From the
Cell migration is a fundamental process of wound repair in
biological systems. In an attempt to identify plasma membrane
glycoconjugates which mediate cell migration, migrating and
nonmigrating rabbit corneal epithelia were analyzed for reactivity with
monoclonal antibodies (mAbs) specific for unsubstituted
N-acetyllactosamine (mAb 1B2), Le
The presence of fully functioning intact epithelium on the
surface of the cornea is essential for the proper functioning and
transparency of this tissue. Re-epithelialization following injury and
various surgical procedures takes place by the migration of adjacent
cells over the injured area
(1, 2) . Delayed
re-epithelialization and failure of the migrated epithelium to remain
adherent to the substratum are fundamental causes of debilitating
clinical conditions such as persistent or recurring epithelial defects
and corneal ulceration
(3, 4) . Although the molecular
events which mediate migration of epithelium over a wound and adhesion
of epithelium to the underlying substratum have not been well defined,
it is generally accepted that plasma membrane glycoconjugates of
corneal epithelium are involved in these
events
(5, 6, 7, 8, 9, 10) .
Various lectins have been shown to either inhibit attachment of corneal
epithelial cells to denuded basal lamina or inhibit the cellular
spreading of the attached cells on the basal lamina
(10) . Other
studies have shown that while some lectins such as Ricinus communis agglutinin I inhibit wound closure
(5) , fucose-specific
lectins such as Ulex europaeus agglutinin I and Lotus
tetragonolobus stimulate re-epithelialization of rabbit corneal
wounds in organ culture
(46) .
A number of studies conducted
using mouse embryo cells, brain cells and lymphocytes have provided
evidence that cell surface glycoconjugates containing neolacto-series
carbohydrate chains are involved in intercellular adhesion as well as
cell-matrix
interactions
(11, 12, 13, 14, 15, 16, 17) .
For example, (i) homotypic interactions between neolacto carbohydrate
chains containing Lewis
To study the migration of corneal epithelium in
vivo, New Zealand rabbits (2-3 kg) (Millbrook Farms,
Amherst, MA) were anesthetized by an intramuscular injection of
ketamine. Proparacaine eye drops were applied to the cornea as a
topical anesthetic, one eye of each animal was proptosed, and the
central cornea was outlined with an 8-mm surgical trephine. The
epithelium within the demarcated area was removed with a Beaver blade,
and the corneas were allowed to heal in vivo for 2 to 3 days.
Re-epithelialization was monitored daily by staining with fluorescein.
Wound size progressively reduced with time (Fig. 1). The
contralateral eye, treated with the topical anesthetic, but unscraped,
served as a control. For immunohistochemical studies, the animals were
sacrificed 24 h after wounding, and the corneas were processed by
preparing 6-µm thick cryosections for staining with anti-GSL
monoclonal antibodies. All animal treatments in this study conformed to
the Association for Research in Vision and Ophthalmology Resolution on
the Use of Animals in Vision Research and the recommendations of the
NIH Guide for the Care and Use of Laboratory Animals.
To
analyze migrating and nonmigrating corneal epithelium in cell culture,
epithelial fragments from 3 to 4 corneas were placed in 100-mm dishes
and incubated in SHEM media
(21) . Within 3 days, approximately
30-50% of each dish was populated with cells that were migrating
away from the explants. These cells were designated
``migrating'' corneal epithelial cells. Within 10 to 12 days,
90-95% of the dish was populated with contact-inhibited,
polygonal cells, designated ``nonmigrating'' corneal
epithelial cells. When ready for harvesting, the cells were washed
extensively with phosphate-buffered saline (PBS), dislodged using a
cell scraper in 0.5 ml of PBS, placed into screw-cap tubes on ice,
lyophilized, and processed for preparation of glycolipids. For
immunohistochemical analyses, cells were cultured on glass slides
instead of in cell culture dishes. Migrating and nonmigrating cells
prepared by tissue culture yielded a protein value of 1.7 mg and 7 mg
per 100-mm dish, respectively.
