©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neolactoglycosphingolipids, Potential Mediators of Corneal Epithelial Cell Migration (*)

Noorjahan Panjwani (1) (2) (3)(§), Zheng Zhao (1) (2), Sameer Ahmad (1) (2), Zhantao Yang (1) (2), Firoze Jungalwala (2) (4), Jules Baum (2)

From the (1) New England Eye Center and Departments of (2) Ophthalmology and (3) Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111 and the (4) E. K. Shriver Center, Waltham, Massachusetts 02254

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 (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.


INTRODUCTION

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 (Le)() antigens (Gal1-4[Fuc1-3]GlcNAc1-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) .


EXPERIMENTAL PROCEDURES

Preparation of Migrating and Nonmigrating Corneal Epithelia

Migrating and nonmigrating corneal epithelia were analyzed using in vivo, organ culture, and cell culture methods.

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.


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.

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.

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-HO 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% HO) (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.

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 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.

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 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.


RESULTS

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.




DISCUSSION

The present study demonstrates that during corneal epithelial cell migration, the level of paragloboside (nLc) 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 (Gal1-4GlcNAc-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) .

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 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.

nLcs also serve as precursors for the synthesis of Le 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.

In the present study, sialosyl Le 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) .

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-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.

The nLc 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.

In brief, the present study has demonstrated that the levels of three neolacto-GSLs, nLc, 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants EY07088 and EY09349. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Ophthalmology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6776; Fax: 617-636-0348.

The abbreviations used are: Le, 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.

N. Panjwani and Z. Zhao, unpublished observations.


ACKNOWLEDGEMENTS

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-GSL standard, and to Dr. Denise K. H. Chou also of the E. K. Shriver Center for providing paragloboside and mAb 7A.


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