Characterization of Glycoconjugates in the Intestinal Mucosa of Vertebrates by Means of Lectin Histochemistry

ACTA HI5TOCHEM. CYTOCHEM. Vol. 22, No. 1, 1989

CHARACTERIZATION OF GLYCOCONJUGATES IN THE INTESTINAL MUCOSA OF VERTEBRATES BY MEANS OF LECTIN HISTOCHEMISTRY

J. F. MADRID, J. BALLESTA*, M. T. CASTELLS, J. A. MARIN AND L. M. PASTOR

Departmentof Cell Biology,Section of Histologyand GeneralEmbryology, Medical School, Universityof Murcia, Murcia, Spain

Received for publication July 13, 1988, in revised form October 24, 1988 and re-revised form December 16, 1988

The glycoproteins of the intestinal mucosa of five vertebrate species including fishes (Sparus auratus), amphibians (Rang perezi), reptiles (Testudo graeca), birds (Gallus gallus) and mammals (Mesocricetus auratus) were studied by means of methods of lectin histochemistry. Goblet cells of the intestine showed different lectin binding patterns depending upon the location in the villi. Thus, goblet cells located in the intestinal glands presented a different reactivity from that of the goblet cells located at the tip of the villi. Differences in lectin stainings between small and large intestines were more marked in higher than in lower vertebrates. WGA binding sites were the most abundant throughout the intestinal mucosa of the animals studied. Golgi areas of the different cell types studied were frequently reactive for DBA and SBA. The results obtained in the Brunner's glands of the hamster were similar to those obtained in other mammals, suggesting a common mucin composition. In the hamster intestinal glands, Con-A stained the Paneth cells.

Intestinal mucins are known to play an important role in the protection of the mucosa against physical and chemical damage (1) and bacterial attacks (4). Thus, the characteristics of mucins in the intestinal mucosa of man and other vertebrates have been widely studied by means of classical methods of histochemistry (10-12, 19, 27, 33, 37, 39). Recently, lectins have been found to be powerful and reliable tools to investigate the characteristics and distribution of glycoproteins in tissues. They have been widely employed to describe the tissue distribution of carbohydrates in the human and rat intestinal mucosa, both at light and electron microscopic levels (3, 7, 8, 13, 14, 17, 25, 26, 28, 30, 34, 35, 41-45). In the intestinal mucosa of non-mammalian vertebrates, however, lectins have been applied only to the chicken colon (40). In the present study, the distribution of glycoproteins in the intestinal mucosa of several mammalian and non-mammalian species has been studied by means of methods of lectin histochemistry in order to establish a possible phylogenetic relation between the carbohydrate composition of the intestinal cellular components.

* To whom offprint requests should be sent .

1 2 Madrid et al.

MATERIALS AND METHODS Preparationof tissues. Five animal species belonging to five different classes of vertebrates were studied (Table 1). Animals of both sexes were sacrificed with an overdose of ether. Digestive tracts of the donor animals were removed and divided into different segments: proximal, middle and distal portions of both small and large intestines. The tissue samples were fixed in either buffered 10% formalin in PBS (0.1 M phosphate buffered saline, pH = 7.4) for 8-12 hr or 6% mercuric chloride in 1% sodium acetate containing 0.1% glutaraldehyde for 3-8 hr. After fixation, the samples were washed over- night in PBS and 70% alcohol respectively. The tissue specimens were processed routinely and 5 pm thick paraffin sections were cut. Prior to staining, sections from the tissues fixed in buffered glutaraldehyde-mercuric chloride were treated with Lugol's solution (1% iodine in 2% potassium iodide) and 5% sodium thiosulfate, so as to remove mercuric deposits. Methods of lectin histochemistry. Sections were routinely deparaffinized in xylene and hydrated in a graded ethanol series. Endogenous peroxidase activity was destroyed by 30 min treatment with 0.3% hydrogen peroxide in TBS (tris buffered saline, pH = 7.4). Sections were washed in three 5 min changes of TBS and then incubated, in a moist chamber, for 2 hr at room temperature in horseradish peroxidase-conjugated lectins diluted in the same buffer (Sigma, St. Louis, M0. Table 2). After three washes in TBS, the peroxidase activity was visualized with TBS containing 0.05% 3,3' diaminobenzidine tetrahydrochloride and 0.015% hydrogen peroxide. When finally reacted, the sections were washed in running tap water (10 min), dehydrated, cleared and mounted in DPX, according to usual manners. The following controls were performed for the lectin stainings: 1) Substitution of lectin-HRP conjugates by TBS. 2) Pre-incubation of the lectins with the corresponding hapten sugar inhibitors (Sigma, St. Louis, M0. Table 2), which were used at a concentration of 0.2 M. To study the lectin reactivity of Paneth cells in the hamster small intestine, serial sections (2 pm) were obtained from the paraffin blocks and stained alternately with a modified Bowie's stain (18) and the lectin-HRP conjugates employed in the present study. The modified Bowie's stain has been reported to stain specifically Paneth cells (18).

