 4 to
5.8 mm from the mucocutaneous junction (Figs. 3
4)
. Five days after the BrdU injection, the forniceal region
(approximately 8–10 mm from the mucocutaneous junction) had the
highest concentration of labeled cells (Fig. 4)
. By 7 days, there was
no identifiable focus of BrdU-labeled cells (Fig. 4)
. There appeared to
be a migration of the peak number of labeled cells/0.4-mm zone at the
lid margin toward the fornix (Fig. 4)
. In the lower lid conjunctiva the
foci of labeling were located progressively greater distances from the
mucocutaneous junction in the animals killed at 1, 3, and 5 days
respectively. The peaks of label at 1, 3, and 5 days were significantly
different from the values at the other zones along the eyelid
conjunctiva. The same pattern of labeling was also found on the upper
lid as the lead edge of labeling moved progressively away from the
mucocutaneous junction over time. No focuses of labeled cells were seen
7 days after the BrdU injection (Fig. 4)
. Although not quantified, in
the short-term BrdU-studies, there appeared to be heavier labeling of
the palpebral conjunctiva than of the bulbar conjunctiva. The lymphoid
tissue also stained heavily for the presence of BrdU at short post-BrdU
survival intervals, indicating that the lymphoid tissue rapidly turns
over within the forniceal conjunctival submucosa.
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Figure 2. Reconstruction of the entire length of the lower lid palpebral and
bulbar conjunctiva in a rabbit who received a single injection of BrdU
one day before death. The black dots are BrdU-positive
nuclei. Paired arrows indicate the peak
focus of label in this eyelid near the mucocutaneous junction. Note
that labeled cells appear along the palpebral conjunctiva, but not in
concentrated foci. Curved arrow indicates lymphoid
tissue, which is plentiful in the forniceal conjunctival submucosa.
Open arrow indicates the mucocutaneous junction. Bar,
0.5 mm.
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Figure 4. Location of BrdU-labeled conjunctival cells. The number of labeled
cells found in full-thickness counts of palpebral conjunctiva at 0.4-mm
increments from the mucocutaneous junction of the lower eyelids 1, 3,
5, and 7 days after a single injection of BrdU on day 0. Note the shift
in peak concentration away from the mucocutaneous junction. To reduce
graph complexity, each point and corresponding line represents the data
from an individual eyelid at each of the postinjection intervals.
*Significantly different from values at other days.
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Figure 3. Reconstruction of the entire length of the palpebral conjunctiva in a
rabbit who received a single injection of BrdU 3 days before death. The
black dots are BrdU-positive nuclei. Paired
arrows indicate the region within which lies the focus of label
in this eyelid. Bar, 0.5 mm.
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In all specimens there was a relatively heavy area of BrdU labeling
that consistently was found within 0.8 mm of the mucocutaneous junction
after all postinjection intervals (Figs. 4
5)
, although it varied quantitatively in animals killed 1, 3, 5, and 7
days after injection with BrdU, with the peak on day 1 (Fig. 5)
.
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Figure 5. BrdU-labeled cells in relation to the eyelid margin. Comparison of the
number of BrdU-labeled cells in the first 0.4-mm zone of the palpebral
conjunctiva starting from the mucocutaneous junction at 1, 3, 5, and 7
days after a single injection of BrdU on day 0 with the corresponding
number for the eighth zone, between 2.8 and 3.2 mm from the
mucocutaneous junction. Note the first zone continues to retain a
relatively high concentration of BrdU-labeled cells. Each point
represents counts from three to four rabbit eyelids. Error bars
indicate SEM.*, **; Pairs of data points with
significant differences at P < 0.05.
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For approximate differentiation of stem cells from transient amplifying
cells and other more mature cells by using location as a criterion,
BrdU-positive cells were counted that were located on or one cell up
from the epithelial layer basement membrane. There was always a focus
of labeled cells at the mucocutaneous junction, but no other peak was
seen along the basement membrane of the palpebral conjunctiva at any of
the post-BrdU survival intervals (Fig. 6)
.