To test the effect of mAbs and GSLs on
corneal epithelial cell migration in a cell culture model of wound
healing, the method developed by Jumblatt and Neufeld
(31) was
used. Primary cell cultures of rabbit corneal epithelium were treated
with Dispase II (Boehringer Mannheim) and were replated in 24-well
Costar plates at a density of 10
To test the effect of exogenous glycolipids on the
rate of re-epithelialization of wounds of injured corneas in organ
culture, corneas were prepared as described earlier except that 5-mm
instead of 8-mm diameter wounds were made. The injured corneas were
incubated in organ culture in 4-well Nunc plates (1 cornea/well; 1 ml
of serum-free media/well) in the presence of nLc
The present study demonstrates that during corneal epithelial
cell migration, the level of paragloboside (nLc
Since higher levels of nLcs were
found in migrating than in nonmigrating epithelia, it was of interest
to determine whether these glycolipids influence migration of corneal
epithelium. A mAb specific for the N-acetyllactosamine
disaccharides of the nLcs, but not several other mAbs, including those
specific for Le
nLcs also serve as precursors for the synthesis of
Le
In the present study, sialosyl Le
Increased levels of nLcs observed in migrating corneal epithelium in
the present study could be due to either blockage of the synthesis of
more complex nLc
The nLc
In brief, the present
study has demonstrated that the levels of three neolacto-GSLs,
nLc
We thank Yanos Kanczler and Rafic Jarrah for technical
assistance. We are also grateful to Drs. Sen-Itiroh Hakomori and Edward
Nudelman of the Biomembrane Institute, Seattle, WA, for providing mAb
2D4, to Dr. Omanand Koul of the E. K. Shriver Center, Waltham, MA, for
providing the Le
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(mAbs 7A and
MMA), and sialyl Le
(mAb CSLEX1) carbohydrate chains of
neolactoglycoconjugates. Immunohistochemical analyses indicated that
regardless of whether the epithelia analyzed were from corneas of
animals in vivo, corneas in organ culture, or cells in tissue
culture, migrating cells stained intensely with mAb 1B2, whereas
nonmigrating cells either did not stain or stained only weakly. mAbs
MMA and 7A stained migrating epithelium as well as basal and middle
cell layers of normal, nonmigrating epithelium. mAb CSLEX1 did not
stain wounded corneas but stained the superficial cell layer of normal
corneal epithelium. Biochemical analyses by TLC immunostaining revealed
the presence of three mAb 1B2-reactive glycosphingolipids (GSL),
neolactotetraosyl-(nLc
, paragloboside), neolactohexaosyl-
(nLc
), and neolacto-octaosylceramide (nLc
) in
migrating epithelia. In contrast, nonmigrating epithelia contained only
trace amounts of these glycolipids. Exogenous addition of
nLc
, but not various other GSLs including a
Le
-GSL (SSEA-1), stimulated re-epithelialization of wounds
in an experimental model of corneal epithelial wound healing. Moreover,
re-epithelialization of wounds was significantly inhibited by mAb 1B2
but not by mAb MMA. The data suggest that neolacto-GSLs of corneal
epithelium may be among the molecules which mediate healing of corneal
epithelial wounds by influencing cell migration.
(Le
)
(
)
antigens
(Gal
1-4[Fuc
1-3]GlcNAc
1-R) have
been shown to mediate intercellular adhesion in early mouse
embryos
(13, 14) , (ii) sialyl Le
molecules
on the surface of lymphocytes serve as ligands for cell adhesion
molecules known as selectins and mediate extravasation of leukocytes
from the circulation to sites of inflammation
(15) , and (iii)
sulfoglucuronylneolactoglycolipids, which are specifically expressed in
the mammalian nervous system, are known to bind to laminin and mediate
cell-matrix interactions
(18) . In the present study, based on
findings that: (i) the levels of polylactosamine GSLs, nLc
,
nLc
, and nLc
(nLcs), are markedly elevated
during corneal epithelial cell migration, (ii) exogenous addition of
nLc
stimulates corneal epithelial cell migration, and (iii)
a mAb specific for nLcs inhibits corneal epithelial cell migration, we
suggest that polylactosamine GSLs of corneal epithelium are among the
molecules which mediate corneal epithelial cell migration following
injury. Part of this work has been reported previously in abstract
form
(47, 48) .
Preparation of Migrating and Nonmigrating Corneal
Epithelia
Migrating and nonmigrating corneal epithelia were
analyzed using in vivo, organ culture, and cell culture
methods.
Figure 1:
Photographs of in
vivo healing corneas used for immunohistochemical studies. The
corneal epithelium from the central 8-mm demarcated area of one eye of
each animal was removed by a blade. The de-epithelialized area was
visualized by fluorescein under a blue light immediately after wounding
(0 h) and after healing in vivo for 27 h and 49 h. To ensure
that all photographs were compared at the same magnification, prior to
taking pictures, a ruler was placed next to the wound. Light areas are cell-free wound areas; dark areas are uninjured
cellular areas. Note that the wound size progressively decreased during
the first 2 days; complete re-epithelialization occurred between days 2
and 3 (not shown). For immunohistochemical studies (see Fig. 2), the
animals were sacrificed 24 h after wounding, and corneas from both eyes
of each animal were excised and processed for preparation of frozen
sections.
To analyze
migrating and nonmigrating corneal epithelia in organ culture, we used
rabbit eyes from Pel-Freez Biologicals (Rogers, AK). The eyes from 8-
to 12-week-old New Zealand rabbits reached our laboratory on wet ice
within 24 h after slaughter. Prior to use, all eyes were examined for
epithelial integrity by fluorescein staining, and approximately 30% of
the eyes lacking an intact corneal epithelium were discarded. For the
preparation of migrating epithelia, the eyes were washed in sterile
saline, the central corneas were demarcated by an 8-mm trephine, and
the epithelium within the trephined area was removed with a No. 15
Bard-Parker blade. The corneas were then excised with a 1- to 2-mm
scleral rim and rinsed with a serum-free medium containing penicillin G
(750 µg/ml), streptomycin (750 µg/ml), amphotericin (12.5
µg/ml), and neomycin (0.44 mg/ml) for 5 to 7 min. The corneas were
again rinsed in saline and incubated in serum-free Eagle's
minimum essential medium containing nonessential amino acids,
L-glutamine, and antibiotics
(19) at 37 °C in a
tissue culture incubator. The healing was monitored by staining the
corneas with methylene blue
(20) . For immunohistochemical
analyses, after a 24-h incubation period, the corneas were processed
for preparing 6-µm-thick cryosections and staining with anti-GSL
mAbs. To prepare epithelia for high performance thin-layer
chromatography (HPTLC)-immunostain analysis, 75 eyes were processed at
a time. After incubating the injured corneas in organ culture for 24 h
as described above, central 8-mm buttons were cut out with a trephine,
and the migrating epithelium was collected by scraping with a No. 15
Bard-Parker blade under a dissecting microscope. The harvested
epithelia were lyophilized and processed for isolation of glycolipids.
