Brush Cells of the Mouse Intestine Possess a Specialized Glycocalyx as Revealed by Quantitative Lectin Histochemistry: Further Evidence for a Sensory Function

Animals

Eleven BALB/c mice of both sexes kept under specified pathogen-free (SPF) conditions, 4-8 weeks old and weighing about 20 g, were anesthetized IP with a mixture of Ketanest (WDT; Garbsen, Germany) and Rompun (Bayer; Leverkusen, Germany). The ileum, including some Peyer's patches, was removed and rinsed in Ringer's solution at 4C for a few seconds to remove feces and mucus. The specimens were cut into small pieces with a razor blade and immediately immersed in the fixative.

Tissue Preparation

The samples were fixed in a solution of 2% formaldehyde and 0.05% glutaraldehyde (grade I; Sigma, Deisenhofen, Germany) in 0.1 M phosphate buffer, pH 7.3, for 4-16 hr at 4C. They were then rinsed in PBS containing 1% L-lysine (Sigma) for 16 hr to block free aldehyde groups. The specimens were partially dehydrated in graded ethanol series (30%, 50%, 70%, 90%) and transferred to LR White resin, medium grade (Plano; Marburg, Germany). The tissue blocks were enclosed in gelatin capsules and allowed to polymerize at 50C for 48 hr. Ultrathin sections were cut and mounted on formvar-coated nickel grids.

Lectin Labeling

The lectin-gold labeling was performed in PBS containing 1.5% bovine serum albumin-c (BSA-c; Biotrend, Cologne, Germany) and 0.1% sodium azide. Free aldehyde groups were blocked for 15 min in a drop of this buffer (PBS-BSA) containing 0.7% L-lysine. After rinsing in PBS containing 5% bovine serum albumin (Serva; Heidelberg, Germany), 0.1% cold water fish skin gelatin (Biotrend), 1% normal goat serum (Sigma), and 0.05% Tween 20 (Serva), the sections were incubated with PBS-BSA containing one of the biotinylated lectins (see Table 1) at 4C overnight. After rinsing, the grids were incubated for 4 hr with a goat anti-biotin antibody (1:50) conjugated to 20-nm colloidal gold (Bio-Cell; Cardiff, UK). Finally, the grids were treated with 2% glutaraldehyde in PBS, washed in distilled water, and stained with uranium acetate and lead citrate. The sections were examined in a Zeiss EM10 electron microscope (Zeiss; Oberkochen, Germany). Controls were carried out by omitting the lectin and by preincubating the lectin with the corresponding mono- or disaccharide overnight (see Table 1).

OPEN IN VIEWER

Table 1

Lectins, specificities, and concentrations a

Lectin	Abbreviation	Nominal specificity	Concentration	Carbohydrate used for controls	Reference
Ulex europaeus b	UEA-I	Fucose	6.25 μg/ml	L-fucose (0.03 M)	Pareira et al. 1978
Lotus tetragonolobus b	LTA	Fucose	100 μg/ml	L-fucose (0.06 M)	Pareira and Kabat 1974
Ricinus communis b	RCA-I	Galactose	6.25 μg/ml	D-galactose (0.03 M)	Nicolson et al. 1974
Vicia villosa b	VVA	N-acetyl-galactosamine	6.25 μg/ml	N-acetyl-D-galactosamine (0.03 M)	Tollefsen and Kornfeld 1983
Maackia amurensis c	MAA	Sialic acid	6.25 μg/ml	N-acetyl-lactosamine (0.04 M)	Knibbs et al. 1991
Triticum vulgaris b	WGA	N-acetyl-glucosamine	25 μg/ml	N-acetyl-D-glucosamine (0.15 M)	Debray et al. 1981
Helix pomatia b	HPA	N-acetyl-galactosamine	25 μg/ml	N-acetyl-D-galactosamine (0.15 M)	Hammarström and Kabat 1971
Canavalia ensiformi b	ConA	Mannose	25 μg/ml	D-mannose (0.15 M)	Debray et al. 1981

^aEight lectins with specificities for all carbohydrates typically found on epithelial cells were used. For each lectin, the concentration that resulted in labeling densities best suited for quantitation by image analysis was selected as the working concentration. The molar concentrations of carbohydrates used for preabsorption controls were adapted to the working concentration of the corresponding lectins.

^bSigma, Deisenhofen, Germany.

^cEY Labaratories, San Mateo, CA.

