Conjunctival Epithelial Cell Differentiation on ... - Semantic Scholar

Conjunctival Epithelial Cell Differentiation on Amniotic Membrane Daniel Meller1 and Scheffer C. G. Tseng1'2 Amniotic membrane (AM)-reconstructed conjunctival surfaces recover the normal epithelial phenotype with a significantly higher cell density than the control. The present study was undertaken to examine how AM modulates rabbit conjunctival epithelial cell differentiation.

PURPOSE.

METHODS. Rabbit conjunctival epithelial cells (RCEs) were cultured on the basement membrane side of dispase-pretreated AM, with or without seeding rabbit conjunctival fibroblasts (RCFs) on the stromal side. After 7 to 12 days, half of the cultures were raised to the air-liquid interface, and the remainder stayed submerged. A small group of air-lifted cultures containing RCFs was treated with retinoic acid. After 1, 2, and 4 weeks, cultures were terminated and processed for immunostaining with antibodies directed against distinct types of mucins (SMC and AM3), glycocalyx (AMEM2), keratin K3 (AE5), and K12 (AK2). Additionally, western blot analysis was performed for K3 keratin expression. Ultrastructural changes were evaluated by transmission electron microscopy.

In general, RCEs grown on AM were uniformly small, negative to AE5 and AK2 antibodies, and positive to AMEM2 and ASPG1 antibodies. Epithelial stratification and cell polarity with prominent microvilli, tight junctions, and hemidesmosomes were more pronounced in air-lifted cultures. RCEs cocultured with RCFs showed scattered AM3-positive goblet cells, which were not increased by retinoic acid. RESULTS.

CONCLUSIONS. RCES cultured on AM primarily exhibit a nongoblet conjunctival epithelial phenotype. Epithelial stratification and cell polarity, features essential for epithelial differentiation, are promoted by air-lifting. This culture model will be useful for studying how growth and differentiation of conjunctival epithelial cells can be modulated further. (Invest Ophthalmol Vis Sci. 1999;40: 878-886)

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pithelia covering the corneal and the conjunctival surfaces are distinctly different. Although both are nonkeratinized, stratified epithelia, the conjunctival but not the corneal epithelium contains goblet cells. Their distinct epithelial phenotypes can be demonstrated by the expression of different keratin pairs and types of mucin and glycocalyx. For example, the corneal epithelium expresses the keratin K3 and K12 pair, which is distinctly different from the conjunctival epithelium, and keratins expressed by the corneal and conjunctival epithelia are distinctly different from those in the keratinized skin epidermis.1'4 Conjunctival goblet cells secrete From the 'Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute, Miami; and the 2 Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida. Supported in part by Public Health Service Research Grant EY06819 from the U. S. Department of Health and Human Services, National Eye Institute, National Institutes of Health, Bethesda, Maryland; an unrestricted grant from Research to Prevent Blindness, New York, New York; and a research fellowship grant (Me 1623/1-1) from the Deutsche Forschungsgemeinschaft (DM), Bonn, Germany. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1998. Submitted for publication September 16, 1998; revised December 3, 1998; accepted December 17, 1998. Proprietary interest category: SCGT has filed a patent for the method of preparation and clinical uses of amniotic membrane. Reprint requests: Scheffer C. G. Tseng, Bascom Palmer Eye Institute, William L. McKnight Vision Research Center, 1638 NW 10th Avenue, Miami, FL 33136.