At the time of harvesting migrating epithelia for both
immunohistochemical and HPTLC-immunostain analyses, approximately 5- to
6-mm wounds remained de-epithelialized. To ensure minimal contamination
with nonmigrating epithelium, care was taken not to let the corneas
fully heal prior to collection of epithelium. To prepare nonmigrating
epithelia, unscraped corneas were processed concomitantly as described
above. Migrating epithelia prepared by organ culture yielded a protein
value of approximately 1 mg from 50 injured corneas. Protein yield from
nonmigrating epithelium was approximately 10 times greater.
Staining of Frozen Sections of Normal and Wounded Corneas
and Cell Cultures with Anti-glycolipid Antibodies
A panel of
four monoclonal antibodies were used for this study. mAbs 1B2
(22) and MMA (23), which are specific for terminal
N-acetyllactosamine and Le chains, respectively,
were from the hybridoma clones obtained from the American Type Culture
Collection; mAb 7A, also specific for Le
, originally
prepared by Drs. Schwarting and Yamamoto
(24) , was provided by
Dr. Chou, and mAb CSLEX1
(25) , specific for sialylated
Le
, was obtained from Becton-Dickinson. Frozen sections and
cell cultures were treated with 3% hydrogen peroxide (37 °C, 10
min) to block endogenous peroxidase activity. The sections were then
immersed in a solution of mouse liver powder (100 µg/ml PBS) for 10
min, rinsed in PBS, and sequentially incubated with the following: a
monoclonal antibody (mAb CSLEX1, 10 µg/ml; all other mAbs,
undiluted hybridoma fluid, 1 h), biotinylated anti-mouse IgM (Vector
Laboratories, Burlingame, CA; dilution 1:200, 1 h), a freshly prepared
complex of avidin D and biotin-peroxidase (Vector Laboratories) for 30
min and then diaminobenzidine-H
O
reagent for 10
min at 37 °C
(26) .
Isolation of Glycolipids from Migrating and Nonmigrating
Corneal Epithelia Prepared by Cell and Organ Culture
Primary
cell cultures from 20 to 30 dishes (100 mm) and epithelia collected
from approximately 15 unscraped and 100 to 200 injured corneas in organ
culture were each pooled. The glycolipids were prepared from migrating
and nonmigrating epithelia using a modified extraction procedure of
Folch et al.
(27) to prepare a total lipid fraction.
Prior to HPTLC analysis, the samples were subjected to methanolysis
with 0.1 N NaOH in methanol for 1 h at 37 °C to eliminate
diacylphosphoglycerides. The samples were then neutralized with glacial
acetic acid and passed through a reversed phase Bond-Elut cartridge
(28) to remove salts. The glycolipids recovered from the
Bond-Elut cartridge were separated on thin-layer chromatography plates
and analyzed by HPTLC-immunostaining. High Performance Thin-layer Chromatography-Immunostaining-To
identify various neolacto-GSLs of migrating and nonmigrating corneal
epithelia, an HPTLC-immunostaining procedure, as described by Yamamoto
et al.
(24) , was used. Aliquots of the total lipid
fractions derived from various corneal epithelia (equivalent to 0.5 to
2 mg of original protein) and various GSL standards including nLc prepared from human red blood cells
(29) and a
Le
-GSL (stage-specific embryonic antigen-1, SSEA-1)
prepared from rat kidney (30) were chromatographed on aluminum-backed
HPTLC plates (HPTLC, Alufolien, Kieselgel 60; EM Science, Cherry Hill,
NJ) using a solvent system consisting of chloroform:methanol:0.25%
CaCl
, 5:4:1. Following chromatography, the chromatograms
were treated with 0.05% polyisobutylmethacrylate in n-hexane
for 1 min to prevent detachment of the silica gel during subsequent
procedures. The plates were then sequentially incubated with 1% bovine
serum albumin in PBS for 2 h to block nonspecific binding, monoclonal
antibody (undiluted hybridoma fluid) for 20 h at 4 °C, and
peroxidase-conjugated goat anti-mouse IgM (Cappel Laboratories, Durham,
NC; 1:200 dilution in 1% bovine serum albumin in PBS) for 2 h. The
chromatograms were then washed with PBS, and the antibody-reactive
glycolipids were detected with a solution of 4-chloro-1-naphthol
containing hydrogen peroxide (1 ml of 0.3% 4-chloronaphthol + 4 ml
of 0.02 M Tris, 0.5 M NaCl, pH 7.5 + 3 µl of
30% H
O
)
(24) .