RESULTS Generalcomments. In general, lectin stainings were slightly more intense in specimens fixed with the buffered glutaraldehyde-mercuric chloride, as compared with the formalin fixed ones. Reactivity to WGA was identified in the goblet cells of both the small and large intestines of all the animals studied. It was also the lectin that showed the strongest affinity towards the intestinal microvilli. The cytoplasm of the intestinal absorptive cells of all the animals studied exhibited affinity towards Con-A. A gradual variation in stainability of goblet cells was noted from the deeper part of crypts to the upper part of villi. However, two exceptions were noted, 1) strongly Glycoconjugates in the Intestinal Mucosa 3

TABLE 1. Animals examined

FIGS. 1a, b. Serial sections of an intestinal gland of the hamster small intestine stained with a modified Bowie's stain (a) and Con-A (b). Paneth cells are intensely stained in the both figures (arrowheads). a) Secretory granules of Paneth cells are stained by the modified Bowie's stain. b) When the same Paneth cells are reacted with Con-A, secretory granules remain unstained, whereas the cytoplasm is heavily reacted. X 650

FIG. 2. Proximal portion of the large intestine of a hamster. Labelled goblet cells are preferentially located in the lower part of the glands. Microvilli of the columnar cells are stained as well (arrowheads). WGA stained. X 150 FIG. 3. Distal portion of the large intestine of a hamster. The microvilli and the Golgi area of the columnar cells are intensely reacted. Goblet cells remain unreactive. SBA stained. X 500 FIG. 4. Small intestine of a chicken. The striated border and the Golgi area of the absorptive cells (arrowheads) are prominently reacted. The staining is likewise observed in the goblet cells. SBA stained. X 550. FIG. 5. Large intestine of a chicken. Only the goblet cells exhibit a positive reaction. WGA stained. X 200 FIG. 6. Small intestine of T. graeca. Only the absorptive cells are reacted. Con-A stained. X 550 FIG. 7. Small intestine of T. graeca. Positive goblet cells are alternated with unreactive ones. It is frequently observed that reactive and unreactive cells occur on different sides of one and the same fold. LTA stained. X 150 4 Madrid et al. TABLE 2. The characteristics o the lectins used

Fuc: fucose Gal: galactose, GalNAc: N-acetY1galactosamine Glc: Glucose G1cNAc: N-acetY1 lucosamine Man: mannose, Methyl-a-Man: methyl-a-mannoPYranoside 6 Madrid et al.