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a gel-forming mucin, MUC5A/C.5 Conjunctival nongoblet epithelial cells and corneal epithelial cells express two types of membranous mucins, MUC16 and sialomucin complex (SMC),7 and conjunctival nongoblet cells express MUC4 mucin.5 In addition, a set of glycoproteins constituting the glycocalyx has also been described for ocular surface epithelia.8"10 The loss of goblet cells,1 x change of keratin expression,3 and loss of mucin/glycocalyx expression12 are invariably found in squamous metaplasia, a hallmark of different forms of dry eye and ocular surface disorders. Therefore, it is important to understand how epithelial phenotypes of mucin expression and nonkeratinization are modulated to endow the ocular surface with sufficient moisture and maintain ocular surface integrity. As a first step toward these objectives, it is necessary to establish an in vitro culture system to facilitate goblet cell differentiation. Studies have shown that growth and differentiation of conjunctival epithelial cells can be modulated by vitamin A,13 by such matrix components as collagen gel and matrigel,14 and by inclusion of conjunctival fibroblasts in an organotypic culture.15 Amniotic membrane (AM), with its thick basement membrane and avascular stromal matrix, has recently been used successfully for ocular surface reconstruction in a variety of ocular surface disorders.16"23 Impression cytology has shown that the reconstructed conjunctival surface fully recovers its normal conjunctival epithelial phenotype with an average 2-fold increase in epithelial cell density and a 10-fold increase in goblet cell density, compared with densities of those cells in control samples.24 We thus wondered whether AM may be an Investigative Ophthalmology & Visual Science, April 1999, Vol. 40, No. 5 Copyright © Association for Research in Vision and Ophthalmology

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ideal matrix substrate to establish an in vitro culture system. Herein, we report how AM was used as a natural substrate to modulate the epithelial phenotype of rabbit conjunctival epithelial cells (RCEs), with or without cocultured rabbit conjunctiva] fibroblasts (RCFs).

MATERIALS AND METHODS

Animals All procedures were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. New Zealand White rabbits of both sexes aged between 4 and 6 months were used in all experiments. Before euthanasia with an intravenous overdose of sodium pentobarbital, they received an intramuscular injection of 50 mg xylazine hydrochloride and 50 mg ketamine hydrochloride.

Materials

FIGURE 1. Experimental design with AM. (A) A side view showing how the AM was fastened to a culture plate insert with a nonabsorbable suture; (B) a view from above the insert with the AM placed in a 24-well tissue culture plate; (C) illustration of the setup of the in vitro reconstruction system for culturing conjunctival epithelial cells on the basement membrane side and their own fibroblasts on the stromal side of the AM.

Dulbecco1 s modified Eagle's medium (DMEM), HEPES-buffer, trypsin-EDTA, amphotericin B, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). Dispase II and fluorescein isothiocyanate (FITQ- conjugated and affinity-purified goat anti-mouse IgM antibody were obtained from Boehringer Mannheim (Indianapolis, IN). The IgG monoclonal antibody AE5, recognizing the 64-kDa basic keratin K3 was purchased from ICN (Costa Mesa, CA). The mouse monoclonal IgG antibody 15H10 directed against ASPGl of SMC was a kind gift from Kermit Carraway, University of Miami, Florida. The FITC-conjugated goat anti-mouse IgG and IgM antibodies ad-

FIGURH 2. Phase contrast images of the in vitro reconstruction system and of air-lifted cultures with RCEs and RCFs 18 days after air-lifting. (A) One clay after seeding RCFs on the stromal side of the AM; (B) low magnification showing RCFs on the stromal side and RCEs on the basement membrane side at day 4. Note the initial formation of small cohesive epithelial islands on the basement membrane of the AM. The same area of the culture is shown at high magnification with different focus on RCFs (C, black arroivbead and on RCEs D, white star). In (E) low-magnification and (F) high-magnification photos, epithelial cells after 18 days of air-lifting are small, round, and compactly organized, liar, (A, C, D, F) 60 ju.ni; (B, E) 300 jmi.

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FIGURE 3- Semithin sections of air-lifted and submerged 50-day-old RCEs cocultured with RCFs (black stars'). Air-lifting was performed for 34 days. (A) Epithelial stratification and cell polarity were more pronounced in air-lifted cultures with small, round, and cuboidal basal cells (black arrowheads). (B) Submerged cultures showed less stratification and cell polarity. Bar, 25 Jim.