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Figure 6. BrdU-labeled nuclei found within one cell thickness of the basement
membrane of the basal layer of the palpebral conjunctival epithelium 1,
3, 5, and 7 days after a pulse BrdU injection. Note there were always
more labeled nuclei at the mucocutaneous junction at all post-BrdU time
intervals. There was never a peak at any other point along the basal
epithelial layer. Each data point represents the mean of four eyelid
counts. Error bars indicate SEM. *Data are significantly different
from values at other locations along the palpebral conjunctiva.
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Long-term retention of BrdU label was demonstrated to reside primarily
in the mucocutaneous junction after a BrdU-free period of 2 months
(Figs. 7
8)
and in the fornix. Small numbers of BrdU-positive nuclei were found
scattered randomly along the length of the palpebral conjunctiva
basement membrane, but never in the quantity found at the mucocutaneous
junction.
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Figure 7. Photomicrograph of a focus of BrdU-labeled nuclei at the mucocutaneous
junction (arrowhead) and scattered positive nuclei
along the length of the palpebral conjunctiva 2 months after 14 days of
daily BrdU administration. Arrow indicates a focus of
long-term labeled cells in the fornix. Bar, 200 µm.
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Figure 8. Long-term retention of BrdU in palpebral conjunctiva, from the
mucocutaneous junction toward the fornix. After 14 days of daily BrdU
administration, animals were allowed to survive for a 2-month wash-out
period. Each point represents the mean of at least four eyelid counts.
Error bars indicate SEM. *Data are significantly different from
values at other locations along the palpebral conjunctiva.
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Discussion
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The mucocutaneous junction of the palpebral conjunctival
epithelium in rabbits appears to be the source of actively dividing
transient amplifying cells. Transient amplifying cells may go through
several cycles of division, ultimately giving rise to terminally
differentiated palpebral conjunctival epithelial cells. The transient
amplifying cells and their daughter cells migrate toward the fornix to
replace the mature cells that are continuously lost (Fig. 9)
. The long-term labeling study confirms that palpebral conjunctival
stem cells must also be located in the mucocutaneous junction region
where the BrdU-labeled transient amplifying cells are first identified.
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Figure 9. Schema of proposed flow of transient amplifying and daughter cells
within the palpebral conjunctival epithelium. Most cells originate at
the basement epithelium of the mucocutaneous junction and move
vertically toward the surface and horizontally toward the fornix, with
maximum transit time of 7 days. Some epithelial cells arise from
randomly dispersed locations along the rest of the basement membrane.
Black dots represent progenitor cells. Black
dots surrounded by open circles represent
transient amplifying cells. Days after BrdU-pulse injection are
indicated at the top of the drawing. MCJ, mucocutaneous
junction.