Effect of Monoclonal Antibodies and Exogenous Glycolipids
on Corneal Epithelial Cell Migration
In an attempt to determine
the role of neolacto-GSLs in corneal epithelial cell migration and
wound closure, experiments were conducted to determine the effects of
anti-GSL mAbs and exogenous GSLs on the rate of re-epithelialization of
wounds in a cell culture model of corneal epithelial wound healing. A
limited number of experiments were also performed to test the effect of
exogenous GSLs on the rate of re-epithelialization of wounds of injured
corneas in organ culture.
cells/well. The cells were
fed SHEM medium every 2 to 3 days. When the cells reached confluency,
5-mm filter discs (HA filters, Millipore) were placed on the surface of
the culture in each well. The discs were gently pressed against the
cultures with a sterile glass rod and then removed with forceps. The
cultures were then incubated in: (i) serum-free media supplemented with
0.4% bovine serum albumin (SFB media), (ii) SFB media containing
nLc
(0.0005-1.0 µg/ml), the Le
-GSL
(0.0005 to 0.1 µg/ml), ceramide trihexoside (CTH,
globotriaosylceramide, Matreya Inc., Pleasant Gap, PA;
0.0005-0.10 µg/ml), or asialo-G
(Sigma; 0.05
µg/ml), (iii) mAb 1B2, MMA, or 2D4 (specific for
asialo-G
) hybridoma fluid, and (iv) hybridoma culture
media. Prior to use, various mAbs (hybridoma fluids) and hybridoma
culture media were dialyzed against MEM at 4 °C for 16 to 20 h and
then against MEM containing nonessential amino acids,
L-glutamine, and antibiotics for 2 h. To prepare media
containing glycolipids, a known amount of test glycolipids in
chloroform:methanol (2:1) were dried with a stream of N
; an
appropriate amount of the SFB media was added to achieve a desired
concentration, and the samples were probe-sonicated using a Branson
sonifer at a 4.5 constant output setting. The wounded cultures were
incubated in the media containing mAbs or exogenous GSLs for 48 h.
Midway through the 48-h incubation period, the media were changed once.
At the end of the 48-h healing period, the media were removed by
suctioning, and the size of the defect in each well was visualized by
staining with full-strength Giemsa. The stained wounds were then
photographed at a standard distance, and the outlines of the wound
areas were traced on paper from projected images of the stained wounds.
These outlines were digitized and quantitated using Sigma Scan
software, and healing rates were calculated in mm
/h. An
unpaired, two-group Student's t test was used to test
for significance of the data. In all experiments, 5-fluorouracil (10
µg/ml) was added to the media to inhibit cell mitosis so that the
effect of the mAbs and glycolipids specifically on cell migration could
be evaluated.
or control
glycolipids, asialo-G
, and CTH (0.05 µg/ml) for 20 to
24 h. The corneas were then stained with methylene blue to delineate
the remaining epithelial defects. The stained wounds were then
photographed, and wound areas were quantitated as described above using
Sigma Scan software. Epidermal Growth Factor (EGF) (100 ng/ml), which
is known to stimulate corneal epithelial cell migration
(19) ,
was used as a positive control.
Healing of Corneas in Vivo and Organ Culture
In vivo (Fig. 1) as well as in organ culture
(not shown), wound size progressively reduced with time. Complete
re-epithelialization of an 8-mm wound in vivo occurred between
60 and 70 h. Re-epithelialization in organ culture was slightly slower
than in vivo and required 72 to 80 h for complete wound
closure.
Immunohistochemical Staining with Anti-glycolipid
Monoclonal Antibodies
Immunohistochemical staining of frozen sections of normal and
healing corneas indicated that the leading edge of the migrating
epithelium of healing corneas stained intensely with mAb 1B2
(Fig. 2C) while the basal cells of unwounded corneal
epithelium did not react with this antibody (Fig. 2D).
However, in some areas, there was equivocal staining of the plasma
membranes of apical cells of nonmigrating epithelium with mAb 1B2 (not
shown). Similar results were obtained regardless of whether the corneas
were allowed to heal in vivo (n = 3) or in
organ culture (n = 12). Like mAb 1B2, mAb MMA also
stained the leading edge of migrating epithelia of healing corneas
(Fig. 2E, n = 6). However, unlike mAb
1B2, which did not stain normal corneal epithelium, mAb MMA stained
basal and middle cell layers of normal corneal epithelium at the site
of cell-cell and cell matrix interactions (Fig. 2F,
n = 6). Similar results were obtained with both mAbs 7A
and MMA except that staining intensity with mAb 7A, in some cases, was
less than that observed with mAb MMA. mAb CSLEX1 did not stain
migrating corneal epithelium of healing corneas
(Fig. 2G, n = 8) and stained only the
apical cell layer of the epithelium of 4 of the 7 corneas analyzed
(Fig. 2H).
Figure 2:
Immunohistochemical staining of frozen
sections of normal and healing corneas with mAbs 1B2, MMA, and CSLEX1.
A, C, E, and G, healing corneas
stained with hematoxylin, mAb 1B2, MMA, and CSLEX1, respectively;
B, D, F, and H, normal corneas
stained with hematoxylin, mAb 1B2, MMA, and CSLEX1, respectively.
Migrating corneal epithelium in vivo and in organ culture (not
shown, staining pattern indistinguishable from corneas in
vivo), stained positively with mAb 1B2 (C); in contrast,
normal nonmigrating epithelium did not react with this antibody
(D). mAb MMA reacted positively with migrating as well as
nonmigrating epithelia; note that mAb MMA stained basal and middle cell
layers at the site of cell-cell and cell-matrix interactions. mAb
CSLEX1 did not stain migrating epithelium and stained only apical cell
layers of 4 of the 7 corneas analyzed. Arrows indicate leading
edge of migrating epithelium (A, C, E, and
G) and the basal cell layer of normal, nonmigrating epithelium
(B, D, F, and
H).