sorbed with human serum proteins, gentamicin, hydrocortisone, mitomycin C, dimethyl sulfoxide, cholera toxin, and insulin-transferrin-sodium selenite media supplement were from Sigma (St. Louis, MO). The mouse monoclonal antibodies AK2 to K12 keratin, 4 ' 25 AM3 to conjunctival goblet cells, 26 and AMEM2 to mucosal epithelial membrane-associated glycocalyx 12 were of the IgM class and were developed in our laboratory. The tissue culture plastic plates (24-well) were from Becton Dickinson (Lincoln Park, NJ). Culture plate inserts used for the three-dimensional reconstruction culture system were from Millipore (Bedford, MA). Isolation and Cultivation of Rabbit Conjunctival Epithelial Cells Immediately after rabbits were euthanatized, the entire sheet of conjunctiva was removed 1 mm from the glandular edge of the tarsal plate and 5 mm from the limbus, and the excessive Tenon's tissue was trimmed off. This sheet was rinsed three times with DMEM containing 50 jug/ml gentamicin and 1.25 jxg/ml amphotericin B, placed on a sterile paraffin sheet, and digested with dispase II (1.2 U/ml in Mg 2+ - and Ca2+-free Hanks' balanced salt solution) for 1 to 2 hours at 37°C in humidified 5% CO 2 . The loosened epithelial aggregates were dispersed from the surface by gentle pipetting several times with the original medium containing dispase II. Loosened RCEs were separated into single cells by a second digestion with 0.1% trypsin and

0.02% EDTA in HBSS for 5 minutes. The enzymatic reaction was blocked with DMEM containing 10% FBS; single RCEs were obtained by slowly passing the cell-containing medium through a 23-gauge needle 7 to 10 times and were collected by centrifugation at 800^ for 4 minutes. Preparation of Rabbit Conjunctival Fibroblasts The remaining stromal conjunctival tissue was dissected into explants of approximately 2 X 2 X 2 mm 3 , placed on 100-mm tissue dishes, and covered overnight with a drop of FBS alone or DMEM containing 10% FBS, the antibiotics listed earlier, and 1 M HEPES buffer. One day later, 10 ml of the same medium was added, and explant cultures were incubated at 37°C in 95% humidity and 5% CO2. The medium was changed every 3 to 4 days. After cells reached subconfluence, RCFs were subcultured with 0.1% to 0.25% trypsin, 0.02% EDTA in HBSS at 37°C with a 1:3 to 4 split for several passages. Three-Dimensional Cultures Using Amniotic Membrane The method of preparation of preserved human AM has been reported. 17 In the present study the tissue samples were kindly provided by Bio-Tissue (Miami, FL). In preliminary experiments it was noted that attachment and growth of RCEs seeded directly on the basement membrane side of the AM were impeded by the devitalized amniotic epithelium. To resolve this problem, the AM was pretreated with dispase (1.2 U/ml in


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Immunostaining Specimens from submerged and air-lifted cultures were removed from the culture insert, and frozen 6-/xm-thick sections were prepared, using ornithine carbamoyltransferase as the embedding medium (Tissue-Tek, Sakura FineTEK, Torrance, CA). Sections were stained with hematoxylin-eosin and periodic acid-Schiff s reagent and subjected to immunofluorescence studies. All sections were preincubated with goat serum (1:1000) to prevent nonspecific staining. After rinsing twice for 5 minutes with PBS, sections were incubated with mouse monoclonal AM3 (1:300), AMEM2 (1:300), AE5 (1:100), AK2 (1:500), and ASGP1 (1:400) for sialomucin complex. After two washings with PBS for 5 minutes, they were incubated with an FITC-conjugated secondary antibody (goat anti-mouse IgG at 1:100 for ASPG1 and AE5; goat anti-mouse IgM at 1:100 for AK2, AM3, and AMEM2). After two additional washings with PBS, sections were mounted with an antifade solution and analyzed with a fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblot Analysis

FIGURE 4. Differences in microvilli of apical cell membrane. Transmission electron microscopy shows that apical microvilli (arroivheacls in A and B, and stars in C) were more pronounced in air-lifted cultures (A) and (B) than in submerged sutures (C). Bars, (A) I fim; (B, C) 0.1

