Histochemistry of glandular metaplasia at the trigone of the urinary bladder in cows

J. Comp.

Path.

1992 Vol.

107, 185-194

Histochemistry of Glandular Metaplasia at the Trigone of the Urinary Bladder in Cows H. Okada,

K. Takehana*,

K. Takahashit

and

K. Matsukawa

Department of Veterinary Pathology, *Department of Veterinary Anatomy and TDepartment of Veterinary Internal Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069, Japan

Summary The epithelium at the trigone of the urinary bladder showed intestinal metaplasia of a colonic type in three Holstein-Friesian cows affected with chronic polypoid cystitis. Except for Ricinus communis (RCA-I), almost all goblet cells in the whole crypt were positive to periodic acid-Shiff (PAS), alcian blue (AB) pH 2.5, AB pH l-0, periodate borohydride-potassium hydroxide-PAS (PB-KOH-PAS), Ulex europaeus (UEA-I), Triticum vulgare (WGA) and Arachis hypogaea (PNA) after neuraminidase digestion. This result indicated that most goblet cells contained acidic and neutral glycoconjugates as 0-acetylated sialomucin, L-fucose, N-acetyl-glucosamine, neuraminic acid residues and sialic acid-galactose dimers and were devoid of 8-D-galactose. The goblet cells at the surface in the upper half of the crypt contained both sulpho- and sialo-mucins with N-acetyl-galactosamine residuesby AB (pH 2*5)-PAS, high iron diamine (HID)-AI3 (pH 2*5), Dolichos bz$orus (DBA) and Glycine maximus (SBA) reactions. On the other hand, the lower goblet cellswere found to contain predominantly sulphated mucins with n-mannose and Dglucose residues by AB-PAS, HID-AB and Concanavalia ensifrmis (Con A) reactivities. This suggestedthat mucin secretedfrom thesecellswas similar to that secreted from the goblet cells of the large intestine in cattle. Introduction Chronic proliferative changes in the urinary tract usually develop in response to cystitis (Stirling and Ash, 1941; Kunze, 1986). These proliferative lesions were classified into von Brunn’s nests, glandular metaplasia (cystitis glandularis), squamous metaplasia, cystitis cystica and papillary (polypoid) cystitis by Pamukcu (1974). Polypoid cystitis is characterized by the finger-like projections or outgrowths of the lamina propria covered with normal urinary transitional epithelium (urothelium) of varying thickness (Brobst and Olson, 1963; Skye, 1975), which can often undergo a “mutinous degeneration” of the urothelium (Brobst, Cottreil and Delez, 197 1). Mucus-secreting goblet cell metaplasia may occur in the urothelium associated with cystitis glandularis (Brobst and Olson, 1963; Gordon, 1963). Focal papillary hyperplasia in the trigone of the urinary bladder has been reported in four Holstein-Friesian cows (Skye, 1975). M ucin histochemical studies in cystitis glandularis have been described for man (Gordon, 1963; Wells and Anderson, 1985; Barresi and 0021-9975/92/060185+

10 $08.00/O

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1992 Academic

Press Limited

H. Okada et al.

186

Marafioti, 1990) and in the cat (Zachary, 1981), but we are not aware of any report of similar studies in cattle. We report here the histochemical properties of mucin in polypoid cystitis at the trigone of the bladder in Holstein-Friesian cows, which was associated with glandular metaplasia of the “intestinal type”. Materials

and

Methods

Case Reports

The Holstein-Friesian dairy cows were reared at different farms in Hokkaido, Japan. Cases1 and 2 were 4 years and case 3 was 10 years old. All of the animals were diagnosedashaving hydronephrosis. Before they were killed, their blood urea nitrogen (BUN) and serumcreatinine in cases1 and 2 were shown to be high (Table 1). Culture of urine and kidney from cases 2 and 3 yielded abundant Escherichia coli and Corynebacterium renale, respectively. Case3 died with chronic renal failure and the others were killed by exsanguination under barbiturate anaesthesia. Light Microscopy and Histochemistry

