J. Nihon Univ. Sch. Dent., Vol. 30, 95-103, 1988

Histochemical Studies on Lectin Binding Patterns in

Postoperative Maxillary Cysts

by

Masahiko GUNGE

(Received 29 February and accepted 18 March 1988)

Key words : histochemistry, lectin, postoperative maxillary cyst, ABC method

Abstract

Up to now, histochemical investigations of the goblet cells found in cases of postoperative maxillary cysts (POMC) have yielded somewhat variable results, while examinations of glycoproteins have been restricted to only a few methods. Recently, lectins, which have specificity for various carbohydrate residues, have been applied for the detection of glycoproteins. However, as far as the author is aware, no studies of POMC using lectin histochemistry have been done. Accordingly, PAS-alcian blue

(pH 2.5) staining was performed on POMC with special reference to goblet cells, and then lectin histochemistry was employed. The specimens examined were sixty cases of POMC, which were obtained from the Department of Oral Surgery, Nihon University School of Dentistry at Matsudo. Goblet cells appeared blue or bluish-

purple, and ciliated cells and columnar cells appeared light purplish-red by PAS-alcian blue staining. The ABC method was employed for lectin staining using seven kinds of lectins, i. e., Con A, WGA, SBA, DBA, RCA-I, PNA and UEA-I. Goblet cells showed positive reactions for WGA, RCA-I, PNA and UEA-I, while ciliated cells and columnar cells were positive for Con A, WGA, SBA, DBA, RCA-I and UEA-I.

Introduction

It is well known that relatively many goblet cells are observed in the walls of POMC, which are often encountered in the field of oral surgery[1-53. However, few histochemical investigations of goblet cells have been done so far. Generally,

goblet cells are positive for the PAS reaction, mucicarmine staining and alcian blue staining, and are suggested to contain mainly glycoproteinsc[6, 7]. On the other hand, TACHIKAWA [1] stated that goblet cells in POMC mainly contained

proteoglycans and a few glycoproteins. Further investigation is therefore needed to settle this question.

Previous histochemical investigations of carbohydrates have mainly been per-

郡家正彦: Nihon University Graduate School of Dentistry at Matsudo, 2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271, Japan.

Originally submitted as a doctor's thesis.

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formed on proteoglycans, and studies on glycoproteins have employed only a few

methods such as PAS and alcian blue staining, with or without digestive methods

using various enzymes [8]. However, it has become possible in recent years to examine

carbohydrates at the monosaccharide level by application of lectins binding to each

of the specific carbohydrate residues and labeling with peroxidase or other agents [9-11]. Detailed studies of glycoproteins in various diseases have been done using these

characteristics [11-13].

In spite of the recent developments made in histochemical techniques, no histo-

chemical investigation of goblet cells in POMC has yet been done. From these view-

points, the ABC method using lectins was employed to investigate carbohydrates, especially those in goblet cells, in the cystic walls of POMC.

Materials and Methods

I. Materials

Sixty specimens of POMC were obtained between May, 1985 and July, 1987,

at the Department of Oral Surgery, Nihon University School of Dentistry at Matsudo. II. Methods

[A] Histopathology All the specimens were fixed in 10% neutral formalin as soon as they had been

removed, and then paraffin sections about 3 ƒÊm thick were made. The sections were

stained with hematoxylin and eosin for histopathological observation, and then with

PAS-alcian blue (pH 2.5).

[B]•@ Lectin staining

The avidin-biotin-peroxidase complex (ABC) method described by HSU et al.

was employed for lectin staining [14, 15].

Paraffin sections were deparaffinized, rehydrated and treated with 0.1% tryspin solution (100mg porcine trypsin II : Sigma, St. Louis, Mo., U. S. A.) dissolved in

Table 1 Lectins used in this study, their sugar specificities and their sugar inhibitors

Abbreviations : D-Man; D-Mannose, D-GlcNAc; N-acetyl-D-Glucosamine, NANA; N-acetyl-

neuraminic acid, D-GalNAc, N-acetyl-D-Galactosamine, D-Gal; D-Galactose, L-Fuc; L-Fucose

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100ml of 0.05 M Tris-HCl buffer, pH 7.6, containing 0.1 % CaCl2. After thorough

washing, the sections were covered with each of the lectins (Vector Laboratories,

Burlingame, Calif., U. S. A.), as shown in Table 1, for 120 min at room temperature.

These were washed in 0.01 M phosphate-buffered saline, pH 7.2, and covered with

ABC for 60 min at room temperature. After a brief rinse, the sections were devel-

oped for 3•`5 min in Tris-HCl buffer, pH 7.6, containing 0.02% 3,3'-diaminobenzi-

dine tetrahydrochloride (Wako, Osaka, Japan) and 0.005 % hydrogen peroxide.

Finally, the sections were counterstained with methyl green and mounted in Malinol.