As was found with corneas prepared by the
in vivo and organ culture methods, migrating cell cultures
reacted intensely with mAb 1B2 (Fig. 3C), whereas
nonmigrating cell cultures reacted poorly with this antibody
(Fig. 3D). mAbs MMA and 7A stained both migrating
(Fig. 3E) as well as nonmigrating
(Fig. 3F) cell cultures. Since basal cells of corneal
epithelium, the in vivo counterparts of cells grown in
culture, did not react with mAb CSLEX1 in all 15 corneas (8 injured, 7
normal), cell cultures were not analyzed for reactivity with this mAb.
Figure 3:
Staining of migrating (sparse) and
nonmigrating (confluent) cell cultures on glass slides with mAbs 1B2
and MMA. A, C, and E, migrating cell
cultures stained with hematoxylin, mAb 1B2, and MMA, respectively.
B, D, and F, nonmigrating cell cultures
stained with hematoxylin, mAb 1B2, and MMA, respectively. Note that (i)
migrating cell cultures (C) stained intensely, whereas
nonmigrating cell cultures (D) stained weakly with mAb 1B2,
and (ii) mAb MMA stained both migrating (E) as well as
nonmigrating (F) cultures.
HPTLC-Immunostaining of Glycolipids
mAb 1B2
Four preparations of total lipid samples
of migrating and nonmigrating epithelial cell cultures were analyzed
for reactivity with mAb 1B2. In the total lipid fraction of migrating
epithelia, three mAb 1B2-reactive glycolipids, nLc,
nLc
, and nLc
were detected (Fig. 4). In
contrast, in nonmigrating epithelium prepared by cell culture, the
largest glycolipid (nLc
) was not detected and the other two
(nLc
and nLc
) were observed only in trace
amounts (Fig. 4). All three mAb 1B2-reactive glycolipids
(nLc
, nLc
, and nLc
) were detected
in higher amounts in migrating than in nonmigrating cell cultures in
all four preparations of epithelia analyzed. HPTLC-immunostaining
experiments of total lipid fractions of epithelia prepared by organ
culture demonstrated that all three mAb 1B2-reactive glycolipids
(components nLc
, nLc
, and nLc
) were
also present in a higher amount in migrating epithelia than in
nonmigrating epithelia (Fig. 4). Two preparations of migrating
and nonmigrating epithelia prepared by organ culture were analyzed with
similar results. By scanning the chromatograms using a Visage 110
computer-assisted scanner (Bio Image, Millipore), it was estimated that
migrating cell cultures contained at least 30 times more nLc
and 10 times more nLc
than nonmigrating cultures.
Migrating epithelia prepared by organ culture contained 5 times more
nLc
and 8 times more nLc
than nonmigrating
epithelia.
Figure 4:
Immunostaining of glycolipids in the total
lipid fractions of migrating and nonmigrating corneal epithelia by mAb
1B2. Migrating and nonmigrating corneal epithelia prepared by both cell
and organ culture techniques were analyzed. Samples derived from 1 mg
and 0.5 mg of original cell protein were chromatographed for mAb 1B2
and orcinol staining, respectively. For standard lanes, 200 ng of
nLc was spotted. Note that regardless of whether the
epithelia were prepared by organ culture or cell culture,
neolactotetraosyl- (nLc
), neolactohexaosyl-
(nLc
), and neolacto-octaosylceramide (nLc
) were
present in a higher amount in migrating epithelium (M)
compared to nonmigrating epithelium (N), whereas the orcinol
staining pattern of migrating and nonmigrating epithelium was similar.
The lower panel shows the densitometric scans of M and N lanes
of the mAb 1B2-stained chromatogram. Broken and solid
lines indicate values of M and N lanes, respectively. Four
immunostained chromatograms of lipid samples from epithelia in cell
culture and two such chromatograms of samples derived from epithelia in
organ culture were scanned, and it was estimated that migrating cell
cultures contained at least 30 times more nLc
and 10 times
more nLc
than nonmigrating cultures. Migrating epithelia
prepared by organ culture contained 5 times more nLc
and 8
times more nLc
than nonmigrating epithelia. (OC,
organ culture; CC, cell culture; S, standard
nLc
.)
mAbs 7A and MMA
Six preparations of total lipid
samples of migrating and nonmigrating cell cultures were analyzed for
reactivity with mAb 7A. In the total lipid fraction of migrating
epithelia, six mAb 7A-reactive components (L1-L6,
Fig. 5
) were detected. All Le-GSLs of corneal
epithelium migrated on a TLC plate slower than a standard
Le
-GSL (SSEA-1) from rat kidney which contains an
oligosaccharide constituted of 8 sugar residues with a globocore
structure
(30) . All six mAb 7A-reactive components were also
detected in the extracts of nonmigrating epithelia but in reduced
amounts compared to migrating epithelia (Fig. 5). For
quantitative analysis, all chromatograms were scanned as described
above, and, on the basis of the average results of the six
immunostained chromatograms, each from a different preparation of cell
cultures, it was estimated that components L1, L2, L3-5, and L6
were present in approximately 6, 2, 3, and 7 times greater amounts,
respectively, in migrating than in nonmigrating epithelia. Lipid
fractions of migrating and nonmigrating epithelia prepared by organ
culture were not analyzed for reactivity with mAb MMA or 7A due to a
limited availability of starting material.
