Complex carbohydrates at the ocular surface of the mouse: An ultrastructural and cytochemical analysis

Ezp. Eye Res. (1984) 39, 19-35

Complex

Carbohydrates An Ultrastructural PETER

Department

of Anatomy,

at the Ocular Surface of the Mouse: and Cytochemical Analysis

A. WELLS

AND LINDA

School of Medicine, Wayne 48201, U.S.A.

D. HAZLETT State University,

Detroit,

Ml

(Received 11 August 1983 and accepted 13 October 1983, New York) Scanning electron microscopy (SEM) of mouse cornea and conjunctiva fixed with picric acidparaformaldehydeglutaraldehyde (PA-P-G) mixture revealed a thin layer of amorphous material covering the microvilli of the cornea1 surface cells. At the transmission electron microscopic (TEM) level, this layer of material stained positively with dialyzed iron, alcian blue and cationized ferritin, all of which are markers for anionic sulfate or carboxyl groups. The cornea1 surface was negative for high iron diamine, which specifically stains sulfate groups. These results indicate that the murine ocular surface is rich in carboxyl groups. Treatment with neuraminidase prior to fixation significantly reduced (P < 9995) cationic ferritin binding, suggesting that most of the carboxyl groups at the ocular surface are associated with sialic acid residues. The cornea1 surface also stained positively at the TEM level when a periodic acid-thiocarbohydrazide-silver protein sequence (PA-T-SP) was applied, This result indicated the presence of periodic acid-Schiff (PAS)-positive glycoprotein and glycolipid at the ocular surface. Key words: cornea; conjunctiva; ocular mucus; precorneal tear film: carbohydrate cytochemistry; electron microscopy.

1. Introduction The precorneal tear film, which covers the anterior surface of the eye, moistens the cornea1 and conjunctival epithelium and may provide a physical and molecular barrier against invading microorganisms and small airborne particles. The tear film consists of three layers: an innermost mucin layer which is secreted by the conjunctival goblet cells, a middle aqueous layer which is secreted by the lacrimal and accessory lacrimal glands and an outer lipid layer which is produced by the meibomian glands (Holly and Lemp, 1977; Friedlaender, 1979). The inner mucin layer is in intimate contact with the microvilli ofthe cornea1 and conjunctival surface cells and aids in maintaining wettability of the surface cells (Holly and Lemp, 1977). A reduction in conjunctival goblet cells and ocular mucus has been observed clinically in cases of cicatricial pemphigoid and Stevens-Johnson syndrome (Ralph, 1975; Holly and Lemp, 1977; Friedlaender, 1979). In these diseases, spreading of the aqueous tear layer is thought to be inhibited by a decrease in goblet cell mucins, and dry eye syndrome may result (Holly and Lemp, 1977; Friedlaender, 1979). Bacterial infection of the cornea and conjunctiva with cornea1 ulceration and perforation may occur as serious complications of dry eye syndrome (Friedlaender, 1979). Recent biochemical studies (Moore and Tiffany, 1979, 1981) have indicated that human ocular mucus consists of high molecular weight glycoproteins which are aggregates of a 200000 MW subunit (GP3M). The GP3M subunit is composed of approximately 70% carbohydrate in the form of oligosaccharide chains covalently linked to a polypeptide backbone rich in serine and threonine (Moore and Tiffany, 1981). Sugar analysis has shown that human ocular mucus contains sialic acid, glucose, galactose, glucosamine, galactosamine, mannose and fucose (Moore and Tiffany, 1981). Please address correspondence to L. D. Hazlett at the above address 9014-4836/84/010019+

17 $03.00/o

0 1984 Academic Press Inc. (London) Limi:ed

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AND

L. I). HA%LE’fT

Mucins produced by gastrointest!inal tract goblet cells have been identified ultrastructurally by staining with alcian blue (Behnke and Zelander, 1970), dialyzed iron (Wetzel, Wetzel and Spicer, 1966) and cationized ferritin (Jersild and Crawford, 1978). Positive staining with these markers is generally attributed to the high sialic acid content of mucins (Spicer and Schulte. 1982; Behnke and Zelander, 1970). In certain organs such as the rectosigmoid colon of the mouse, goblet cell granules have been positively stained with a high iron diamine solution which is specific for sulfated mucins and glycosaminoglycans (Spicer, Hardin and Setser, 1978). Conjunctival goblet cells have been stained at the light microscope (LM) level with the periodic acid-Schiff (PAS) method (Spicer and Meyer, 1960; Srinavasan, Worgul, Iwamoto and Merriam, 1977), alcian blue (Spicer and Meyer, 1960; Srinavasan et al., 1977) and dialyzed iron (Srinavasan et al., 1977). Conjunctival goblet cells are not stained at the LM level with aldehyde-fuchsin which is specific for sulfated mucins (Spicer and Meyer, 1960; Srinavasan et al., 1977). Norn (1972) has shown vital staining of ocular mucus in the inferior conjunctival fornix of man using alcian blue. In the present study, a fixative containing picric acid, paraformaldehyde and glutaraldehyde (PA-P-G) in an acetic acid/Na-acetate buffer (Accinni, Hsu, Spiele and DeMartino, 1974; Lillie, 1965) was employed to preserve ocular mucus for morphological and cytochemical studies. The PA-P-G mixture appears to precipitate the innermost mucin layer of the tear film, and adequately preserves the epithelial surface cells; however, it does not fix the aqueous or oily layers of the precorneal tear film. Murine cornea and conjunctiva fixed with this mixture were observed with scanning electron microscopy (SEM) and subjected to a battery of cytochemical procedures in order to analyze complex carbohydrates (polysaccharides and glycoconjugates) at the ocular surface.