Prior to the investigations, the specificity of each lectin was confirmed by treat-

ment with 0.2 M hapten sugars, as shown in Table 1.

Observations with regard to lectin binding were recorded as follows :

(〓) Strongly positive Reaction demonstrating a dark brown to black color.

(+) Moderately positive

Reaction demonstrating a brown color.

(±) Weakly positive

Reaction demonstrating a light brown color.

(-) Negative

Reaction demonstrating no change in color.

Results

[A] Histopathology

The inner surfaces of POMC were mainly covered with ciliated epithelium, and

partly with columnar epithelium lacking cilia. Furthermore, cases which had round or polygonal cells at the basal side of the epithelium were sometimes identified.

Goblet cells were stained mainly blue and partly bluish-purple by PAS-alcian blue,

whereas ciliated cells and columnar cells were stained mainly light purplish-red

(Fig.1) Round or polygonal cells at the basal side of the epithelium showed no

reaction for PAS-alcian blue staining.

[B] Lectin staining ―Con A―

Goblet cells revealed no reaction. There were two types of reaction in ciliated cells; one was a moderately to strongly positive reaction at the apical border, while the other was a uniformly. and moderately positive reaction throughout the whole of the cell. Some columnar cells showed a moderately positive reaction throughout the whole cell. Round or polygonal cells were mostly negative (Figs. 2 and 3). ―WGA―

Goblet cells showed a weakly to moderately positive reaction with a reticular

pattern. Binding sites in ciliated cells and columnar cells were similar to those of Con A, but the degrees of binding were weaker (Fig. 4). Other cells showed a mostly negative reaction. ―SBA―

Goblet cells were negative. Reactions in ciliated cells, columnar cells and other

cells were similar to those at the WGA binding sites and were also of similar inten-

sity.

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Fig. 1 Goblet cells mainly revealing blue, and partly bluish-purple staining. Ciliated cells and

columnar cells show light purplish-red staining (PAS-alcian blue (pH 2.5). Original mag-

nification •~ 200)

Fig. 2 Strongly positive reaction identified at the apical border of ciliated cells (Con A, original

magnification •~ 200)

Fig. 3 Ciliated cells revealing a moderately positive reaction throughout the whole cell (Con A,

original magnification •~ 200)

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Fig. 4 Goblet cells showing a reticular and weakly to moderately positive reaction, while ciliated cells and columnar cells show a weakly positive reaction (WGA, original magnification ×400)

Fig. 5 Goblet cells revealing a moderately positive reaction with a reticular or spotted pattern

while ciliated cells and columnar cells show a weakly to moderately positive reaction

(RCA-I, original magnification •~ 200)

Fig. 6 Goblet cells revealing a weakly to moderately positive reaction with a reticular or spotted

pattern (PNA, original magnification •~ 400)

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―DBA―

Goblet cells were negative. Reactions in ciliated cells, columnar cells and other

cells were slightly different from those at the SBA binding sites and were also of

similar intensity.―RCA-I―

Goblet cells revealed a moderately positive reaction with a reticular or spotted

pattern. Ciliated cells showed a weakly to moderately positive reaction overall except at the apical border. Columnar cells were wholly and weakly to moderately

positive (Fig. 5).―PNA―

Goblet cells showed a moderately to strongly positive reaction with a reticular or spotted pattern. Ciliated cells, columnar cells and other cells were negative (Fig. 6).―UEA-I―

A few goblet cells revealed a weakly positive reaction with a reticular pattern, but most were negative. Ciliated cells demonstrated a weakly positive reaction at the apical border, as with Con A, WGA, SBA and DBA. Some columnar cells showed a weakly positive reaction throughout the whole cell. Other cells were nega-tive.

Discussion

Despite the many clinical and histopathological investigations that have been conducted on POMC, hardly any histochemical investigations of this cyst are de-scribed in the literature [1-5, 16-23], and only examinations using mucicarmine staining, PAS reaction and alcian blue staining have been performed for goblet cells found in POMC.

Generally, goblet cells have been identified in the epithelia of the respiratory mucosa and intestinal mucosa [7, 24, 25], and have been confirmed histochemically to contain glycoproteins with strong PAS and alcian blue positivity.

On the other hand, NAKAMURA [26] stated that PAS reactivity changed from

positive to negative in goblet cells of the sinus mucosa associated with experimental inflammation in rabbits. IMAMURA [27] reported that acid mucopolysaccharides were increased in goblet cells in cases of sinusitis. TACHIKAWA [1] found large amounts of acid mucopolysaccharides in goblet cells of POMC using the PAS reaction, and alcian blue and toluidine blue staining. Therefore it was suggested that there were some differences in carbohydrate content between goblet cells in normal tissue and those in pathological tissue.