Mg2+- and Ca2+-free HBSS) for 15 to 30 minutes, followed by gentle scraping with a rubber policeman. With a nonabsorbable suture, AM of approximately 1,5 X 1,5 cm2 was sutured onto a culture plate insert with the basement membrane facing up and was placed in a 24-well tissue culture plastic plate (Fig. 1). RCEs were seeded at a density of 1.0 to 1.6 x 105 cells/ culture insert on the basement membrane of the AM. In a subgroup RCFs were seeded at a density of 1.0 X 105 cells/ culture insert on the stromal side of the AM 24 hours before seeding with RCEs. Cells were then cultured in a medium of equal volume of HEPES-buffered DMEM containing bicarbonate and Ham's F12 supplemented with 0.5% dimethyl sulfoxide, 2 ng/ml mouse epidermal growth factor, 5 /xg/ml insulin, 5 jug/ml transferrin, 5 ng/ml selenium, 0.5 /Lig/ml hydrocortisone, 30 ng/ml cholera toxin A subunit, 5% FBS, 50 jLLg/ml gentamicin, and 1.25 jLtg/ml amphotericin B. The cultures were incubated at 37°C in 5% CO2 and 95% air, and the medium was changed every 2 to 3 days. After RCEs reached subconfluence, half of the cultures were air-lifted by lowering the air-liquid interface to the level of the cultured epithelial cells. After cultures were air-lifted, the medium was changed daily. In a subgroup, retinoic acid prepared at a final concentration of 10~H M in dimethylsulfoxide under amber light was applied, with the same cautious light condition used each time the medium was changed.

After 4 weeks, air-lifted and submerged epithelial cultures, with or without cocultured RCFs, were rinsed with PBS for 3 minutes and homogenized in 1 ml 0.5% Triton in 50 mM Tris/HCI (pH 7.4). The insoluble precipitate collected by centrifugation was extracted with 0.5 ml 9 M urea in 50 mM Tris/HCI (pH 7.4), containing a mixture of protease inhibitors. The same volume of the urea-soluble fraction was mixed with 4% sodium dodecyl sulfate (SDS) and 0.6 M dithiothreitol. The samples were subjected to 7% SDS-polyacrylamide gel electrophoresis and immunoblotted with the monoclonal antibody AE5. Normal rabbit corneal epithelium and rabbit conjunctiva! epithelium cultured in air-lifted organotypic cultures4 expressing K3 keratin were used as positive control samples. Amniotic membrane without cultured cells was used as a negative control.

Transmission Electron Microscopy Selected specimens were fixed in 2% glutaraldehyde and processed for conventional transmission electron microscopy. Samples were rinsed in 0.1 M phosphate buffer (pH 7.3), postfixed in 1% osmium tetroxide, and embedded in Epon. Ultrathin sections were cut and conventionally stained with uranyl acetate and lead citrate and examined with an electron microscope (model 420; Philips, Eindhoven, The Netherlands). Semithin sections were stained with 1% methylene blue, 1% azure II, and 1% borax.

RESULTS

Epithelial Morphology RCFs attached to the stromal side of the AM within 4 to 24 hours and exhibited typical morphology (Fig. 2A). Most RCEs also attached to the basement membrane side within 24 hours, predominantly as single cells. After 3 to 4 days, a cohesive cell sheet was formed by small and uniform epithelial cells (Fig, 2B). At high magnification, RCEs and RCFs could be distinguished by their characteristic cell shapes (Figs. 2C, 2D, respectively, when viewed at the same site on different focal

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FIGURE 5- Differences in intercellular contacts. Transmission electron microscopy shows that intercellular contacts among epithelial cells in the stratified layers were eiilianced in air-lifted cultures (A), (B) compared with those in submerged cultures (C), which showed wide intercellular spaces. Intercellular digitation (arrows) and desmosome formation {arrowheads) contributed to a cohesive, compactly organized epithelial sheet in air-lifted cultures. Bars, (A) 0.1 /am; (B, C) 1 fxm.

