ACTA OPHTHALMOLOGICA 64 (1986) 445-450

Detection of mucus g lycocon jugates in human conjunctiva

by using the lectin colloidal gold technique in TEM I. A quantitative study in normal subjects

P. Versura, M. C. Maltarello, F. Bonvicini, R. Caramazza and R. Laschi

Center of Biotechnological and Clinical Research in Ophthalmology (Head: R. Caramazza), Departments of Ophthalmology and Becfron Microscopy, University of Bologna and IOR-Galileo, Italy

Abstract. We applied a specific cytochemical reac- tion to characterize the glycoconjugates produced by goblet and non-goblet epithelial cells of normal human conjunctiva. For this purpose we utilized the lectins, proteins of vegetal origin, which are extremely sensitive in binding glycosidic residues. In particular, we used WGA, PNA, SBA and ConA conjugated with colroidal gold as ultrastructural marker for Transmission Elec- tron Microscopy. This technique allowed us also to perform a quantitative analysis, by counting colloidal gold particles present on mucus granules. In this way we analyzed the content both of goblet and non-goblet epithelial cells. In the former, WGA, PNA, SBA and ConA receptors, here reported in decreasing density, were present. In the latter WGA was always positive, SBA and PNA sometimes were negative, ConA was always negative. We speculate the different contribu- tion to mucus production by these two sources may be important in evaluating tear film stability alterations occurring in those diseases in which non-goblet epithel- ial cell vesicles increase.

Key words: glycoconjugates - goblet cells - non-goblet epithelial cells - normal human conjunctiva - lectin- colloidal gold - electron microscopy - quantitative analysis.

It is well assessed that mucus layer maintains the tear film stability by making the ocular surface wettable (Lemp et al. 1970). This mucus is pro- duced by conjunctival goblet and non-goblet epi- thelial cells (Takakusaki 1969; Srinivasan et al.

1977; Greiner et al. 1979, 1980; Greiner & Allan- smith 1981); it is extruded outwards, spreading over conjunctival and corneal microprojections.

Conjunctival mucus consists mainly of glyco- protein molecules which have been widely investi- gated by different points of view. Biochemical studies on mucus clots obtained by scraping-off have been performed by Iwata & Kabasawa (1971) and Lee & Robinson (1980) in rabbit and by Dohlman et al. (1976) and Moore & Tiffany (1979) in man. Collecting samples in such a way permits evaluation of the whole carbohydrate content of the tear film. In fact there are other sources, besides goblet cells, for example the lacri- mal gland.

Histochemical investigations have been carried out by several authors (Spicer & Meyer 1960; Matsumoto & Mimura 1974). Nichols et al. (1985) and Lee et al. (1981) used tannic acid and ruthen- ium red to stain the mucus layer at the ultrastruc- tural level. Histochemical stainings on paraffin sections in Light Microscopy (LM) were applied by Srinivasan et al. (1977), who detected in con- junctival goblet cells non-sulphated mucopolysac- charides containing sialic acid, by Greiner et al. (1985) who found neutral, sialo and sulphomucins in non-goblet epithelial cell secretory vesicles. A new approach was introduced by Kawano et al. (1984) who studied goblet cell carbohydrates on human conjunctiva by means of fluorescein-con-

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juga ted lectins a t LM. Lectins are proteins, mainly of ~ e g e t a l origin, which very specifically bind glycosidir residues (Goldstein & Hayes 1978). In t h e present work we aimed t o better characterize t h e glyc oproteins produced by conjunctival goblet and rion-goblet epithelial cells. In this respect, we used f o u r lectins: Wheat Germ Agglutinin (WGA) specific for N-acetylglucosamine and N-acetyl- new aniinic acid, Arachis Hipogea (Peanut Lectin, PNA) specific for galactosyl-fl-( 1-3)-N-acetylgalac- tosatnine and non-reducing terminal galactose, Soybean Agglutinin (SBA) specific f o r a-N-acetyl- D-gdac tosamine and Concanavalin A (ConA) spe- cific for trimannosidic cores substituted by two N-ac etylglucosaminyl residues (Roth 1978).

We have conjugated these lectins with colloidal gold as an ultrastructural marker , in order to study t h e presence and distribution of their glyco- sidic receptors, in Transmission EM (TEM). I n this way, it is possible t o detect even small amount of glycoprotein, eventually not evidenced by fluorescence microscopy and , a t the same time, to per form an accurate quantitative analysis by count ing colloidal gold particles present over the mucus granules.