Quantitative Analysis

To quantify the labeling density of gold particles on the brush border and cytoplasm, electron microscopic photographs were taken at a standardized magnification of 12,500-fold. The negatives were digitized in transmitted light mode using a Duoscan scanner (Agfa; Leverkusen, Germany) at a resolution of 1000 dots per inch. The 8-bit gray scale datasets, each containing about 8 megabytes, were stored and analyzed on standard PCs using the image analysis software UTHSCSA ImageTool, version 2.0 alpha, which is freely available in the internet (http://ddsdx.uthscsa.edu). An ImageTool program (a so-called “script”) was written by the authors which allowed a region of interest (ROI) to be drawn that contained the brush border or a part of the cytoplasm of individual cells. The program then determined the area of the ROI and detected and counted gold particles of a defined size, e.g., particles with an area of 314 ± 100 nm² for 20-nm colloidal gold.

Labeling densities measured by this method for the glycocalyx of the brush border may not only depend on the composition of glycoconjugates detected by the lectins but are also influenced by the size and arrangement of the microvilli. Because the geometry of the brush borders of the two cell types investigated was relatively constant, a mathematical model was developed to determine the influence of the different microvillous diameters and distances between brush cells and enterocytes (Figure 1). In cross-sections (Figure 2), the average diameter of enterocyte microvilli was 95 nm (SD 10 nm; n = 50) and the minimal distance between neighboring microvilli was 20 nm (SD 7 nm; n = 34). The diameter of brush cell microvilli was 139 nm (SD 17 nm; n = 58), and their minimal distance was 18 nm (SD 9 nm; n = 30). Given a hexagonal arrangement of microvilli, the relative length of microvillous membranes per area was calculated for each cell type using the formulas shown in Figure 1. The quotient of the two values was 1.29, i.e., the length of the apical membrane and glycocalyx in a defined area of a thin section was 29% greater in enterocytes than in brush cells. This correction factor was then used to calculate labeling densities (“corrected densities”) that were no longer affected by the differing geometries of the two cell types. Corrected labeling densities were compared using the Mann-Whitney rank sum test (U-test) at a significance level of 0.05.

OPEN IN VIEWER

Figure 1

The microvilli of brush cells and enterocytes are arranged in approximately hexagonal patterns, but differ in their diameters (d) and in the minimal distances between the microvilli (a). A cross-sectional model was used to determine the relative length of membrane (L) in a defined region of interest (rectangle). The relative membrane length of enterocytes exceeds that of brush cells by 29%, so that the (raw) densities measured for brush cells were multiplied with a correction factor of 1.29. Hence, the resulting “corrected densities” are only marginally affected by the geometrical differences of the two cell types.

Brush Cells

In LR White ultrathin sections of the small intestine, brush cells were identified and distinguished from enterocytes by their typical ultrastructure. The microvilli of the brush cells were thicker and more electron-dense than those of neighboring enterocytes (Figure 2). They contained microfilament rootlet bundles which extended deeply into the apical cytoplasm; many electron-lucent vesicles and tubules lay between these filament bundles (Figure 3). At the periphery of the cell apex and in the remaining cytoplasm, bundles of intermediate filaments lay close to the lateral plasma membrane, where they inserted at many desmosomes. The perinuclear cytoplasm of the brush cells possessed the typical pear-like shape and, in contrast to enteroendocrine cells, lacked electron-dense granules. The brush cells comprised only a very small portion of the intestinal epithelium and appeared to be more numerous on the villi than in the crypts. Interestingly, the Peyer's patches contained considerably more brush cells than distant villi. Whereas seven section profiles of Peyer's patch domes counted for this purpose contained 17 brush cells and 2805 other epithelial cells (0.61%), the section profiles of 12 villi contained only 2 brush cells and 3596 other epithelial cells (0.06%). The Peyer's patch and the villous brush cells did not apparently differ in structure or lectin binding characteristics.

Gold Labeling and Quantitation

The binding of eight lectins to enterocytes and brush cells of the intestinal epithelium was determined using on-section gold labeling and quantitative image analysis. Depending on the binding specificity and the avidity of the lectin used, the labeling densities of the brush border differed considerably (Figure 4). Because high labeling densities of colloidal gold may cause spatial hindrance, some of the lectins (UEA-I, RCA-I, VVA, MAA) were used at a fourfold higher dilution than our standard concentration of 25 μg/ml. Because LTA at this standard concentration showed labeling densities near zero, it was used for quantitative evaluation at a fourfold higher concentration. To exclude the possibility that quantitative differences between the two cell types were due to variation in the labeling procedure, on each photograph of a brush cell an adjacent enterocyte was included in the quantitation. In additional tests, consecutive ultrathin sections running through the same cells were separately labeled, photographed, and analyzed. However, the differences in the labeling densities between brush cells and enterocytes of the same ultrathin section typically exceeded that of the same brush cell found in adjacent but separately labeled sections by 500% or more. To compare the labeling density in the glycocalyx of brush cells with that of enterocytes, a correction factor was used which minimized the influence of the differing brush border geometries (Figure 1). This correction did not influence the statistical significance of the differences found for seven of the lectins. In principle, visual examination of the gold labelings in the electron microscope closely corresponded with the densities determined by computer-based image analysis.