Generally, mucoidal materials are classified into proteoglycans (acid mucopoly-saccharides) and glycoproteins. Histochemical investigations of mucoidal materials have mainly centered on proteoglycans because of the lack of adequate investiga-tive methods for glycoproteins. For this reason, KUSUHARA [28] employed lectin his-tochemistry for identifying the localization of carbohydrates in the lingual gland.

It has now become possible to examine the types, structures, arrangements, binding patterns and configurations of polysaccharide molecules by utilizing the

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histochemical features of lectins, which have a superior ability to bind to specific

carbohydrates [9,10].

Goblet cells in the large intestine of humans and rats have been stained with

WGA, UEA-I, SBA and DBA [29, 30]. Those in the rat small intestine were also stained

with WGA, DBA and RCA-I, and their binding patterns varied according to the

site [31]. This was considered to be due to differences in carbohydrate content with

goblet cell location.

FUKAMI [7] studied the lectin binding sites on goblet cells in the upper respiratory

airways, such as normal nasal, larynx and trachea mucosa, using six kinds of lec-

tins. In spite of common binding of PNA, RCA-I and WGA in these mucosae,

strongly positive UEA-I binding in the nasal mucosa and moderate SBA and DBA

bindings in the trachea mucosa were identified. MAZZUCCA et al. [32] found strongly

positive reactions for SBA, WGA and PNA, and a weakly positive reaction for

UEA-I binding in the trachea mucosa, being mostly identical to the results of FUICAMI.

MATSUYAMA [33] recognized a remarkable WGA reaction in goblet cells at the time

of sinusitis, and mentioned that this reaction was due to sialic acid production by

goblet cells, large amounts being present in the inflammatory pituita.

Goblet cells, ciliated cells and columnar cells in POMC occurred mainly at the

reactive binding sites for each lectin, despite the various degrees of staining they

showed.

It has been suggested that goblet cells contain D-GalNAc, NANA, D-Gal(1•¨

3)-D-GalNAc and L-Fuc. TACHIKAWA [1] indicated that the invasion of the nasal

mucosa into the sinus was one of the pathogenetic features of POMC. In comparison

with the results obtained for goblet cells in the normal nasal mucosa, those in POMC

revealed a tendency for L-Fuc to be diminished, irrespective of the levels of D-

GlcNAc, NANA and D-Gal(1•¨3)-D-GalNAc.

A strong positive reaction for RCA-I and a weakly to moderately positive

reaction for DBA, SBA, UEA-I and WGA at the apical border of the ciliated cells

appearing in normal nasal and sinus mucosa were identified by HYUN et al. [34] as

lectin-binding sites in ciliated cells. In comparison with the results obtained for nasal

and sinus mucosa, the apical border of the ciliated cells in POMC showed a Con

A-positive reaction, whereas RCA-I was negative. It was therefore considered that

the apical border contained D-Man, D-GlcNAc, NANA, D-Gal and L-Fuc. Re-

ports on lectin binding patterns in ciliated cells other than those in the apical border

and columnar cells have not been available in the past, and therefore will be needed

in the near future.

Conclusions

PAS-alcian blue (pH 2.5) staining and lectin staining were performed to inves-

tigate the cells composing the cystic walls of POMC, and the results were as follows :

(1) The inner surfaces of POMC were mainly covered with ciliated epithelium,

and partly with columnar epithelium lacking cilia. Furthermore, cases in

which round or polygonal cells were present at the basal side of the epi-

thelium were sometimes identified.

(2) Goblet cells were stained mainly blue and partly bluish-purple by PAS-

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alcian blue. Ciliated cells and columnar cells also showed mainly light

purplish-red staining.

(3) Histochemical staining using the avidin-biotin-peroxidase (ABC) method with seven kinds of lectins (Con A, WGA, SBA, DBA, RCA-I, PNA and UEA-I) revealed a positive reaction in goblet cells in POMC for WGA, RCA-I, PNA and UEA-I. However, the UEA-I reaction was weaker than that in normal nasal mucosa. Goblet cells in POMC showed a tendency for L-Fuc to be reduced.

(4) The apical border of ciliated cells in POMC showed the appearance of

D-Man and the disappearance of D-Gal in comparison with nasal and

sinus mucosa. Other ciliated cells contained D-GlcNAc, D-GalNAc and

L-Fuc.

Acknowledgements

I am very grateful to Dr. Hirotsugu Yamamoto for teaching me the basics of

histopathology and lectin histochemistry and for providing constructive criticism

during my work. I also wish to thank Prof. Hirotsugu Izumi and Prof. Shinichiro

Umemura for their enthusiastic direction of this study, and Prof. Goro Hirai, Prof.

Ichiro Matsue, Prof. Shunsuke Furuyama, and Prof. Hiroshi Katayama for aiding

the compilation of this manuscript. The technical assistance of the staff of the De-

partment of Oral Surgery is greatly appreciated.

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