Materials and Methods

Biopsy specimens have been obtained from the inner lower tarsal conjunctiva of ten informed patients (6 males and 4 females, medium age 51), undergoing surgery for diseases not involving the anter ior segment, selected on the basis of smooth conjunctival surface.

T h e biopsies were mounted on ashless filters, briefly washed in saline, fixed in 2.5% glutaralde- hydc - 1.6% p-formaldehyde in 0.1 M cacodylate buffer for 3 h at 4°C. They were then washed overnight in 0.1 M cacodylate buffer plus 2.7% saccarose, dehydrated i n a graded series of etha-

nol and embedded in Epon. Ultrathin sections were obtained with the Reichert UM3 ultrotome and collected on niche1 grids covered with a thin film of formvar.

Cytochemical reactions WGA, ConA, PNA and SBA were purchased from Sigma Co, St. Louis as well as their respective inhibitor sugars. Colloidal gold (size of 17 nm) was prepared by reduction of 100 ml of 0.01 % HAuC14 with 4.2 ml of 1 % Na-citrate according to Frens (1973). The optimum amount of lectin was evaluated by serial dilutions ac- cording to Horisberger (1981).

On serial sections, the reactions were performed as follows:

etching in 3% NaOH in absolute ethanol for 35”. washing in TBS 0.02 M pH 7.00 for WGA, PNA and

SBA and pH 7.2 for ConA. incubation for 1 h in AU17 conjugated WGA, PNA,

SBA diluted 1 : 10 in 0.02 M TBS pH 7.00; Au17 con- jugated ConA was used undiluted.

washing in distilled water with stirring. Control sections were incubated in a solution contain-

ing the same concentration of colloidal gold conjugated lectin as described above plus 0.5 M specific haptenic sugar, except for PNA, whose inhibitor (D-+-galactose) was added 1.0 M. All sections were counterstained with uranile acetate and lead citrate, and observed at Zeiss EM9 and Jeol 100 B transmission electron microscope.

Quantitative evaluation The density of the labelling over mucus granules of goblet cells and subsurface vesicles of the non-goblet epithelial cells was evaluated for each of the four lectins and for their respective controls. A total of 150 micro- graphs (15 each specimen) for each lectin were taken at the original magnification of 9500 X. A carbon grating replica with 2160 lines per mm was used for calibration. The surface area occupied by mucus granules was expressed in pm2. The number of gold granules was counted by means of a projector unit which increases the original magnification of the film negative by 5. The density of the labelling was calculated according to the formula: density = number of gold granulesisurface area in pm2.

Figs. 1 -4 . Fig. 1 Normal conjunctiva. Goblet cell labelled by Au17-WGA. The gold particles (small black points) heavily label the niucus granules (arrows), in some areas they appear to be lacking (asterisks). Bar = 1 pm. TEM X 13 800. Fig. 2. Normal conjunctiva. Goblet cell labelled by Aul7-PNA. The intensity of the labelling is less than in Fig. 1. The cytoplasm surrounding the mucus granules is quite free from gold particles (arrows). Bar = 1 pm. TEM X 16 150. Fig. 3. Normal conjunctiva. Goblet cell labelled by Au17-SBA. The labelling appears decreased as respect to the preiious figures, as it is also demonstrated by the quantitative analysis in Table 1. Bar = 1 pm. TEM X 13 800. Fig. 4 . Normal conjunctiva. Goblet cell labelled by Au17-ConA. The lectin slightly marks the mucus granules. The cytoplasm is completely free from labelling. Bar = 1 pm. TEM x 24 200.

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Resu I ts

Fixation of the tissue with only glutaraldehyde and p-formaldehyde without osmification re- sulted in a good preservation of the cellular struc- tures. The labelling over the cytoplasm and the cellular compartments was low and rather con- stant for all the four lectins studied. The labelling over the mucus granules was heavy and had a non-uniform pattern. In fact some areas of the granules appeared to be free of gold particles, and in some cases even a whole granule was negative. These observations refer to all the four lectins. Aul7-WGA, Au17-PNA, Auly-SBA and Au17- ConA distributions are shown respectively in Figs. 1, 2, 3 and 4. The quantitative estimation of the labelling over mucus granules of goblet cells is sunimarized in Table 1.

The subsurface vesicles of the non-goblet epi- thelial cells were always labelled by Aul7-WGA (Fig. 5a) with a minimum of 3 to a maximum of 15 gold particles each vesicle. Aul7-SBA and Au17- PNA were not present in all the vesicles we ob- served (Fig. 5b and 5c), when present there were 1

Table 1. Density o f labelling over the mucus

granules (gold particles per pm2 2 SEM).