OPEN IN VIEWER

Figure 2

Longitudinal (A) and oblique (B) sections of brush cells in the small intestine of the mouse. Compared to enterocytes, the brush cells possess a tuft of thick microvilli which contain dense bundles of microfilaments (arrows). They extend deep into the apical cytoplasm and are associated with many electron-lucent vesicles (arrowheads). The oblique section shown in B runs across enterocyte microvilli (emv), across brush cell microvilli (bmv), and across the microvillous filament bundles located in the apical cytoplasm (ac) of the brush cells. The lectins VVA (A) and MAA (B) are specific for N-acetyl-galactosamine and sialic acid, respectively, and bind more avidly to the brush border of enterocytes than to that of brush cells.

OPEN IN VIEWER

Figure 3

Brush cell (bc) and adjacent enterocytes (e) labeled for α-L-fucose residues using the lectin UEA-I and 20-nm colloidal gold. Whereas the brush border of enterocytes remains almost unlabeled, the glycocalyx of the brush cell intensely binds the lectin. Note that the membranes of vesicles (arrowheads) lying between filament bundles in the apical cytoplasm are densely labeled. Quantitative image analysis revealed that the labeling densities of these cytoplasmic regions were closely correlated with that of the brush border.

Lectin Binding Properties of Brush Cells and Enterocytes

The lectins used in this study preferentially bound to the brush border of the gut epithelium. They differed considerably in their binding to the two cell types investigated (Figure 4). Quantitative analysis enabled three groups to be distinguished: lectins that preferentially bound to the glycocalyx of brush cells, lectins that preferentially bound to that of enterocytes, and a lectin that bound to the two cell types with densities that were not statistically different. These lectin binding patterns were uniformly found in all of the 11 mice investigated.

OPEN IN VIEWER

Figure 4

Quantitation of the labeling densities on brush cell (B) and enterocyte (E) microvilli as determined by image analysis. Each circle represents the labeling density in the region of interest of an individual cell; black rhombs indicate medians. Statistically significant differences, as determined by the Mann-Whitney rank sum test at a significance level of 0.05, are indicated by asterisks. The densities measured on the microvilli of brush cells have been corrected according to the geometrical model (see Figure 1). This allows influences of the microvillous geometry on the densities measured to be reduced or eliminated. Significantly higher labeling densities on brush cells than on enterocytes are detected for UEA-I and LTA. In contrast, the lectins RCA-I, VVA, MAA, WGA, and HPA bound more avidly to enterocytes. No differences were found for ConA. Note that the absolute values of the corrected densities considerably differed among the eight lectins, so that the y-axis has been scaled for each lectin to display the full range.

Preferential binding to the glycocalyx covering the microvilli of brush cells compared to that of enterocytes was found for the lectins UEA-I (Figure 3) and LTA (Figure 5). The absolute labeling density of UEA-I, however, was sevenfold higher than that of LTA (compare Figures 3 and 5), even at the 16-fold higher dilution of UEA-I used for quantitation. In addition to the brush border, UEA-I and LTA also bound to the small electron-lucent vesicles lying between the filaments that extended from the microvilli into the apical cytoplasm (Figures 3 and 5). Because of the differing geometries of microvilli and vesicles, the labeling densities on the glycocalyx and on the vesicle-containing cytoplasm could not be compared directly. Quantitation, however, revealed that the labeling densities of the two regions correlated closely (Figure 6). The basal cytoplasm, the basolateral membrane, and the nuclei of both brush cells and enterocytes remained almost unlabeled.

Preferential binding to the brush border of enterocytes was found for RCA-I, VVA, MAA, WGA, and HPA (Figure 4). Of these lectins, RCA-I and VVA bound most avidly to the enterocytes with labeling densities of 30 and 20 particles/μm², respectively (Figure 2A). MAA revealed the clearest difference between the two cell types, showing an approximately eightfold higher binding to enterocytes than to brush cells (Figure 2B). The lectins WGA and HPA revealed only small but statistically significant differences in the corrected labeling densities.

Although ConA labeled the enterocytes at slightly higher (raw) densities, the corrected values did not allow statistical differences between brush cells and enterocytes to be detected. In contrast to all other lectins studied, ConA bound to the cytoplasm of the epithelial cells to roughly the same extent as to the brush border.

Controls

Preabsorption of the lectins with their corresponding saccharides (see Table 1) resulted in drastic reductions in lectin binding to all tissue elements. The binding of most lectins (UEA-I, LTA, RCA-I, WGA, HPA, and ConA) was reduced by 99% or more. The lectins VVA and MAA were reduced by preabsorption with their corresponding saccharides by 93% and 71%, respectively. Replacing the lectin with buffer during the labeling procedure reduced the densities measured over lectin-positive tissue elements by 99% or more (not shown).