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The results of the present study are not necessarily at variance with
the results of the previous studies,1
2
but they diminish
the relative importance that they assign the forniceal region in
palpebral conjunctival epithelial homeostasis. This study did not
completely exclude the possibility that some stem cells could be
randomly and diffusely located throughout the palpebral conjunctiva,
but it appears that this is not the predominant pattern. The present
study differed from prior studies in that the absolute number of
labeled cells was counted in a continuous sequence of zones measuring
0.4 mm from the mucocutaneous junction, whereas previous studies used a
labeling index defined as the percentage of labeled cells per 1000
nuclei. The present strategy of counting labeled nuclei along the
entire length of the palpebral conjunctiva in equally sized regions
also differed from the previous analysis of three distinct regions, the
bulbar, fornix, and palpebral conjunctiva, spatially separated from
each other and not necessarily in a continuous sagittal plane. The
morphometric analysis of the entire palpebral conjunctiva allowed the
detection of movement of the foci of labeled cells from the
mucocutaneous junction toward the fornix over several days. The change
in location of the peak of BrdU-labeled cells strongly supports the
hypothesis that large numbers of palpebral conjunctival calls are
produced at the mucocutaneous junction at the eyelid margin and migrate
toward the fornix. As in epithelium in general, the labeled cells from
the basal layer of the epithelium move to the more superficial layers
of the epithelial sheet. It is well accepted that epithelial cells
migrate, both vertically within the epithelium and horizontally within
the plane of the epithelial sheet. Tissue culture studies have
demonstrated that this horizontal migration can be
substantial.8
9
An overall movement of conjunctival cells
of between 8.4 and 9.2 mm in 5 days would indicate a migration rate of
between 1.68 and 1.84 mm/d. This is compatible with migration rates of
approximately 1.5 mm/d determined by in vitro studies.9
The migration rate of proliferating cells from the corneal limbus in
normal cornea is approximately 100 µm/d.10
After injury,
progenitor cells, many of which may be resting at the limbus, can cover
a denuded cornea within 24 hours,11
a rate of movement of
approximately 6 mm/d.
Some BrdU-labeled epithelial cells remained near the mucocutaneous
junction in all specimens, even 7 days after BrdU pulse labeling. It
may be that some proportion of the transient amplifying cells mature or
migrate more slowly than others or even may not migrate toward the
fornix, and such processes would be reflected by the retention of
labeled cells near the mucocutaneous junction. Stem cells, because they
are slow-cycling cells compared with other cells, retain tritiated
thymidine or BrdU labeling, so the observed stationary focus of
BrdU-labeled cells near the mucocutaneous junction may include some
stem cells. The presence of foci of PCNA labeling in this area suggests
that there is a population of activated stem and transient amplifying
cells in this region. Long-term retention of BrdU-labeling at the
mucocutaneous junction supports the concept that this region, in fact,
contains palpebral conjunctival stem cells.12
It is interesting that 7 days after BrdU pulse labeling no specific
focus of labeling was seen along the length of the palpebral
conjunctiva. There are several possible explanations for the
disappearance of the foci of labeled cells. First, cells may continue
to divide, and this continued division could result in sufficient
dilution of the BrdU label within the nuclei to levels below detection
by antibody staining. However, the duration of the cell cycle in
actively dividing epithelial cells has been demonstrated to be
approximately 28.4 hours.13
This means that in 7 days
approximately 6 cycles of division would have occurred. It is possible,
however, that the duration of the cell cycle could be much shorter in
this region of epithelium. The span of epithelial cell cycle duration
determined in vitro or in tissue-stripping experiments has been
postulated to be between 2014
and 39 hours.15
A cell cycle duration of 20 hours would allow eight cycles of division
within 7 days, which would certainly substantially dilute the BrdU
label and result in its becoming undetectable. This assumes, however,
that all epithelial cells continue to cycle at the same rate as in the
basal layer. Other in vivo studies suggest that in adult epidermis, for
example, the cell cycle time is on the order of 4.8 days.3
If this were the case in the palpebral conjunctiva, only a single cell
cycle would have occurred during this same period. It is more likely
that the loss of the migrating focus of labeled cells is due to their
transit time—that is, their movement upward and diagonally through the
thickness and along the surface of the conjunctival epithelium to the
time when they are sloughed. This contention is supported by the fact
that the focus of labeled cells at 3 and 5 days after a pulse BrdU
injection is not found along the basement membrane (Fig. 6)
. Thus, the
cells that had divided on day 0 had a transit time of between 6 and 7
days and could not be recognized beyond the completion of their transit
time. This transit time is shorter than that of skin, which has been
shown to have a transit time of between 10 and 14 days.16
However, in contrast to skin, the palpebral conjunctiva is exposed to a
great deal of friction that occurs during repeated eye blinks. The
average eye-blink rate is 8 to 12 per minute. This constant rubbing of
two opposing epithelial layers is a relatively unique feature of the
eyelids and could easily result in an increased sloughing rate compared
with other epithelial sheets.