WGA 608.80 _t 20.55 PNA 235.99 ? 11.02 SBA 147.76 f 8.47 ConA 74.65 _t 5.12

up to 4 gold particles each vesicle for SBA and 2 up to 9 for PNA. The vesicles were completely negative for Aul,-ConA (Fig. 5d). As in normal subjects these vesicles are not present in great quantity, we had at disposal only few micro- graphs; for this reason we did not quantitatively analyzed their density each square unit.

Addition of specific haptenic sugar of each lectin lead to a drastic reduction in the presence of gold particles (Figs. 6 and 7). The quantitative analysis of these results is summarized in Table 2.

Figs. 5a -d. Nornral conjunctiva. Non-goblet epithelial cell vesicles labelled by: a) Aun-WGA, 3 up to 15 gold granules are prescnt over the vesicles (arrows). Bar = 1 pm. TEM x 42 000. b) Aul7-SBA, some vesicles are not marked by this lectiii (large arrows), only one appears positive (small arrow). Bar = 1 pm. TEM X 41 800. c) Aul7-PNA, two vesicles .ire negative for the labelling (arrows), in the third vesicle 8 gold granules are present. Bar = 1 pm. TEM X

147 400 d) Au17-ConA, the vesicles are completely free from labelling (arrows). Bar = 1 pm. TEM X 121 800.

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Figs. 6 and 7. Fig. 6. Normal conjunctiva. Control section incubated with Au17-WGA plus the inhibitor sugar. Goblet cell. The labelling intensity has enormously decreased as respect to Fig. 1. Bar = 1 pm. T E M X 38 950. Fig. 7. Normal conjunctiva. Control section incubated with Aul,-SBA plus the inhibitor sugar. Goblet cell. The number of gold granules is less than in Fig. 3. Bar = 1 pm. TEM x 24 960.

Discussion

The functional integrity of mucus layer depends primarily upon its molecular composition. If some components change in quality and/or quantity, biophysical properties can consequently be altered.

In our study we employed four lectins (WGA, PNA, SBA and ConA) conjugated with colloidal gold as ultrastructural marker. in Transmission EM. With this cytochemical technique, whose spe- cificity is also well demonstrated by the quite complete lack of background label, we have also been able to detect small quantities of glycosidic

Table 2. Density of labelling over the mucus

granules (gold particles per pmZ k SEM).

WGA 35.53 k 3.70 PNA 17.86 k 2.87 SBA 16.03 k 2.48 ConA 12.64 k 1.14

receptors inside both goblet cells and non-goblet epithelial cells. These latter were not evidenced in LM by Kawano et al. (1984), as they are very small.

Our results on the presence and distribution of glycoconjugates are quite in agreement with Ka- wan0 et al. (1984). We found, as they did, a good positivity in all goblet cells observed, for WGA, PNA and SBA; Concanavalin A receptors (i.e. mannose) which have not been detected in fluor- escence microscopy by those authors, are con- versely present in goblet cells as we evidenced in TEM. The count of the colloidal gold particles over a data area, in our case over mucus granules, permits the presence of each lectin to be pointed out with accuracy as it is clearly shown in Table 1.

The glycoprotein content of the non-goblet epithelial cell vesicles have been studied by Grei- ner et al. (1985). We have detected in all the micrographs a positivity of these vesicles for sialic acid and N-acetyl-glucosamine. N-acetyl-galacto- samine and galactose-N-acetyl-galactosamine are present only in some vesicles and not in others. Mannose was always found to be absent.

An important result seems to be that non-goblet

449 29 Acfa Ophthal. 64.4

epithelial cells contribute to the mucus layer com- position in a different manner in respect to goblet cells. This could be explained by the fact that the non-goblet epithelial cells are not specifically dif- ferentiated for mucus secretion and so, probably, they partially lack in the enzymes for its produc- tion.

These data suggest that an increase in the number of non-goblet epithelial cell vesicles, and consequently of the glycoconjugates they pro- duce, may lead to a change in the mucus composi- tion as a whole. This hypothesis could explain the mucus alteration during some pathological condi- tions in which the non-goblet epithelial cell ves- icles are markedly increased, such as giant papil- lary conjunctivitis and vernal conjunctivitis.

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Received on April 8th, 1986.

Author’s adress:

P. Versura, Department of Ophthalmology, University of Bologna, Via Massarenti 9, 1-40138 Bologna, Italy.

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