planes). RCFs were spindle-shaped and were found on the stromal side, whereas RCEs were small and round and were located on the basement membrane side. Under all conditions tested, confluence of epithelial cells was usually observed within 7 to 14 days, when air-lifting was introduced. Air-lifted cultures showed a higher proliferation rate than did submerged cultures, especially when retinoic acid was added (not shown). Stratified cell layers developed primarily from small epithelial cell islands noted in the early stage in air-lifted cultures. Submerged cultures with or without fibroblasts were comparably less proliferative and did not reveal obvious stratification under phase contrast microscopy. The morphology of RCEs did not change significantly in 18 days (Figs. 2E, 2F) or even up to 41 days (not shown) after air-lifting. Most of the RCEs remained uniformly small and showed little signs of senescence (i.e., intracytoplasmic vacuolation or desquamation) for up to 60 days (the longest time tested) of culturing in all groups. In general, RCEs were stratified into more cell layers when air-lifted compared with stratification in those that remained submerged. Furthermore, air-lifted cultures with RCFs exhibited more stratification than those without RCFs. A representative sample of air-lifted and submerged cultures with RCFs after 50 days of culturing is shown in Figure 3. In these samples air-lifting was performed for 34 days after 16 days of submerged culturing. Besides the visualization of more epithelial cell layers, it was noteworthy that basal cells of the air-lifted culture were more cuboidal than those of the submerged cultures.

Transmission electron microscopy showed that air-lifted cultures showed more prominent cell polarity in formation of microvilli on the apical cell membrane, the extent of intercellular interdigitations, and the density of intracellular desmosomes and hemidesmosomes (Figs. 4, 5, 6). Microvilli coated with glycocalyx materials were more pronounced in air-lifted cultures (Figs. 4A, 4B) than were those found in submerged cultures (Fig. 4C). Intercellular contacts among epithelial cells in the stratified layers were enhanced in air-lifted cultures (Figs. 5A, 5B) compared with contacts seen in submerged cultures (Fig. 5C), which showed wide intercellular spaces. This was attributed to an increase in intercellular digitation and desmosome formation that resulted in a cohesive and compactly organized epithelial sheet in air-lifted cultures. Adjacent to the basement membrane of the AM, the air-lifted culture showed more hemidesmosomes (Fig. 6A, 6B) than did the submerged cultures (Fig. 6C, 6D). Furthermore, in air-lifted cultures, basal epithelial cells were typically cuboidal to round shaped, and contained a large number of mitochondria and endoplasmic reticulum (Fig. 6B). Suprabasal cells also had a high content of these organelles (Fig. 5A). Intermediate to superficial cells had a denser amount of cytoplasmic filaments than did basal cells (cf. Figs. 5A and 6A). Superficial epithelial cells became flattened and less electron dense, and in some areas became round to oval shaped with multiple vesicles (Fig. 3A). The latter cell type was not found in the submerged cultures. In air-lifted and submerged cultures, no goblet cells with characteristic ultrastructural features were found.


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J.J FtGURE 6. Differences in formation of hemidesmosomes. Transmission electron microscopy shows that hemidesmosomes (arrows) were more pronounced in air-lifted cultures (A, B) than in submerged cultures (C, D). Bars, (A, C) 0.1 /xm; (B, D) 1 fim.