2. Materials Primary

and Methods

aldehyde jixation

Swiss-Webster mice (eight to 12 weeks old) were anaesthetized with sodium pentobarbital (40 mg kg-‘) and the eyes enucleated and immersed in a fixative consisting of 618 y0 pi&c acid, 2 0/0paraformaldehyde, 1 o/0glutaraldehyde and 61 M Na-acetate buffer pH 7.0 (PA-P-G fixative) (Lillie, 1965; Accinni et al., 1974). Whole eyes were fixed for 1 hr at 25°C. SEM After the primary PA-P-G fixation, whole eyes were washed with 61 M Na-acetate buffer and postfixed 3-5 hr with 1% 0~0, in 61 M Na-cacodylate. The eyes were dehydrated with a series of ethanol solutions, washed with Freon 113 (E. I. DuPont de Nemours and Co.) and critical-point dried using Freon 13. The eyes were mounted on aluminum stubs using silver paint, and a thin layer of gold (approx 15 nm) was evaporated on to the eyes with a sputter coater. The eyes were viewed and photographed with an ETEC Autoscan scanning electron microscope operating at 10 kV. Dialyzed

iron staining

Following the PA-P-G fixation, whole eyes were washed with @l M Na-acetate buffer pH 7-O and incubated with a dialyzed iron reagent (Wetzel et al., 1966). To prepare this reagent, three parts of a dialyzed iron stock solution are diluted with one part glacial acetic acid. Whole eyes were incubated for 3 hr at 25°C in the dilute iron solution, pH 2.0. Control eyes were incubated with @9 o/0NaCl diluted with glacial acetic acid (three parts NaCl : one part glacial acetic acid). After treatment with dialyzed iron, the eyes were washed with 61 M Na-acetate and postfixed with 1 o/0 090, in 61 M Na-cacodylate buffer.

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Cationized ferritin After the primary PA-P-G fixation, eyes were washed with 605 M Na-phosphate-buffered saline (PBS) pH 7.4 and treated with 200 mM glycine in 0.1 M Na-phosphate buffer pH 7.4 for 30 min to block any unreacted aldehyde groups. The eyes were washed with PBS and incubated with cationized ferritin (Miles Laboratories, Inc.) 933 mg ml-’ in PBS for 1 hr at 25°C. Controls were incubated with native horse spleen ferritin (Polysciences, Inc.) in the same manner. Following incubation with ferritin, the eyes were washed with PBS, fixed for 30 min with 1.5 o/0 glutaraldehyde in 61 M Na-cacodylate buffer, washed with 61 M Na-cacodylate and postfixed with 1 y0 050,. Alcian

blue staining

Whole eyes were washed with 61 M Na-cacodylate buffer pH 7.2 following the PA-P-G fixation and treated for 3 hr at 25°C with 1 y0 alcian blue 8GX (Sigma Chemical CO.), 2 9,; glutaraldehyde in 91 M Na-cacodylate buffer pH 65 according to the method of Behnke and Zelander (1970). Control eyes were incubated in 2% glutaraldehyde, 61 M Na-cacodylate buffer pH 65 for 3 hr at 25°C. After treatment with alcian blue, the eyes were washed with 91 M Na-cacodylate buffer and postfixed with 1 oA 0~0, in 61 M Na-cacodylate. High iron diamine

staining

Following the primary PA-P-G fixation, whole eyes were washed with 91 M Na-acetate buffer pH 7-O and incubated 24 hr at 25°C with 924 7’ N.N-dimethyl-m-phenylene diamine (HCl), 094 y0 N,N-dimethyl-p-phenylene diamine (HCl) and 1.12 ‘YOFeCl,, pH 1.4 (Spicer et al., 1978). Control eyes were incubated 24 hr in the diamine solution pH 1.4 without FeCl,. After the high iron diamine incubation, the eyes were washed with 61 M Na-acetate buffer and postfixed 3-5 hr with 1 oh 090, in 61 M Na-cacodylate. PA-T-SP

staining

Whole eyes were fixed 1 hr with PA-P-G as outlined above, washed with 91 M Na-acetate buffer pH 7.0, dehydrated with a graded series of ethanols, infiltrated with Epon-Araldite (Mollenhauer, 1964) and propylene oxide, and embedded in Epon-Araldite mixture. Following polymerization, ultrathin sections (approx 90 nm) were cut and mounted on stainless steel grids. These sections were treated with periodic acid. thiocarbohydrazide and silver protein as outlined by Thiery (1967). This is a modification of the light microscopic PAS reaction for TEM. Sections were floated on drops of 1 y0 periodic acid for 45 min at 25’C, washed with distilled water (D. H,O), floated on drops of 62 y0 thiocarbohydrazide in 20 y0 acetic acid for 40 min at 25°C rinsed with 10 y0 acetic acid followed by a D H,O rinse, floated on drops of 1 y0 silver protein in the dark for 30 min at 25°C and washed with D H,O. All reagents were filtered through 62 pm Millipore filters mounted in Swinnex adapters. Control grids were processed in the same manner except that oxidation with 1 o/0 periodic acid was omitted. Digestion