TABLE3. Lectinbinding patterns in the small (anterior)intestine

WGA-reactive and unreactive goblet cells were diffusely distributed throughout all the areas of the hamster intestine, 2) In the small intestine of T. graeca, reactive and unreactive goblet cells were detected on different sides of one and the same folds (Fig. 7). The cytoplasm of the intestinal absorptive cells from all the animals studied showed affinity towards Con-A. Marked differences in lectin binding patterns between the small and large intestines have been noted in the higher vertebrates studied; however, in the lower vertebrates species (frog and fish) the particular differences were slight (Tables 3, 4). Differences in lectin bindings were most distinguished between the absorptive cells, whereas the reactivity of goblet cells was more uniform. Glycoconjugates in the Intestinal Mucosa 7

TABLE 4. Lectin binding patterns in the large (posterior) intestine

Keys to the symbols of Tables 3 and 4: C: cytoplasm; CM: cell cytoplasm except for mucous granules; G: Golgi area; LS: luminal surface; M: mucous granules; MC: microvilli. The intensity of stainings: from - to -f-H-f.Symbols separated by a slash (/) indicate gradual variations of the staining intensity. *: indicates that no gradual variation of the staining intensity was noted. Cells were either strongly reactive or unreactive.

Mesocricetusauratus (Hamster). SMALL INTESTINE. All the lectins tested showed reactivity in the Brunner's glands (Table 3). In the villi, the affinity of goblet cells towards DBA was moderate and no binding of the lectin was noted in the intestinal glands. The cytoplasm of the columnar (absorptive) cells stained with WGA and Con-A; however, the latter did not react with the terminal web. Few Con-A negative microvilli were detected in the jejunum and il- eum. The Golgi area of the columnar cells located at the tip of the intestinal villi fail- 8 Madrid et al. ed to bind to PNA, and the Golgi area of the columnar cells located in the intestinal glands did not stain with DBA either. Paneth cells could be identified in specimens subjected to a modified Bowie's stain and the affinity of these cells towards Con-A was demonstrated in serial sections (Figs. la, b). Con-A bound preferentially to the basal region of Paneth cells (Fig. lb). Other lectins tested gave rise to negative results in these cells. LARGE INTESTINE. A mixture of Con-A, WGA, DBA and SBA reactive- and unreactive-goblet cells were diffusely distributed throughout the large intestine of the hamster (Figs. 2, 3). WGA and DBA exhibited a variable affinity towards the columnar cells, whereas unreactive cells predominated in the proximal parts of the large intestine (Fig. 2). The Golgi area of the columnar cells was labelled by DBA, except for the posterior portion of the colonic mucosa. In contrast, PNA showed little affinity towards the Golgi area except for the rectum. PNA-unreactive microvilli were predominantly found in the rectum.

Gallus gallus (Chicken). SMALL INTESTINE. Only a small population of goblet cells located at the posterior portion of the small intestine was unreactive to WGA. Goblet cells of the intestinal glands reacted to all the lectins except Con-A. This lectin, however, exhibited a weak affinity towards the goblet cells of the villi. PNA, and LTA bound to the columnar absorptive cells of the villi, but not to those in the intestinal glands. The Golgi area of all the columnar cells stained with WGA, DBA and SBA (Fig. 4). LARGE INTESTINE. WGA bound vividly to the goblet cells located at the tip of the folds and less intensely to those of the glands (Fig. 5). The binding of DBA to the columnar cells occurred mainly in the basal portion of the cells and only occasionally

FTC. 8. Large intestine of T. graeca. A vivid reaction is detected in mucous granules of surface mucous cells. WGA stained. X 500 FIG. 9. Large intestine of T. graeca. Lectin binds to the non-mucous part of the cells, whereas mucin contents remain unstained. The luminal surface of the cells is vividly stained. Epithelial cell aggregations of the lamina propria exhibit reactive materials as well (arrows). PNA stained. X55O FIG. 10. Small intestine of R, perezi. Absorptive cells show strong positive reactions in the striated border and the Golgi area. A positive reaction is likewise seen in goblet cells. WGA stained. X 150 FIG. 11. Small intestine of R. perezi. Both the striated border and the goblet cells are intensely stained. A weak reaction is detected in the Golgi area of the columnar cells (arrow). DBA stained. X250 FIG. 12. Anterior intestine of S. auratus. Microvilli are intensely reacted. The apical portions of the cytoplasm of the columnar cells are likewise heavily stained, even though a clear negative area (terminal web) can be seen immediately below the striated border. Goblet cells remain unreactive (arrows). Con-A stained. X 450 FIG. 13. Anterior intestine of S. auratus. Both goblet and columnar cells are reacted for the lectin. DBA stained. X450 FIG. 14. Anterior intestine of S. auratus. Both reactive and unreactive goblet cells can be noted (arrows). The apical region and microvilli of the columnar cells are intensely reacted, even though a clear unstained band is visualized between these areas. WGA stained. X 600 FIG. 15. Posterior intestine of S. auratus. Only goblet cells show affinity towards lectin. DBA stained. X 700 Glycoconjugates in the Intestinal Mucosa 9 10 Madrid et al. in the supranuclear region. PNA reacted with the microvilli of columnar cells of the folds, but not with those of the glands.