Epithelial Phenotype To verily the epithelial phenotype, we used a panel of monoclonal antibodies to keratins, mucins, and glycocalyx glycoproteins in immunofluorescence staining. To verify their antigenic epitopes, we first stained normal rabbit ocular surface epithelia in vivo. As reported,27 AE5, which recognizes K3 keratin, stained the full thickness of the corneal epithelium (Tig. 7B), but not the conjunctival epithelium (Fig. 7A). AE5 antibody did not stain the RCEs cultured on the AM, no matter whether air-lifting was conducted or whether RCFs were included (Tig. 8B). This result was further confirmed by western blot analysis (Fig. 8A). Normal in vivo corneal epithelium (labeled as CO in Fig. 8A) and RCEs grown in air-lifted organotypic cultures with RCFs (Fig. 8A, OT and A+F+) were strongly positive for 64-kDa keratin 3, and submerged organotypic cultures were weakly positive for keratin 3 (labeled as Fig. 8A, OT and A—F+). Thisfindingwas consistent with those previously reported/' In contrast, no AM cultures, with or without air-lifting and RCFs, expressed keratin 3 (Fig. 8A). Immunostaining for K12 keratin by AK2 was negative in the normal conjunctival epithelium in vivo and in AM cultures (not shown). ASGP1 recognizes SMC, a newly found transmembrane mucin expressed by corneal epithelium and conjunctival nongoblet epithelial cells in vivo in rats.7 This antibody stained nongoblet epithelial cells and the membranous portion of the goblet celts of the rabbit conjunctival epithelium (Fig. 7C) and most of the RCEs grown on AM (Fig. 8C). AMEM2 recognizes

the mucin-like glycoconjugates found in the glycocalyx of all wet mucosal epithelia, including ocular surface epithelia.10 AMEM2 stained nongoblet epithelial cells of the rabbit conjunctiva (Fig. 7D) and stained most epithelial cells of RCEs grown on AM (Fig. 8D), a pattern resembling that of ASGP1 (Fig. 8C). AM3 recognizes conjunctival goblet cell-secreted mucins and stains rabbit conjunctival goblet cells in vivo (Fig. 7E). On cross sections AM3 stained no cells (not shown) but detected a few scattered goblet cells grown on AM when prepared as flatmounts (Figs. 8E, 8F). Air-lifting or the addition of fibroblasts or retinoic acid did not significantly affect the latter observation (not shown). Collectively, these results indicate that the resultant phenotype of RCEs grown on AM retained conjunctival origin and was predominantly that of the nongoblet epithelial cell. Goblet cell differentiation was not frequently observed.

DISCUSSION The major finding reported is that RCEs grown on AM maintain a conjunctival nongoblet epithelial phenotype. Epithelial differentiation and cell polarity are promoted by air-lifting with cocultured RCFs. We think that such a culture system will be useful for exploring how goblet cell differentiation can be promoted further. New information thus derived will be valuable for understanding the mechanism by which squamous metaplasia develops in various ocular surface and tear disorders.

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IOVS, April 1999, Vol. 40, No. 5 in which a native type I collagen gel impregnated with RCFs was used.4 Although the exact mechanism by which air-lifting triggers epithelial stratification remains unknown, a stark contrast in the resultant epithelial phenotype is noted between AM cultures and reported organotypic cultures. Stratified RCEs in air-lifted AM cultures exhibited a genuine conjunctival phenotype with negative AE5 staining and absence of K3 keratin expression by western blot analysis (Fig. 8) and negative AK2 staining (not shown). In contrast, as reported earlier,4 stratified

CO OT

OT

AM

AM

AM AM AM

A+F+ A-F+ A+F+ A-F+ A+F- A-F- no 71 K d -

FIGURE 7. Fmmunostaining using monoclonal antibodies AE5, ASGP1, AMEM2, and AM3 in the normal rabbit ocular surface. AE5 did not stain the conjunctival epithelium (A) but stained the full-thickness corneal epithelium (B). ASGP1 stained the superficial epithelial cells of nongoblet epithelial cells and the membranous portion of some goblet cells (C). AMEM2 stained the superficial cell layers of all nongoblet epithelial cells (D). AM3 stained selectively all goblet cells (E). Bar, (A, B, C, D, E) 100 ju,m.