with neuraminidase

The eyes of pentobarbital-anesthetized (40 mg kg-‘) Swiss-Webster mice were enucleated and placed in PBS pH 55 containing neuraminidase 2.5 mg ml-’ (1.25 U ml-i) (Sigma Chemical Co., type V, purified from Clostridium perfringens) Control eyes were placed in PBS pH 5.5 without enzyme. All eyes were incubated for 20 min at 37”C, washed with PBS pH 55 and then fixed 1 hr at 25’C with the PA-P-G fixative. Following this fixation, the eyes were washed with PBS, pH 7.4, quenched with glycine and incubated with cationized ferritin 933 mg ml-’ as outlined above. The neuraminidase used in these experiments was checked for protease activity using Bio-Rad Laboratories (Richmond, CA) protease detection kit. This assay is based on the diffusion of proteases during a 16-24 hr incubation period in a 1 y0 agar gel containing casein (Bjerrum, Ramlau, Clemmese, Inglid and Boghanse, 1976). In this system, diffusable proteases cause clear zones in the gel, whose diameters are directly related to protease concentration. Protease from Streptomyces griseus (Sigma Chemical Co. type XIV.

P. A. WELLS

AND

L. D. HAZLETT

FIG. 1. Agar cell containing casein used for the detection of proteases. Wells l-3 were filled with 9 pl of PBS pH 7.5 containing 6.25 pg ml-‘, 312 Fugml-’ and 1.56 pg ml-’ bovine pancreatic trypsin. Wells 44 contained 9 gl of PBS with 75 pg ml-‘, 375 pg ml-’ and 18.7 pg ml-’ of protease from Streptmycea gviseus. Well 7 contained 9 pl of neuraminidase 125 mg ml-’ (6F5 U ml-‘) dissolved in PBS. Well 8 was filled with 9 yl of PBS pH 7.5 (buffer blank). The gel was photographed after an 18 hr incubation at room temperature.

58 U mg-i) and bovine pancreatic trypsin (Sigma Chemical Co. type III, iO(OO6U mg-i) were used as standards for this assay. The neuraminidase was found to be free of protease activity at concentrations up to 125 mg ml-i (62.5 U ml-‘), which is 50 times the concentration used for incubations with whole eyes (Fig. 1). Embedding

and sectioning

Following postfixation in 1 y0 OsO,, whole eyes were dehydrated with a series of ethanol solutions, infiltrated with Epon-Araldite and propylene oxide and embedded in Epon-Araldite mixture. Thin sections (approx 90 nm) were cut with a diamond knife mounted in a Sorvall MT-2B ultramicrotome. Sections cut from dialyzed iron, alcian blue or cationized ferritin blocks were stained with 2 y0 aqueous uranyl acetate for 20 min. Sections cut from high iron diamine blocks were examined and photographed unstained. Transmission electron micrographs were taken with a JEM-1OOCX electron microscope operating at 60 kV. Light microscopy of 2 pm thick plastic sections was accomplished with a Zeiss photomicroscope using Kodak Panatomic-X film.

3. Results SEM of whole eyes fixed with the PA-P-G mixture revealed light, medium and dark cells at the cornea1 surface (Fig. 2). These cells were categorized based upon their relative brightness in the image formed by the secondary electrons, light cells being the brightest. Some of the dark cells had lifted edges and appeared to be desquamating (Fig. 2). The dark cells had only a few short microvilli visible and looked as though they were coated with a layer of amorphous material (Figs 2 and 3). At higher magnification, dark smooth areas were visible on the medium and light cells (Fig. 4). These areas were variable in shape and consisted of a thin layer of material which adhered to and obscured the microvilli of the underlying cell (Fig. 4). This material

Fm. 2. Low-power SEM showing the peripheral cornea after PA-P-G fixation. The cornea1 surface is composed of polygonal cells which appear light (L), medium (M) or dark (D) in the secondary electron image. Numerous fine strands of mucus are seen on the medium and light cells. Strands of mucus often cross from one cell to another. Several desquamating cells are indicated by arrows. x 1500. FIG. 3. Higher magnification of two dark cells which are coated with ocular mucus. Blebs of mucus are present at the edges of the dark cell on the left. x 2500. FIG. 4. High magnification of ocular mucus on a medium cell. Where the mucus is thin (arrow), the surface cell’s microvilli are only partially hidden by the mucin layer. In other areas where the mucus is thicker (*), the underlying microvilli are completely obscured. x 55(w).

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FIG. 5. Swiss-Webster mouse cornea incubated 3 hr at 25°C with dialyzed iron and glacial acetic acid (3: 1) pH 20. Dialyzed iron staining is seen as a band 56140 nm thick covering the microvilh of the surface cells, Aqueous uranyl acetate-stained section. x 42090. FIG. 6. Dialyzed iron staining shown at higher magnification covering a cornea1 surface cell. The staining appears continuous and often spans the spaces between adjacent microvilli (arrows). Aqueous uranyl acetate staining. x 81000. FIQ. 7. Control cornea incubated with 69 y0 NaCI and glacial acetic acid (3: 1) pH 2.0 for 3 hr at 25°C. Aqueous uranyl acetate stain. x 42009.