Figure 5:
Immunostaining of glycolipids in the lipid
fractions of migrating and nonmigrating primary cell cultures of
corneal epithelium with mAb 7A. Samples derived from 2.0 mg of original
cell protein were chromatographed on TLC plates, and the GSLs
containing Le epitope were visualized by immunostaining the
chromatograms with mAb 7A. Following immunostaining, the same plate was
stained with orcinol. Six mAb 7A-reactive components
(L1-L6) were detected in the lipid fractions of
migrating cell cultures of corneal epithelium; components L1-L6
were present in higher amounts in migrating than in nonmigrating cell
cultures, whereas orcinol staining patterns of migrating and
nonmigrating cell cultures were similar. Lower panel shows
densitometric scans of M and N lanes of the mAb 7A stained
chromatogram. Broken and solid lines indicate values
of samples of migrating and nonmigrating cells, respectively. Six
immunostained chromatograms, each from a different preparation of cell
cultures, were scanned, and it was estimated that components L1, L2,
L3-5, and L6 were present in approximately 6, 2, 3, and 7 times
greater amounts, respectively, in migrating than in nonmigrating
epithelia. Le
, Le
-glycolipid (SSEA-1);
S, glycolipid standards; CD, ceramide dihexoside;
CT, ceramide trihexoside; aGM
,
asialo-G
.
Effect of Monoclonal Antibodies and Exogenous
Glycolipids on the Rate of Corneal Epithelial Wound Closure
In the cell culture model, mAb 1B2 significantly inhibited
the rate of wound closure (Fig. 6, top panel). In
contrast, mAb MMA and 2D4 had little effect. Since mAb 1B2 was found to
inhibit the rate of wound closure, experiments were conducted to
determine whether the exogenous addition of nLc would
stimulate corneal epithelial cell migration. NLc
stimulated
whereas the Le
-GSL inhibited the rate of wound closure
(Fig. 6, bottom panel). Two control glycolipids, CTH
(Fig. 6) and asialo-G
(not shown) had little effect.
The stimulatory effect of nLc
on the rate of corneal
epithelial wound closure was dose-dependent with the maximal
stimulation at 0.1 µg/ml (Fig. 6). Use of a higher nLc
concentration up to 1.0 µg/ml, did not further stimulate the
rate of corneal epithelial wound closure (data not shown). NLc
was found to stimulate the wound closure rate regardless of
whether the assays were performed in the presence or absence of
5-fluorouracil, a drug that inhibits cell mitosis. As expected from
published studies
(19) , compared to the cultures incubated in
media alone, wound closure rate was significantly faster in the
cultures incubated with EGF which was used as a positive control.
Figure 6:
Effect of anti-glycolipid mAbs and
exogenous GSLs on the corneal epithelial wound closure rate in a cell
culture model. Primary cultures of rabbit corneal epithelium were
treated with Dispase and subcultured in 24-well Costar plates. Cultures
were allowed to reach confluency, and 5-mm-diameter wounds were made as
described in the text using filter discs. The cultures were then
incubated in the presence or absence of monoclonal antibodies (top
panel) or glycolipids (bottom panel) for 48 h, and the
remaining acellular wound areas were visualized by staining with Giemsa
stain. For calculation of healing rates, wound areas were photographed
and quantified using Sigma Scan software. A value of 1.0 was assigned
to the healing rate of culture wounds incubated in media alone. The
values for culture wounds incubated in media containing mAb or
glycolipids are expressed as change in healing rate with respect to
control cultures. Wound cultures incubated in media containing EGF (100
ng/ml) served as a positive control. Mean values ± S.E. are
shown. The wound closure rate was significantly inhibited by mAb 1B2
(p < 0.05, n = 13) but not by mAb MMA
(n = 13) or 2D4 (n = 3). nLc stimulated the wound closure in a dose-dependent manner in the
concentration range of 0.005 to 0.1 µg/ml (solid bars,
lower panel, n = 8 in each group). The
stimulating effect of nLc
reached maximum at 0.1 µg/ml;
no further increase in the rate of wound closure was detected up to 1.0
µg/ml nLc
(not shown). The Le
-GSL was
inhibitory (dot bars, lower panel, n = 4 in each group) and CTH had no effect (shaded
bars, lower panel, n = 4 in each group).
All values reported in this figure are of the experiments carried out
in the presence of 5-fluorouracil. nLc
was found to
stimulate wound closure both in the presence or absence of this drug.
Representative photographs of wound areas from various groups (EGF, 100
ng/ml, PG (nLc
), CTH, and the Le
-GSL, 0.05
µg/ml) after a 48-h incubation period are shown in the lower
panel above the bar graph. Light areas are
remaining acellular wound areas; dark areas are uninjured
cellular areas. 0 h indicates photograph of the wound immediately after
injury. PG, paragloboside
(nLc
).
Having determined that nLc stimulates the
re-epithelialization of wounds in cell culture, it was of interest to
determine whether this glycolipid would also stimulate the
re-epithelialization of injured corneas in organ culture. In all five
experiments, nLc
(0.05 µg/ml) consistently stimulated
wound closure of rabbit corneas (0.64 ± 0.01 mm
/h,
n = 25, p < 0.05). Neither asialo-G
(0.57 ± 0.03 mm
/h, n = 10) nor
CTH (0.57 ± 0.03 mm
/h, n = 6) was
found to influence the corneal epithelial wound closure rate in organ
culture (Fig. 7).