It has been recognized that the basement membrane facilitates migration of epithelial cells,28 reinforces adhesion of basal epithelial cells,29'30 promotes epithelial differentiation,-^'~3'' and prevents epithelial apoptosis.3536 Collectively, these effects may explain why we observed that RCEs cultured on AM were uniformly small and round and could be maintained for up to 50 days in vitro without notable signs of senescence (Fig. 2). This finding is consistent with the clinical data from impression cytology that AM-reconstructed conjunctival surfaces contain uniformly small basal epithelial cells with twice the cell density of age- and sex-matched normal control samples.24 We are currently testing the hypothesis that the AM can preserve epithelial progenitor cells and prolong their life span. Increased epithelial stratification and cell polarity was observed when RCEs cultured on AM were further exposed to the air-liquid interface, an effect termed air-lifting (Fig. 3). This finding resembles that noted earlier in an organotypic culture,

FIGURE 8. Immunoblotting for AE5 and immunostaining for AE5, ASGP1, AMEM2, and AM3 in RCEs grown on AM (A). Normal corneal epithelium in vivo (CO) and organotypic cultures (OT) with RCFs and air-lifting (A+F+) and OT with RCFs but without air-lifting ( A - F + ) were used as positive control samples. All expressed K3 (64 kDa) keratin. In contrast, all AM cultures with or without air-lifting (A+, A—) or with or without RCFs (F+, F—) did not express K3 keratin. Amniotic membrane alone (no, last lane) was used as a negative control sample. AE5 was negative (B). ASGP1 (C) and AMEM2 (D) stained nearly all epithelial cells of frozen sections intensely. AM3 stained some scattered goblet cells in a flatmount preparation (E, F). Bar, (B, C, D, E) 100 /xm; (F) 25 jam.


RCEs in air-lifted organotypic cultures showed positive AE5 staining and K3 keratin expression, and submerged organotypic RCEs cultures showed weakly positive keratin expression,findingsconsistent with those in our previous report.4 No AM cultures expressed K3 keratin (Fig. 8) or K12 keratin (not shown). Although the K3 and K12 keratin pair has been considered the cornea-type differentiation marker,2'27 our reported data point out that K12 keratin is more specific than K3 keratin in this regard.4 K4 and K13 keratins, which are thought to be specific for nonkeratinized stratified epithelia, are also expressed in the conjunctival epithelium more abundantly than in the corneal epithelium.37'38 They were not investigated in the present study. Taken together, these results indicate that AM cultures permit RCEs to express more conjunctival epithelial phenotype than do organotypic cultures. This difference may be attributable to the unique property of the basement membrane of the AM. The basement membrane has been described by Kurpakus et al.33 to influence the phenotype and cytokeratin expression of ocular surface epithelia. We also observed that air-lifting further promoted expression of such ultrastructural features as microvilli of the apical membrane, intercellular junctions, and density of desmosomes and hemidesmosomes (Figs. 4, 5, 6). Because the resultant epithelial phenotype responded positively ASGP1 and AMEM2 antibodies (Fig. 8), markers for nongoblet epithelial cells, we think that such morphologic changes of epithelial stratification and polarity are therefore correlated with the biochemical phenotype of the nongoblet conjunctival epithelial phenotype. Future studies are needed to determine which component or components in the basement membrane are responsible for controlling the genetic expression of such an epithelial phenotype. In the currently defined culture conditions, goblet cell differentiation marked by positive AM3 staining was occasionally found (Fig. 8). It has been recognized that in vitro differentiation of goblet cells requires stringent culture conditions in different epithelial tissues (for review see reference 15). Soluble factors such as calcium and thyroxine promote epithelial differentiation in general.39"0 Vitamin A13 and factors increasing cyclic adenosine monophosphate production, such as cholera toxin, are known to induce an increased secretion of mucin.4' In contrast, hydrocortisone has been shown to inhibit the differentiation of goblet cells in duodenal explants.40 In the present study, addition of retinoic acid did not stimulate goblet cell differentiation. Besides soluble factors, the extracellular matrix may play an important role. When the AM is used as a natural substrate, cultured tracheal epithelial cells predominantly express cilia without goblet cells.42 This result, resembling that described in the present study, indicates that the basement membrane alone may not be sufficient to induce goblet cell differentiation. Because AM-reconstructed conjunctival surfaces actually show a 10-fold increase in goblet cell density compared with density in the control sample, determined by impression cytology,24 we wonder whether additional mesenchymal elements are needed to promote goblet cell differentiation. This view is suggested by studies of tracheal epithelial cells, which lose goblet cell differentiation when cultured on plastic dishes but reexpress goblet cells when reseeded on the denuded and devitalized tracheal lumen.43'44 This finding strongly suggests that stromalfibroblastsmay play an important role in modulat-