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was presumed to be ocular mucus because of its adherence to the surface cells. The mucus sometimes formed strands and blebs on the apical surface of the light, medium and dark cells (Figs 2 and 3). Strands of mucus often crossed from one cell to the next, spanning the cell-to-cell junctions (Fig. 2). Whether the strands and blebs occurred in vivo or were artifacts of fixation is not known. At the TEM level, using dialyzed iron, cationic ferritin and alcian blue staining, a layer of material 25-250 nm in thickness was visualized overlying the cornea1 surface cells. Collectively, these markers are small cationic probes which label carboxyl (-COO-) and sulfate (-G-SO,-) groups. After fixation with PA-P-G and incubation with dialyzed iron, a layer of positively stained material 50-140 nm thick was observed by TEM covering the microvilli of the cornea1 surface cells (Fig. 5). At higher magnification, the dialyzed iron-stained material appeared continuous and often crossed from the tip of one microvillus to the next, bridging the spaces between adjacent microvilli (Fig. 6). Control corneas incubated with 0.9 y0 NaCl and acetic acid pH 2.0 did not show any electron-dense staining at the ocular surface (Fig. 7). Whole eyes which had been fixed with PA-P-G and incubated with cationized ferritin (933 mg ml-‘), exhibited a band of ferritin particles 49-250 nm thick at the ocular surface (Fig. 8). The cationized ferritin particles formed a network overlying the apical plasma membranes of the surface cells (Fig. 9). In many instances, branched patterns were observed within the network and extending 100-200 nm outward from the surface ccl microvilli (Figs 8 and 9). Corneas which were incubated with native ferritin (633 mg ml~1)showedalayerofamorphousmaterialwithsomeelectron-opaque granules along the cornea1 and conjunctival surface ; a few native ferritin particles were observed embedded in the amorphous material (Fig. 10). The electron-opaque granules were larger in diameter and irregular in shape compared to the ferritin particles. This allowed the ferritin particles to be distinguished from the granular material on a morphological basis. Eyeswhich were incubated with 1 y0 alcian blue and 2 y0 glutaraldehydeafter PA-P-G fixation displayed a band of stained material 25-90 nm thick covering the microvilli of the surface cells (Fig. 11). Light microscopic observations on unstained 2 pm thick plastic sections showed a thin layer of blue-staining material along the cornea1 and conjunctival surface. Goblet cells located in the bulbar conjunctiva also stained positively with alcian blue [Fig. 11(B)]. At higher magnification with TEM (Fig. 12) the alcian blue-stained material appeared continuous and often bridged the spaces between adjacent microvilli, as did the dialyzed iron-stained material (compare Figs 12 and 6). Control eyes incubated in the glutaraldehyde without alcian blue did not exhibit staining in either the cornea or conjunctiva (Fig. 13). To differentiate sulfate esters from carboxyl groups, whole eyes were fixed with PA-P-G and incubated with a high iron diamine solution. Unstained 2 pm thick plastic sections, examined by LM, showed only positively stained mast cells in the conjunctiva and sclera [Fig. 14(B)]. The granules in the mast cells appeared purple at the LM level. Goblet cells in the bulbar conjunctiva were not stained by the high iron diamine solution. At the TEM level, the granules in the mast cell cytoplasm were more electron opaque than the heterochromatin in the nucleus of the cell (Fig. 14). This density was attributed to the binding of iron by the sulfated glycosaminoglycans (heparin) in the granules. At the LM and TEM levels, the surface of the cornea and conjunctiva was negative after incubation with high iron diamine (Fig. 15). A periodic acid-thiocarbohydrazide-silver protein staining sequence was applied to sections of peripheral cornea and conjunctiva cut from blocks fixed with the PA-P-G

I’. il. WELLS

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L. D. HAZLETT

FIQ. 8. Swiss-Webster mouse cornea, incubated 1 hr at 25°C with cationized ferritin (CF) 0.33 mg ml-i. The CF particles stained a layer of material 4&250 nm thick overlying the cornea1 surface cells. Branched patterns (arrows) were often seen in the CF-stained material. Aqueous many1 acetate-stained section. x 42000. FIQ. 9. Higher magnification of a cornea1 surface cell incubated with CF (0.33 mg ml-i). CF particles are shown clustered near the apical plasma membrane of the cell Aqueous many1 acetate stain. x 81000. Fro. 10. Control eyes were incubated 1 hr at 25°C with native horse spleen ferritin (0.33 mg ml-‘). A layer of amorphous material with a few ferritin particles (5-8 nm in diameter) and some larger granules was observed at the cornea1 surface. The granules are easily differentiated from the ferritin particles on the basis of size and shape. Aqueous uranyl acetate staining. x 42000.