Testudograeca (Greek Tortoise). SMALL INTESTINE. Con-A and LTA merely stained a small population of goblet cells; however, WGA, DBA and SBA showed a strong affinity towards the whole population (Figs. 6, 7). Staining of goblet cells with WGA and DBA was more intense at the top than at the bottom of the folds. WGA and SBA bound preferentially to the cytoplasm and microvilli.of certain absorptive cells in the distal portion of the small intestine. LARGE INTESTINE. The large intestine of this animal species did not contain any columnar absorptive cells (Figs. 8, 9) according to the authors' observations in light and electron microscopy. The surface epithelium of the large intestine consisted merely of mucus-secreting cells exhibiting strong positive reactions with WGA, DBA and LTA (Fig. 8). PNA and SBA stained the luminal surface of these cells (Fig. 9). In the lamina propria of the large intestine, small groups of epithelial cells were found. These epithelial aggregations reacted with all the lectins employed in the present study (Fig. 9). Rana perezi (Lake frog). SMALL INTESTINE. SBA and LTA showed affinity towards a small population of goblet cells. All the goblet cells were stained with WGA and DBA (Figs. 10, 11). The PNA-reactive columnar cells gradually increased in number in an anal direct- ion. SBA reacted with the Golgi area of the columnar cells located at the middle portion of the small intestine. PNA stained exclusively those microvilli belonging to columnar cells, of which cytoplasm was reactive for this lectin as well. LARGE INTESTINE. Con-A binding of goblet cells gradually increased in intensity from the proximal portion where all the cells were negative, to the distal region where most of them were reactive. SBA stained the Golgi area of the columnar cells located in the proximal portion of the large intestine. PNA-reactive microvilli were visualized only in the caudal portion of the large intestine.

Sparus auratus (Gilthead sea bream). ANTERIOR INTESTINE. In the fish, the terms "anterior and posterior intestine" are currently used instead of small and large intestine (6). Con-A and WGA stained both the microvilli and the cytoplasm of the columnar cells, whereas they failed to stain the terminal web (Figs. 12,14). Several granules immediately below the terminal web were identified with WGA (Fig 14). DBA-reactive goblet cells were predominantly located in the proximal portion of the anterior intestine (Fig 13). POSTERIOR INTESTINE. Goblet cells were stained by PNA, WGA, DBA and SBA (Fig. 15). WGA and SBA reacted more intensely in those goblet cells located at the tip of the intestinal folds. Columnar cells were found to be either strongly reactive or unreactive for WGA. In the posterior intestine, Con-A only stained the cytoplasm of the columnar cells, whereas no reactivity was noted in the terminal web and microvilli of these cells. All the results obtained are summarized in Tables 3 and 4. Glycoconjugates in the Intestinal Mucosa 11