The mucocutaneous junction plays a vital role in the embryologic
development of the eye and is a very active site during all stages of
eyelid development. The leading edge of the developing eyelid in a
mouse at embryonic day 15 forms from a loose aggregation of cells
growing out of each future lid across the corneal surface. This is the
future site of the mucocutaneous junction and supports the concept of
cell migration from this area.17
18
Stem cells are usually located in relatively thick areas of epithelium
in those regions where there is variation in thickness. The
mucocutaneous junction epithelium is relatively thick (approximately
seven cell layers) compared with the rest of the conjunctiva. In some
epithelial surfaces of the human body, it has been shown that the stem
cells responsible for producing daughter cells that go on to replace
lost epithelium reside in certain clusters or zones deep within that
epithelium, rather than having random distribution throughout the
epithelium. Examples of this architecture occur in the rete ridges of
the palmar epithelium,4
the follicular epithelium at the
bulge area of hair follicles,19
20
the epithelium of the
corneal limbus,5
6
and the base of the crypts in the
epithelium of the small intestines.21
Also, stem cells
tend to be located in areas of increased pigmentation. Similar to the
corneal limbus, the mucocutaneous junction is more highly pigmented
than the remainder of the conjunctiva, and this pigmentation may help
to protect the stem cells from the harmful effects of UV radiation, as
has been postulated for other areas that contain epithelial stem
cells.6
The transient amplifying cells demonstrated in the present study point
to the mucocutaneous junction as the location of the stem cells for the
palpebral conjunctiva. Long-term retention of BrdU label after multiple
injections followed by a 2-month BrdU-free chase period resulted in
foci of BrdU-labeled nuclei in the mucocutaneous junction in these
eyelids.12
This confirms the presence of slow-cycling
cells at this location. The corneal limbus is thought to harbor stem
cells for both the cornea and the perilimbal conjunctiva. Most squamous
cell tumors of the conjunctiva arise presumably from a single clone of
transformed stem cells in the perilimbal region corresponding to the
interpalpebral fissure.22
Moreover, the much rarer
squamous cell tumors (dysplasia, carcinoma in situ, and invasive
squamous cell carcinoma) of the palpebral conjunctiva invariably
involve the mucocutaneous junction,7 thus supporting the
concept that the mucocutaneous junction is the predominant location of
the stem cells for the palpebral conjunctiva.
The clinical significance of these observations requires further study.
There are a number of disorders for which replacement conjunctiva is
needed. Currently such replacement is obtained as autografts of healthy
conjunctiva or other mucous membranes.23
24
New
technologies make possible the in vitro production of artificial (skin)
epithelial and fibroblast replacement materials. It is possible that
the mucocutaneous junction might provide a therapeutically significant
source of replacement conjunctival cells. It may be that these cells
could be stimulated to be more productive in situ or used as a source
of stem and transient amplifying cells for the in vitro production of
artificial conjunctiva.
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Acknowledgements
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The authors thank E. Lee Stock for critical reading of the
manuscript.
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Footnotes
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Supported in part by Grant EY07935 from the National Eye Institute, the
Minnesota Lions and Lionesses, and an unrestricted grant from Research
to Prevent Blindness to the Department of Ophthalmology, University of
Minnesota.
Submitted for publication March 10, 1999; revised June 28, 1999; accepted July 14, 1999.
Commercial relationships policy: N.
Presented in part at the annual meetings of the Association for
Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May,
1995, and May, 9–14, 1999, and at the annual meeting of the
American Ophthalmologic Society, May, 1997.
Corresponding author: Jonathan D. Wirtschafter, Department of
Ophthalmology, University of Minnesota, Room 374 LRB, 2001 Sixth Street
SE, Minneapolis, MN 55455. E-mail: wirtsch{at}maroon.tc.umn.edu
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Abstract
Introduction