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FIG. Il. Murine cornea1 epithelium incubated 3 hr at 25°C with 1 y0 alcian blue and 2 y0 glutaraldehyde. A layer of electron-opaque material 2590 nm thick is observed covering the microvilli of the surface cells. Aqueous uranyl acetate stain. x 42000. (B) Light micrograph of a 2 pm thick unstained plastic section showing positively stained goblet cells in the bulbar conjunctiva of an eye which had been incubated with alcian blue and glutaraldehyde. The goblet cells appeared blue in unstained sections. Conjunctival epithelium (CE). x 410. Fro. 12. Higher magnification of the cornea1 surface showing following treatment with alcian blue and glutaraldehyde. The alcian blue-stained material often bridged the space between adjacent microvilli (arrow). Aqueous uranyl acetate staining. x 81000. Fm. 13. Cornea1 surface cell from an alcian blue control which was incubated with 2 % glutaraidehyde. Electron-opaque staining was not observed along the ocular surface. Aqueous uranyl acetate-stained section. x 42000.

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Fm. 14. TEM of a mast cell (MC) located in the sclera of an eye incubated with high iron diamine for 24 hr at 25%. The mast cell granules are more electron opaque than the nuclear heterochromatin of the cell. A portion of a fibroblast (F) is also shown. Unstained section. x 12999. (B) Light micrograph of a mast cell (MC) in the sclera of an eye incubated with high iron diamine. The mast cell granules appeared dark purple in the section. Unstained 2 pm thick p&tic section. x 925. Fm. 15. TEM showing the cornea1 surface after incubation with the high iron diamine solution. Electron-opaque staining was not observed along the microvilli of the surface cells. Unstained section. x 42999.

mixture. This technique is analogous to the PAS method of light microscopy in that staining occurs over polysaccharide residues with vicinal 1,2-glycols (i.e. glucose and galactose residues). With the p eriodic acid-thiocarbohydrazide-silver protein technique, positive staining occurred over the apical and basal plasma membranes of the cornea1 surface cells (Fig. 16). Silver protein deposits were also localized over membrane vesicles in the cytoplasm of the surface cells (Fig. 16). In the wing and basal

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Fm. 16. Thin (96 nm) plastic section of unosmicated Swim-Webster mouse cornea stained with periodic acid-thiocarbohydrazide-siver protein. Electron-opaque deposits are shown over the apical plasma membrane (apm), basal plasma membrane (bpm) and membrane vesicles (v) in the cytoplasm of the cornea1 epithelial cells. The heaviest staining occurred over the apical membranes of the surface cells. x 42666. Fm. 17. Higher magnification EM showing silver protein deposits along the apical plasma membrane of a surface cell. Thin section stained with periodic acid-thiocarbohydraside-silver protein sequence. X81000. Fm. 18. Control thin section of unosmicated mouse cornea stained with thiocarbohydrazide followed by silver protein. Electron-opaque deposits were not observed when periodic acid oxidation was omitted. x 42666.

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cell layers of the cornea1 epithelium, positive staining occurred over the plasma membranes and over glycogen and membrane vesicles in the cytoplasm of these cells. Labelling along the apical plasma membrane of the surface cells was qualitatively heavier than labelling over the basal membranes (Figs. 16 and 17). Control sections, incubated in thiocarbohydrazide followed by silver protein, did not show any electron-opaque deposits over the cornea1 epithelium (Fig. 18).

TABLE 1

Summary

of ocular surface cytochemistry

Methods Periodic acidthiocarbohydrazidesilver protein Dialyzed

iron

Cationized fenitin Alcian blue High iron diamine

Specificity

Results

Vicinal 1,2-glycols in neutral sugars (glucose and galactose), also amino alcohols (ethanolamines and sphingomyelin) Sulfate esters (-O-SO,-) * Carboxyl groups (COO-)i Sulfate esters (-O-SO,-) Carboxyl groups (COO-) Sulfate esters (-O-SO,-) Carboxyl groups (COO-) Sulfate esters ((O-SO,-)

+

+ + + -

* Sulfate esters are found on sulfated glycoproteins and the glycosaminoglycans, dermatan sulfate, keratan sulfate, heparan sulfate and chondroitin sulfate. t Carboxyl groups are associated with sialic acid, glucuronic acid and iduronic acid residues of glycoproteins, glycolipids and glycosaminoglycans.

TABLEII

Quantitation

of cutionized ferritin

Treatment Control Neuraminidase

(CF) particles

after treatmed

CF particles per pm of cornea1 surface

with neuraminidase

Areas observed n*

Range

x

Student’s t test P value

4 4

214-353 28-97

292 49

P < ooo5t

* Each area consisted of a 34pm length of cornea1 surface chosen at random and examined at 70000 x total magnification. t A Student’s t test using separate estimates for the variances showed that the neuraminidsse-treated mean was significantly different from the control mean.

In summary, results from the cytochemical studies demonstrated that the ocular surface is rich in carboxyls groups and PAS stainable material (i.e. glycoproteins and glycolipids) but possesses few sulfate groups (Table I). In order to determine whether sialic acid (N-acetyl-neuraminic acid) was contributing to the large number of carboxyl groups at the ocular surface, whole eyes were incubated with neuraminidase 25 mg ml-l (1.25.U ml-l) for 20 min at 37°C. These eyes were then fixed with PA-P-G and incubated with cationized ferritin (CF) @33 mg ml-l to localize any remaining carboxyl groups. Control eyes, incubated in

COMPLEX

FIQ. 19. Cornea1 surface pH 56, fixed with PA-P-G, acetat,e stain. x 81000. FIG. 20. Cornea1 surface in PBS pH 55, fixed with stain. x 81000.