Various organs and tissues were fixed in 10 per cent formalin solution. Tissue specimenswere dehydrated in a graded ethanol seriesand embedded in paraffin wax. Sectionswere cut at 4 pm thicknessand stained with haematoxylin and eosin(HE) and by the foIlowing histochemical procedures: periodic acid-Schiff (PAS) for neutral glycoconjugates, alcian blue (AB) pH 2.5 for acidic glycoconjugates, AB pH l-0 for sulphated glycoconjugates, AB (pH 2*5)-PAS for demonstrating acidic and neutral glycoconjugates, and high iron diamine (HID)-AI3 (pH 2.5) for differentiating sulphated and sialic acid mucins. 0-acetylated sialic acid was demonstrated by a sequence of periodate oxidation, borohydride treatment and alkaline hydrolysis (saponification) in 1 per cent potassiumhydroxide and PAS reaction (PB-KOH-PAS) (Reid, Culling and Dunn, 1973; Culling, Reid, Clay and Dunn, 1974; Lapertosa, Baracchini, Fulcheri and Tanzi, 1985; Lapertosa, Baracchini, Fulcheri and Tanzi, 1986). Lectin Histochemistry

Sections were soaked in 0.5 per cent periodic acid solution to block endogenous peroxidase activity. They were then incubated with the following seven biotinylated lectins (Vector, Burlingame, CA, U.S.A.) for 30 min at room temperature; Arachis hhypogaea (PNA), Concanuvalia ensiformis (Con A), Ricks communis (RCA-I) Dolichos bijorus (DBA), Glycine maximus (SBA), T&cum vulgare (WGA) and Ulex europaeus (UEA-I), followed by reaction with an avidin-biotin peroxidase complex (Dakopatts,

Haematological

findings

Table 1 for cases 1 and 2 just killed

before

they

Factor

Case I

Case 2

Ht (per cent) Hb (g per dl) RBC (10’ per ~1) WBC ( lo2 per ~1) BUN (mg per dl) Creatinine (mg per dl)

32 NE 796 54 61.1 6.9

35 12.1 720 170 89.8 5.2

NE = not examined:

Ht = haematocrit.

were

Histochemistry

of Bovine

Polypoid

187

Cystitis

Copenhagen, Denmark) for 30 min at room temperature. Finally, sections were treated with 3-3’diaminobenzidine, containing hydrogen peroxide, for 5 to 10 min. They were counterstained with methyl green. Control sections were reacted with the same lectins in a solution containing 0.2 M of their corresponding inhibiting sugar. The lectins used, their optimal concentrations, sugar specifications and binding inhibitors are summarized in Table 2. Samples of normal proximal and distal colon from each of five Holstein-Friesian dairy cows aged between 4 and 10 years were used as control tissues to demonstrate the normal distribution of lectin binding sites in the goblet cells of the large bowel. Neuraminidase Digestion To detect sialic acid residues in the metaplastic lesions, sections were digested with neuraminidase from Arthrobacter ureafaciens (Nakarai Chemicals Co., Kyoto; Japan) 1 unit per ml in acetate buffer pH 5.3 containing CaCl, at 40°C for 16-h prior to staining with AB pH 2.5 and PNA. Results Macroscopic Findings

In each affected case, the mass measured 3 to 5 cm in diameter, consisted of numerous papillae or polyps 2 to 10 mm in length and was positioned at the trigone of the urinary bladder. The kidneys were enlarged and firm and the capsules were thickened and adherent. The cortices of the kidneys were pale yellowish-brown in colour and appeared atrophic when the cut surface was examined. The renal pelvis and the ureters were dilated. Calculi, 1 to 5 mm in diameter, were also observed in the dilated renal pelves of cases 2 and 3. These changes occurred bilaterally in each case. Microscopic

Findings

The proliferated

papillae

at the trigone

of the urinary

bladder

were covered

with

hyperplastic urinary epithelium of various thickness. Solid cell nests, the so-called von Brunn’s nests, grew down into the lamina propriae continuous

with the epithelium,

and appeared

Lectins Lectin

Table 2 used in this

(abbreviation)