Figure 7:
Effect of exogenous glycolipids on corneal
epithelial wound closure of rabbit corneas in organ culture. The
corneal epithelium from the central 5-mm circular area was removed from
each eye, and the corneas were excised and allowed to heal in
serum-free media containing various glycolipids (0.05 µg/ml) or EGF
(100 ng/ml). After a 24-h healing period, the corneas were stained with
methylene blue to visualize the remaining wounds, and the wound areas
were photographed and quantitated. A value of 1.0 was assigned to the
healing rate of control corneas incubated in media alone, and the
values for corneas incubated in media containing glycolipids are
expressed as change in healing rate with respect to control corneas.
Injured corneas incubated in media containing EGF served as a positive
control. For media, media + EGF, and media + nLc groups, mean values ± S.E. of 5 experiments are indicated.
For media + asialo-G
, and media + CTH groups,
mean values ± S.E. of 2 and 1 experiments, respectively, are
indicated. In each experiment, either a group of 4 or 6 corneas were
used. The wound closure rate was significantly faster in corneas
incubated in media containing nLc
than in media alone
(p < 0.05); percent increases in healing rate in the 5
individual experiments were 15%, 21%, 16%, 11%, and 12%. In contrast,
CTH and asialo-G
, were found to have no influence on the
corneal epithelial wound closure rate. Arrows indicate
representative photographs of wound areas of the healing corneas from
different groups after a 24-h incubation period. Photographs of corneas
from the CTH (not shown) and asialo-G
groups were
indistinguishable.
) and two
related GSLs, nLc
and nLc
, are markedly
elevated. Higher levels of the nLcs (nLc
, nLc
,
nLc
) were found in migrating than in nonmigrating epithelia
regardless of whether the epithelia were prepared by in vivo,
organ culture, or cell culture techniques and whether the analyses were
performed on isolated lipid fractions by HPTLC-immunostaining or on
frozen sections of the cornea by immunohistochemistry. The nLcs belong
to the neolacto family of glycolipids which are characterized by the
presence of N-acetyllactosamine disaccharides
(Gal
1-4GlcNAc-R) in their oligosaccharide
chains
(32) . With the exception of human
erythrocytes
(33) , neutrophils
(34) , and tissues of the
nervous system (35), which contain small amounts of the nLcs, these
glycolipids have thus far not been found in chemically detectable
levels in normal tissues. They are, however, found in high levels in
tumor cells as well as in sera from patients with
cancer
(36, 37) .
and asialo-G
, inhibited
re-epithelialization of wounds in a cell culture model of corneal
epithelial wound healing. Comparison of the rate of corneal epithelial
cell migration in two different models of corneal epithelial wound
healing, in the presence and absence of glycolipids, suggests that the
addition of exogenous nLc
to the culture media stimulates
corneal epithelial wound closure. Many studies have shown that
exogenous glycolipids, when added to media, are taken up by cells,
become constituents of their plasma membranes, and function like those
synthesized by cells
(38) . It is thus likely that the
stimulatory effect of exogenous nLc
on corneal epithelial
wound closure observed in our study may be due to the incorporation of
the added glycolipid into the plasma membranes of the epithelial cells
which in turn may have augmented the effect of the endogenous
glycolipid. It would be of interest to determine whether exogenous
addition of nLc
-derived GSLs such as nLc
and
nLc
, which are not readily available in the purified form,
would also stimulate corneal epithelial sheet migration. In fact,
nLc
and nLc
may have a more potent effect than
nLc
itself because they contain longer carbohydrate chains
and are, therefore, less likely to encounter stearic hindrances. It
should be noted that, although exogenously added glycolipids get
incorporated into the cell membrane, they cannot directly serve as
precursors for the synthesis of complex glycolipids because
glycosylation usually takes place intracellularly in Golgi. However, it
is possible that a feedback mechanism exists in which when elevated
levels of nLc
are present in plasma membranes, newly
synthesized nLc
in Golgi is not transported to the
membranes, but is utilized for the synthesis of complex
nLc
-derived glycolipids including nLc
and
nLc
.
GSLs which have been shown to mediate cell-cell
interactions
(11, 12, 13, 14, 15, 16, 17) .
In the present study, migrating as well as nonmigrating corneal
epithelia were found to react positively with anti-Le
mAbs.
Although biochemical analysis by TLC-immunostaining revealed that
migrating cell cultures contained elevated levels of
Le
-GSLs compared to nonmigrating corneal epithelium, in
immunohistochemical study, migrating corneal epithelium in organ or
cell culture was not found to stain more intensely than nonmigrating
corneal epithelium. This may be due to the presence of
Le
-glycoproteins in higher levels in nonmigrating than in
migrating epithelium. The Le
-GSLs of corneal epithelium are
likely to contain relatively long carbohydrate chains because they
migrated on the TLC plates slower than a standard Le
-GSL
(SSEA-1) from rat kidney which contains an oligosaccharide constituted
of 8 sugar residues with a globocore structure
(30) . The
Le
-GSLs of corneal epithelium may also contain
oligosaccharides with globocore structures especially since they did
not react with mAbs FH4 and FH5 (ATCC, Rockville, MD) which are
specific for di- and trifucosyl Le
(39) ,
respectively.