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cell from an enzyme control which was incubated 20 min at 37°C with PBS and then incubated with cationized ferritin (CF) 0.33 mg ml-‘. Aqueous umnyl cell from an eye incubated 20 min at 37°C with neuraminidase (1.25 U ml-‘) PA-P-G and then incubated with CF (0.33 mg ml-‘). Aqueous uranyl acetate

PBS pH 5.5 without added enzyme, showed normal amounts of CF particles at the ocular surface (Fig. 19). & uantitation of the CF particles in randomly-selected areas from the controls gave an average of 292 ferritin particles per ,um of cornea1 surface (Table II). Eyes which were incubated with neuraminidase prior to fixation showed less CFat the ocular surface (Fig. 20). Quantitation of the CF particles in neuraminidasetreated eyes showed an average of 49 ferritin particles pm-’ of cornea1 surface (Table II). This corresponded to an 83 y0 reduction in CF l&belling after neuraminidase treatment. A Student’s t test using separate estimates for the variances showed that the neuraminidase-treated mean was significantly different (P < 0905) from the control mean (Table II). 4. Discussion

SEM of corneas fixed with PA-P-G demonstrated light, medium and dark surface celIs. Similar patterns of light and dark cornea1 surface cells have been observed with SEM in mouse pups (Hazlett, Spann, Wells and Berk, 198Oa), adult mice (Hazlett,

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Wells, Spann and Berk, 1980b) and in rabbits, cats, rats, monkeys and dogs (Pfister, 1973). In these studies, the ocular mucus was removed either with N-acetylcysteine (Pfister, 1973) or by extensive washing (Hazlett et al., 1980b) after an initial fixation with glutaraldehyde. In mouse pups (two to 10 days postpartum), whose eyelids are not yet opened, mucus was not observed over the cornea1 epithelium (Hazlett et al., 1980a). It has been suggested that the brightness of the cornea1 surface cells in these preparations is related to the number and length of the microvilli on their apical surfaces (Pfister, 1973). A more convoluted surface (i.e. many long microvilli) gives rise to more secondary electrons and appears brighter in SEM, while a smoother cell surface (i.e. a few short microvilli) gives rise to less secondary electrons and appears darker. In eyes fixed with PA-P-G as well as in eyes where the ocular mucus was removed (Pfister, 1973; Hazlett et al., 1980b), the dark cells were judged to be mature or hypermature surface cells. Desquamating cells in PA-P-G fixed corneas generally belonged to the dark cell population. The light cells possessed the morphological characteristics of newly emerging cells, while the medium cells, which were the most numerous, were termed intermediate in age. SEM observations of corneas fixed with PA-P-G showed variable amounts of mucus on the apical surface of light, medium and dark cells. The dark cells possessed the most mucus of the three cell types. These cells appeared dark in the SEM because of the smooth mucin coat which obscured the surface microvilli and not because of the length or number of microvilli. Mucus on the light and medium cells either formed flat sheets or appeared as a network of fine strands covering the surface-cell microvilli. TEM of cornea and conjunctiva incubated with either dialyzed iron, alcian blue or cationized ferritin revealed a layer of material covering the cornea1 surface cells. The layer of stained material was variable in its thickness, depending on the cytochemical technique employed. The alcian blue-stained material was 25-90 nm in thickness, while the cationized ferritinand dialyzed iron-stained layers were 40-250 nm and 50-140 nm respectively. The decrease in thickness which occurred with alcian blu+utaraldehyde may be attributed to a cross-linking and precipitation of mucin macromolecules by this mixture. As a group, dialyzed iron, cationized ferritin and alcian blue are all small cationic probes which bind to negatively charged carboxyl groups and sulfate esters. These markers label sialylated and sulfated glycoproteins and glycolipids as well as sulfated and non-sulfated glycosaminoglycans (Behnke and Zelander, 1970; Spicer and Schulte, 1982). Of the glycosaminoglycans, heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate and dermatan sulfate possess both carboxyl groups (n-glucuronic acid and L-iduronic acid residues) and sulfate esters. Keratan sulfate contains only sulfate esters, while hyaluronic acid possesses only carboxyl groups. Sulfate esters were not localized at the ocular surface using a high iron diamine technique. Light microscopic histochemical studies (Spicer sulfated and Meyer, 1960; Srinavasan et al., 1977) have also failed to demonstrate mucins in conjunctival goblet cells. These studies, in addition to the ultrastructural evidence presented here, suggest that sulfated glycoproteins, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate and keratan sulfate are not present at the ocular surface. Cationized ferritin staining was sensitive to digestion with neuraminidase, indicating that ocular mucus consists of sialylated glycoprotein. This result is in agreement with biochemical studies (Moore and Tiffany, 1981), which established that ocular mucus is rich in sialic acid residues. Most of the sialylated glycoproteins found in ocular mucus are synthesized and