Major

Concanavalia ensifrmis (Con Arachis hypogaea (PNA) Ricinus communis (RCA-I) Dolichos biJIrus (DBA) Glycine maxima (SBA) ‘Triticum vulgare (WGA) Ulex eurojxzeus (UEA-I) Man, mine;

as small islands in the lamina

mannose; NeuNAc,

A)

Glc, glucose; Gal, N-acetyl-neuraminic

galactose; acid.

study spec$c

a-o-Man, D-Gal-p-( $-~-Gal a-o-GalNAc a-D-GalNAc, /3-D-GIcNAc, a+Fucose

3 12 6 12 12 3 12 GalNAc,

propriae.

sugar

Binding

a-D-Glc I-3)-D-GalNAC

a-Methyl-D-Man D-Gal D-Gal D-GalNAc wGal-NAc D-GlcNAc L-Fucose

a-D-Gal NeuNAc

N-acetyl-galactosamine;

inhibitor

GlcNAc,

N-acetyl-glucosa-

The

H. Okada et d.

188

cystic spaces observed in the thickened epithelium and von Brunn’s nests contained a spherical eosinophilic substance. Some cysts, especially in the lamina propriae, were lined by one to several layers of cuboidal or columnar cells. Columnar cells replaced the transitional epithelial cells associated with von Brunn’s nests and frequently formed crypts. The columnar cells had undergone metaplasia to mucus-secreting goblet cells which resembled the mucosa of the large intestine (Fig. 1A). The lamina propriae of the papillae were markedly oedematous and included a moderate number of lymphocytes and plasma cells. No remarkable changes were observed in the tunica muscularis or tunica adventitia of the urinary bladders. In the kidneys, marked fibrous proliferation and lymphocytic infiltration were seen in the interstitium. The renal pelvis was dilated and the pelvic epithelium was hyperplastic. A large number of lymphocytes and plasma cells had infiltrated in the oedematous lamina propriae of the pelves. Pyelonephritis was present in all the kidneys. Histochemical and Lectin Histochemical Findings

All goblet cells in metaplastic lesions were stained magenta with PAS and PBKOH-PAS (Fig. lB), and blue with AB pH 2.5 (Fig. 1C) and AB pH 1.0. By AB-PAS and HID-AB stainings, the goblet cells of the upper half of the crypt and cyst differed from those of the lower half segment. The goblet cells located at the surface and upper part of the crypts were stained bluish purple and/or blue with AB-PAS and blue and/or black with HID-AB (Fig. 1D). On the other hand, the lower goblet cells were stained predominantly blue with ABPAS and black with HID-AB (Fig. lD), respectively (Table 3). Except for RCA-I and PNA, the lectins used in this study bound to goblet cells. However, the staining pattern was somewhat different for the upper and Histochemical

reactions

Table of goblet

3 cells

in the

metaplastic

lesion

Staining procedures

Lower c@t

Surface and upper cypt

PAS AB pH 2.5 N-AB pH 2.5 AB pH 1.0 AB pH 2.5-PAS HID-AB pH 2.5 Con A UEA-I DBA SBA RCA-I WGA PNA N-PNA

3M 3B I-2B

3M 3B l-2B 3B 3BP/3B 3B/3Bl 0 2-3Br 0-1Br O-1Br 0 2-3Br 0 2-3Br

3B;3B 3Bl/3B 1Br 2-3Br 23Br 2-3Br 0 2-3Br 0 2-3Br

Number indicates intensity of staining reaction: 3=intense, 2 = moderate, 1 = weak, 0 = negative. Colour of reaction: M, magenta; B, blue; BP, bluish-purple; Bl, black; Br, brown; N, neuraminidase. Symbols separated by / in&ate a mixture of cells with different mucin.