(
)
As described above,
Le
-related glyconjugates have been shown to mediate
cell-cell interactions. In mouse embryos, the SSEA-1 antigen appears at
the 8-cell stage, i.e. at the time of onset of compaction, and
then disappears at the blastocyst stage
(11) . That the Le
determinant present in SSEA-1 antigen plays an essential role in
cell-cell interactions leading to cell compaction during embryogenesis
has been shown by the studies demonstrating that multivalent
lacto-N-fucopentaose III-lysyllysine conjugates, but not those
of other closely related oligosaccharides, induce individual
blastomeres to round up and decompact the embryos
(13) . Studies
aimed at identifying the mechanism by which cell surface
glycoconjugates containing Le
determinants may mediate
cell-cell interactions suggest that the Le
recognizing
molecules in adjacent cells is Le
itself
(14, 17) , and it is believed that
Le
-Le
interactions occurring between opposing
homotypic cell surfaces mediate cell recognition during early
development. It would be important to determine whether the
Le
-Le
-mediated recognition system is operative
in the cornea. Based on the strategic location of the Le
antigen in normal corneal epithelium at the site of the cell-cell
junction, it appears likely that Le
-glycoconjugates of
corneal epithelium may be among the molecules which mediate cell-cell
adhesion and contribute to the structural integrity and cellular
polarity of normal corneal epithelium. In the studies described herein,
the exogenous addition of the Le
-GSL was found to inhibit
the corneal epithelial wound closure. It is known that as the cells
become migratory in response to injury, intercellular adhesion is
disrupted and the breakage of the intercellular contact is thought to
be important for initiating the stage of re-epithelialization. Thus, if
the exogenous Le
-GSL is capable of inducing the cell-cell
contact in the cell culture model used in this study, an inhibitory
effect on the rate of corneal epithelial wound closure would be
expected. It is tempting to speculate that the nLcs by stimulating and,
the Le
-GSLs by inhibiting, the cell migration may work in
concert to dictate the rate of re-epithelialization of wounds and that
maintaining a fine balance in the relative levels of the two types of
glycolipids may be essential for the healing of the corneal epithelial
wounds.
antigen was
detected only in the apical cell layer of normal corneas suggesting
that the Le
-glycoconjugates we detected in normal corneal
epithelium, at the site of cell-cell and/or cell-matrix interactions,
most probably do not undergo sialylation. The positive reaction of
anti-sialosyl Le
with the apical cell layer of corneal
epithelium may have been caused by mucin-like glycoproteins which are
known to be present in the superficial cell layers of normal corneal
epithelium
(40) . Various nonocular studies have identified
mucin-bound sialosyl Le
carbohydrate chains
(41) .
-derived glycolipids or the expression of a
precursor which is either not found or found only in low levels in
normal nonmigrating corneal epithelium. Sundsmo and Hakomori
(42) compared cell surface glycolipids of polyoma-transformed
NIL cells (NILpy) with those of nontransformed NIL cells and found that
while membranes of transformed cells contain nLc
, those of
nontransformed cells contain sialylated nLc
instead. The
authors suggested that nLc
accumulates in NILpy cells as a
precursor due to blocked synthesis of
sialyl-nLc
(42) . On the other hand, studies by
Holmes et al.(43) have provided evidence suggesting
that the expression of the Le
antigen in fetal colon
epithelium and in adenocarcinomas, but not in normal tissues, is due to
the expression of precursors of Le
(e.g. paragloboside oligosaccharide) which are not found in normal adult
tissues. With regard to the mechanism by which the nLcs may accumulate
in various tissues, recently it has been demonstrated that the
expression of neolacto-GSLs in leukocytes
(44) and in brain (45)
is regulated by the activity of the enzyme
-1-3-N-acetylglucosaminyltransferase which
catalyzes the synthesis of lactotriaosylceramide. It remains to be seen
whether the expression of the N-acetylglucosaminyltransferase
is also up-regulated in corneal epithelium during cell migration.
stimulation of the corneal epithelial wound
closure rate observed in this study is more likely due to increased
cell migration rather than cell mitosis because the stimulatory effect
of this glycolipid on the healing rate was detected even when the
assays were performed in the presence of 5-fluorouracil, a drug that
inhibits cell mitosis. Furthermore, it is well established that
following corneal epithelial injury, mitosis ceases at the wound
periphery and the wound closes largely by migration of the epithelial
sheet over the defect; after the wound closes, mitosis resumes and a
mature stratified epithelium is then formed.
, nLc
, and nLc
, are elevated
during corneal epithelial cell migration. We propose that these
glycolipids, by influencing cell-cell and cell-matrix interactions,
play a role in corneal epithelial cell migration and wound healing. We
further propose that Le
-GSLs of corneal epithelium which
are strategically located at the site of cell-cell junctions are most
likely among the molecules which mediate cell-cell adhesion and
contribute to the structural integrity and cellular polarity of normal
corneal epithelium.
, Lewis
; CTH, ceramide trihexoside; HPTLC,
high performance thin-layer chromatography; mAb, monoclonal antibody;
PBS, phosphate-buffered saline; GSL, glycosphingolipids; SFB,
serum-free medium supplemented with 0.4% bovine serum albumin; SSEA,
stage-specific embryonic antigen; EGF, epidermal growth factor.
-GSL standard, and to Dr. Denise K. H. Chou
also of the E. K. Shriver Center for providing paragloboside and mAb
7A.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.