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SURFACE

secreted by conjunctival goblet cells (Ralph, 1975; Holly and Lemp, 1977; Moore and Tiffany, 1979). Biochemical investigations (Chao, Vergnes, Freeman and Brown, 1980) using [14C]-glucosamine, have demonstrated that human lacrimal gland explants synthesize and secrete tear film glycoproteins in vitro. The newly synthesized glycoproteins were identified immunologically as the plasma glycoproteins : immunoglobulin A (IgA), immunoglobulin G (IgG), albumin, transferrin and ceruloplasmin. Mucin-type glycoproteins were not synthesized and secreted by the lacrimal gland explants (Chao et al., 1980). Immunofluorescence studies (Moore and Tiffany, 1979) using a rabbit antiserum against human ocular mucus glycoprotein GP2 have localized ocular mucus glycoprotein in conjunctival goblet cells but not in the lacrimal gland of humans. Tiffany and Bron (1978) have suggested that conjunctival goblet cell mucins are highly branched polymers which have a ‘ bottle-brush ’ appearance. These conjunctival cell mucins are thought to be adsorbed to the surface cell microvilli and under normal conditions would be relatively insoluble in the aqueous tear layer (Holly and Lemp, 1977; Edwards, 1978). In addition to sialylated mucins, sialylated membrane glycoproteins and glycolipids belonging to the surface cell apical plasma membranes would also be stained with alcian blue, dialyzed iron, cationized ferritin and the periodic acid-thiocarbohydrazide silver protein sequence. Thus the positively stained material at the ocular surface in this study most likely consists of sialylated mucins and glycocalyx belonging to the surface cells. The cytochemical staining techniques employed here do not differentiate between membrane-bound sialic acid and sialic acid associated with mucin glycoproteins. Previous studies in the guinea pig (Nichols, Dawaon and Togni, 1983; Dawson, Togni, Lauricella and Nichols, 1980) have localized a layer of ruthenium red-positive material overlying the cornea1 and conjunctival epithelium. After staining with tannic acid and uranyl acetate, this material was visualized as a weblike network extending 200-300 nm from the apical plasma membrane of the surface cells (Nichols et al., 1983). The authors interpreted the tannic acid-uranyl acetate-stained material as glycoealyx belonging to the surface cells. However, because of the intimate association between the surface cells and the ocular mucus, it is possible that the positively stained material consisted of glycocalyx in combination with mucin glycoprotein. In addition to the localization of sialylated glycoproteins, IgA has been localized at the ocular surface of the mouse using Sternberger’s peroxidase anti-perioxidase (PAP) technique (Hazlett, Wells and Berk, 1981). Edwards (1978) has proposed that there may be some affinity of IgA for mucin macromolecules, and so some secretory IgA molecules may be associated with the innermost mucin layer of the precorneal tear film, In addition to binding IgA antibodies, the mucus layer maintains wettability of the surface cells (Holly and Lemp, 1977) lubricates the surface of the cornea and lids (Tiffany and Bron, 1978) and provides protection against invading microorganisms. Experimental studies involving mouse pups (five days post partum) have shown that the cornea1 epithelium of the unopened eye lacks morphologically detectable mucus (Hazlett et al., 1980a), and is easily penetrated by Pseudomonas aeruginosa (Hazlett, Wells, Spann and Berk, 198Oc; Hazlett, Wells and Berk, 1982). In contrast, the cornea1 surface of adult mice which has detectable surface mucin (Hazlett et al., 1980a) is not penetrated by P. aeruginosa unless cornea1 scarification precedes topical application of the bacteria (Hazlett, Rosen and Berk, 1978). Escherichiu c&i pili have been shown to possess receptors for D-mannose which function in the adherence of E. c&i to epithelial cell surfaces (Ofek, Mirelman and Sharon, 1977; Schaeffer, Amundsen and Jones, 1980). Similar receptors for carbohydrates may exist on Vibibrio chderae, 2