DISCUSSION Some of the lectins tested in the present study for Brunner's glands of the hamster have likewise been applied to the Brunner's glands of the cat, monkey and human, and similar results were obtained (13, 32, 38, 45). These findings suggest a common carbohydrate composition, and probably similar functions of the mucins elaborated and secreted by Brunner's glands in all the mammals. It has been previously recorded that WGA bound to goblet cells of the rat, monkey and human small intestine and rat and guinea pig large intestine, however, failed to do so in the cells of the cat small intestine (3, 7, 8, 13, 14, 17, 31, 38, 41, 44, 45). In the present study, WGA-staining of the goblet cells was noted in both the small and large intestine from the five animals studied. These findings appear to indicate that the presence of G1cNAc and/or sialic acid residues is a common feature of the intestinal mucins. In the present work, differences in intensity of the reactivity of the goblet cells to the lectins, depending upon their positions in villi, folds or intestinal glands, have been detected. A similar variation in stainability of goblet cells has been reported to occur in human and monkey intestines with methods of both lectin and conventional mucin histochemistry (12, 20, 31, 32, 45). These findings suggest a direct relation between the location of goblet cells and their carbohydrate contents. However, Tsuyama et al. (41, 43) have reported the presence of goblet cells at the same depth of the rat intestinal glands which showed different stainabilities. A similar finding has been obtained in the hamster and tortoise small intestines reacted with WGA and LTA respectively in the present study. Thus, the precise relation between the location of goblet cells and their glucidic contents is to be considered with caution. The Golgi area of the hamster absorptive cells contained Gal(1-3)Ga1NAc, G1cNAc and/or sialic acid and GalNAC residues, in view of the binding specifities of the lectins used in the present study. The Golgi area of the columnar cells of the rat large intestine exhibited a similar reactivity to that of the hamster (41); however, in the cat and man merely G1cNAc and/or sialic acid residues have been identified (38, 44). WGA was the lectin that showed the strongest affinity towards the intestinal microvilli in the animals studied. Similar results have been obtained in other species (7, 8, 44). This appears to indicate a wide distribution of G1cNAc and/or sialic acid residues in glycoconjugates involved in the microvilli. Mucosubstances have been described in the Paneth cells of mice (neutral and acidic mucins), baboon and human (neutral mucins) and gerbil and guinea-pig (neutral and sialo-mucins) (37). However, the lectin reactivity of Paneth cells in any animal species has not yet been reported. In the present study, in contrast, the hamster Paneth cells were found to be reactive for Con-A, suggesting the presence of mannose residues. The results obtained in the present study on the chicken colon are different from those by previous authors (40). An intense labelling with PNA was noted in the goblet cells of the present chicken colon, whereas Suprasert et al. (40) described no affinity of the goblet cells towards the same lectin. Previous studies have revealed certain differences in lectin binding patterns of goblet cells in developing rat colons (5). Thus, such a disagreement may be related to the different ages of the animals studied, 12 Madrid et al. the animals used in the present study being younger. The presence of abundant vesicles implicated in pinocytotic processes immediately below the terminal web of the intestinal absorptive cells of S. auratus, has previously been described by electron microscopy (6). In keeping with this, WGA-positive granules have been visualized in such a region in the present study. These findings may support the concept that in fishes the absorption of carbohydrates occurs in the anterior intestine (9, 36). The differences noted in lectin binding patterns between the small and large intestine in the higher vertebrates studied appear to indicate a progressive specialization ranging from the anterior to posterior regions of the intestine, depending upon the evolution of the animal species. Differences in lection reactivity of the mucosa between the small and large intestines have likewise been reported in the pig (24). In conclusion, the present results are taken to substantiate that G1cNAc and/or sialic acid and GalNAc are the most common carbohydrate residues in the digestive tract. The prominent affinity of DBA and SBA towards the Golgi areas of the intestinal epithelial cells indicates the presence of GalNAc in these structures. The mucin content of all the mucous cells in the fish digestive tract did not show any affinity towards LTA and Con-A, suggesting the absence of mannose and fucose residues in the carbohydrates involved.

ACKNOWLEDGEMENTS

We are greatly indebted to Ms. C. Aroca, Ms. C. Navarro, Mrs. C. Gonzalez and Mr. J. Martinez for their technical assistance. We wish to thank Ms. R. Vicente for typing the manuscript. This work was supported by a grant from DGICYT PB 87-0701 (Spain).

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