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ing goblet cell differentiation. Our preliminary data indicate that RCFs added to the same side of the basement membrane promoted goblet cell differentiation much more than when added to the stromal side (Meller and Tseng, unpublished results). Therefore, future studies will be directed toward studying whether the thick basement membrane of the AM may have impeded the epithelial-mesenchymal interaction needed to support goblet cell differentiation. Acknowledgments The authors thank Karl Meller from the Department of Cytology, Institute of Anatomy, Bochum, Germany, for preparing the specimen for ultrastructural analysis and Kermit Carraway from the Department of Cell Biology and Anatomy, University of Miami, Florida, for providing the monoclonal antibody to the sialomucin complex ASGP1.

References 1. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins. Patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:ll-24. 2. Galvin S, Loomis C, Manabe M, Dhouailly D, Sun T-T. The major pathways of keratinocyte differentiation as defined by keratin expression, an overview. Adv Dermatol. 1989;4:277-299. 3. Tseng SCG, Hatchell D, Tierney N, Huang AJW, Sun T-T. Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. J Cell Biol. 1984;99:2279-2286. 4. Chen WYW, Mui M-M, Kao W\V-Y, Liu C-Y, Tseng SCG. Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K12 keratin is restricted to corneal epithelium. Curr Eye Res. 1994;13:765-778. 5. Inatomi T, Spurr-Michaud SJ, Tisdale AS, Zhan Q, Feldman ST, Gipson IK. Expression of secretory mucin genes by human conjunctival epithelia. Invest Ophthalmol Vis Sci. 1996;37:l6841692. 6. Inatomi T, Spurr-Michaud SJ, Tisdale AS, Gipson IK. Human corneal and conjunctival epithelia express MUC1 mucin. Invest Ophthalmol Vis Sci. 1995;36:1818-1827. 7. Price-Schiavi SA, Meller D, Jing X, Carvajal ME, Tseng SCG, Carraway KL. Sialomucin complex at the rat ocular surface: a new model for ocular surface protection. Biochem J. 1998;335:457463. 8. Jones DT, Monroy D, Pflugfelder SC. A novel method of tear collection: comparison of glass capillary micropipettes with porous polyester rods. Cornea. 1997; 16:450-458. 9. Watanabe H, Fabricant M, Tisdale AS, Spurr-Michaud SJ, Lindberg K, Gipson IK. Human corneal and conjunctival epithelia produce a mucin-like glycoprotein for the apical surface. Invest Ophthalmol Vis Sci. 1995;36:337-344. 10. Pflugfelder SC, Tseng SCG, Yoshino K, Monroy D, Felix C, Reis BL. Correlation of goblet cell density and mucosal epithelial membrane mucin expression with rose bengal staining in patients with ocular irritation. Ophthalmology. 1997;104:223-235. 11. Tseng SCG. Staging of conjunctival squamous metaplasia by impression cytology. Ophthalmology. 1985;92:728-73312. Pflugfelder SC, Tseng SCG, Sanabria O, et al. Evaluation of subjective assessments and objective diagnostic tests for diagnosing tearfilm disorders known to cause ocular irritation. Cornea. 1998; 17: 38-56. 13- Clark JN, Marchok AC. The effect of vitamin A on cellular differentiation and mucous glycoprotein synthesis in long-termrattracheal organ cultures. Differentiation. 1979;l4:175-183. 14. Tsai RJF, Tseng SCG. Substrate modulation of cultured rabbit conjunctival epithelial cells. Invest Ophthalmol Vis Sci. 1988;29: 1565-1576. 15. Tsai RJF, Tseng SCG, Chen CK. Conjunctival epithelial cells in culture-growth and goblet cell differentiation. Prog Retinal Eye Res. 1997;l6:227-24l.

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