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34

P. A. WELLS

AND

L. D. HAZLETT

Mycwplasma and Neisseria gonorrhoeae (Beachey, 1981). Bacterial adherence to epithelial cell surfaces is generally regarded as the first step in bacterial penetration and colonization (Gibbons, 1977). In the normal eye, receptor-mediated bacterial adherence to the ocular surface cells may be blocked either physically or biochemically by carbohydrate moieties on mucin macromolecules. ACKNOWLEDGMENTS This study was supported by United States Public Health Service grant EY 02986 and Core Vision grant EY 04068 from the National Eye Institute. REFERENCES Accinni, L., Hsu, K. C., Spiele, H. and DeMartino, C. (1974). Pioric acid formaldehyde fixation for immunoferritin studies. Hi&o&m. 42, 257-64. Beachey, E. H. (1981). Bacterial adherence : adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Die. 143, 32545. Behnke, 0. and Zelander, T. (1970). Preservation of intercellular substances by the cationic dye alcian blue in preparative procedures for electron microscopy. J. Ultr&ruct. Res. 31, 424-38. Bjerrum, 0. J., Ramlau, J., Clemmese, I., Inglid, A. and Boghanse, T. C. (1975). Artifact in quantitative immuuoelectrophoresis of spectrin caused by proteolytic activity in antibody preparations. Sea&. J. Zmmunol. (Suppl. 21, 81-8. Chao, C. W., Vergnes, J. P., Freeman, I. L. and Brown, S. 1. (1980). Biosynthesis and partial characterization of tear film glycoproteins. Incorporation of radioactive precursors by human lacrimal gland explants in vitro. Exp. Eye Res. 30, 41 l-25. Dawson, C. R., Togni, B., Lauricella, L. and Nichols, B. (1980). Surface coats of conjunctiva and cornea. In Zmmu~logical Diseases of Mucous Membranes (Ed. O’Connor, G. R.). Pp. 216. Masson Publishing, New York. Edwards, P. A. (1978). Is mucus a selective barrier to macromolecules? Br. Med. Bull. 34, 55-6. Friedlaonder, M. H. (1979). Allergy and Zmmundogy of the Eye, Pp. 55-105. Harper and Row Publishers, Hagerstown, MA. (Ed. Schlesinger, Gibbons, R. J. (1977). Adherence of bacteria to host tissue. In Microbiology D.). Pp. 395406. American Society for Microbiology, Washington DC. Hazlett, L. D., Rosen, D. D. and Berk, R. S. (1978). Age-related susceptibilty to Pseudomonas aeru+osa ocular infections in mice. Infect. Zmmun. 20, 25-9. Hazlett, L. D., Wells, P. and Berk, R. S. (1981). Immunocytochemical localization of IgA in the mouse cornea. Exp. Eye Res. 32, 97-104. Hazlett, L. D., Wells, P. and Berk, R. S. (1982). The mouse cornea as a model for Pseudomonas infection : further studies on penetration and lysis of the unwounded eye. In The Structure of the Eye (Ed. Hollyfield, J. G.). Ch. 29, Pp. 279-96. Elsevier North-Holland, Amsterdam. Hazlett, L. D., Spann, B., Wells, P. and Berk, R. S. (1980a). Desquamation of the cornea1 epithelium in the immature mouse : a scanning and transmission microscopy study. Exp. Eye Rea. 31, 21-30. Hazlett, L. D., Wells, P., Spann, B. and Berk, R. S. (198Ob). Epithelial desquamation in the adult mouse cornea, a correlative TEM-SEM study. Ophthalmic Res. 12, 315-23. Hazlett, L. D., Wells, P., Spann, B. and Berk, R. S. (19800). Penetration of the immature mouse cornea and conjunctiva by Pseudonwws: preliminary SEM analysis. Znved. Oph&almol. Vis. Sci. 19, 694-7. Holly, F. J. and Lemp, M. A. (1977). Tear physiology and dry eyes. Surv. Ophthdnol. 22, 69-87. Jersild, R. A. and Crawford, R. W. (1978). The distribution and mobility of anionic sites on the brush border of intestinal absorptive cells. Am. J. Anatomy 152, 287-306. Lillie, R. D. (1965). Histopatk&gical Technique and Practical Histochemistry (3rd en). Pp. 506-15. The Blakiston Division, McGraw-Hill Book Co., New York.

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Mollenhauer, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-14. Moore, J. C. and Tiffany, J. M. (1979). Human ocular mucus. Origins and preliminary characterization. Exp. Eye Res. 29, 291-301. Moore, J. C. and Tiffany, J. M. (1981). Human ocular mucus. Chemical studies. Exp. Eye Res. 33, 203-12. Nichols, B., Dawson, C. R. and Togni, B. (1983). Surface features of the conjunctiva and cornea. Invest. Ophthalmol. Vis. Sci. 24, 57&6. Norn, M. 8. (1972). Vital staining of cornea and conjunctiva. Acta Ophthdmol. (Suppl.) 113, 166. Ofek, I., Mirelman, D. and Sharon, N. (1977). Adherence of Escherichia wli to human mucosal cells mediated by mannose receptors. Nature (London) 265, 6235. Pfister, R. R. (1973). The normal surface of cornea1 epithelium: a scanning electron microscopic study. Invest. Ophthalmol. 12, 654-68. Ralph, R. A. (1975). Conjunctival goblet cell density in normal subjects and in dry eye syndromes. Invest. Ophthalmol. 14, 299-302. Schaeffer, A. J., Amundsen, S. K. and Jones, J. M. (1980). Effect of carbohydrates on adherence of Escherichia coli to human urinary tract epithelial cells. Infect. Immun. 30, 531-7. Spicer, S. S. and Meyer, D. B. (1960). Histochemical differentiation of acid mucopolysaccharides by means of combined aldehyde fuchsin-alcian blue staining. Am. J. Clin. Path&. 33, 453-60. Spicer. S. S. and Schulte, B. A. (1982). Ultrastructural methods for localizing complex carbohydrates. Hum. Path.&. 13, 343-54. Spicer, S. S., Hardin, J. H. and Setser, M. E. (1978). Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with high iron diamine procedure. His&hem. J. 10, 435-52. Srinivasan, B. D., Worgul, B. V., Iwamoto, T. and Merriam, G. R. (1977). The conjunctival epithelium. II. Histochemical and ultrastructural studies on human and rat conjunctiva. Ophthalmic Res. 9, 65-79. Thiery. J. P. (1967). Mise en evidence des polysaccharides sur coupes fines en microscopic Blectronique. J. Microscopic 6, 98771018. Tiffany, J. M. and Bron, A. J. (1978). Role of tears in maintaining cornea1 integrity. Trans. Opht?udmol. Sot. U.K. 98, 335-8. Wetzel, M. G., Wetzel, B. K. and Spicer, S. S. (1966). Ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains. J. Cell Biol. 30,299315.