Histochemistry

Fig . I.

of Bovine

Polypoid

Cystitis

189

Serial sections of the bladder of case 2. (A) Bladder mucosa shows mucin-secreting goblet cells in the large intestine. HE X 60. (B) Goblet ccl 11sthroughout the crypt stain magenta. PB-KOH-PAS x 60. (C) All of the goblet cells throughout the crypt strongly positive. AB pH 2.5 x 60. (D) Some goblet cells in the upper half of the crypt sta tin blue indicating that they contain sulphomucin, and those deeper in the crypts stain black indic :ating sialomucin. HID-AB X 60. (E) The AB pH 2.5 staining of goblet cells decreased compare d with that shown in Fig. 1C. AB pH 2.5 following digestion with neuraminidase X 60. (F) I ‘NA reactivity of goblet cells became positive. PNA following digestion with neuraminidase x 6IO.

190

Fig. 2.

H. Okada et al.

Serial sections of the bladder of case 1. (A) Most of the goblet cells of the whole crypt stain. UEA-I X 120. (B) Goblet cells throughout the crypt are positive. WGA X 120. (C) Only goblet cells at the surface and in the upper half of the crypt are strongly positive. SBA x 120. (D) The deeper goblet cells in the crypts show only weak staining. Con A x 120.

Histochemistry

Histochemical

reactions

Staining procedures

Proximal

Lower mypt

PAS AB pH 2.5 AB pH 1.0 AB pH 2.5-PAS HID-AB pH 2.5 Con A UEA-I DBA SBA RCA-I WGA PNA See Table

3 for explanation

3M 3B 3B 3BP/3B/2RB 3B1/3B l-2Br 2-3Br 3 3 0 2 0 of abbreviations.

of Bovine

Polypoid

Table 4 of the goblet

cells

of the normal

colon

Surface and UPPer ctypt 3M 3B 3B 3B/3BP/lRB 3B/3Bl 0 2-3Br 0 3 0 2 0

191

Cystitis

colon Distal

colon

Lower nypt

Surface and UPPer*YPt

3M 3B 3B 3B/3BP 3Bl l-2Br 2-3Br 3 3 0 2 0

3M 3B 3B 3BP/3B 3B/3BI 0 2-3Br 0 1 0 2 0

RB, reddish-blue.

lower half of the crypts. After neuraminidase digestion, the staining by AB pH 2.5 decreased somewhat (Fig. lE), whereas the reactivity for PNA increased significantly (Fig. 1F) in almost all goblet cells of the lesions. The lectinstaining pattern of PNA after enzyme digestion was closely similar to that of UEA-I and WGA. Almost all goblet cells through the crypts and cysts were intensely positive to UEA-I (Fig. 2A) and WGA (Fig. 2B). The goblet cells at the surface and in the upper part of the crypts were markedly positive to SBA (Fig. 2C) and DBA, whereas those at the lower crypt stained weakly with Con A (Fig. 2D) (Table 3). The distribution of lectin binding sites in the goblet cells of the normal colon is shown in Table 4. No specific staining was observed in any of control sections by the addition of appropriate sugars to the lectin solutions. Discussion

Chronic proliferative lesions in the urinary bladders of all three affected cases were characterized by the presence of von Brunn’s nests, cystitis cystica, cystitis glandularis and polypoid cystitis with metaplastic reactions. Non-neoplastic proliferation of the urinary epithelium can be induced by excessive stimulation, that is by urotoxic chemical agents, foreign bodies, mechanical injury, chronic infection or irradiation (Kunze, 1986). Papillary hyperplasia is considered to represent a chronic proliferative lesion of the bladder in which the mucosa is elevated into blunt papillae by oedema, chronic exudate or granulation tissue (Stirling and Ash, 1941; Brobst and Olson, 1963; Skye, 1975; Kunze, 1986). In cases where there is histological evidence of chronicity, the ureters may be found to have been partially obstructed for a long time (Skye, 1975). Either hydronephrosis or pyelonephritis seem to result from the partial

192

H. Okada

et al.

obstruction of the ureters and urethra by papillomatous proliferation at the trigone of the bladder (Skye, 1975). I n our cases, it is possible that chronic inflammation stimulated the papillary proliferation at the trigone of the urinary bladder. In all three cases, the goblet cells in the areas of glandular metaplasia showed similar staining patterns. Goblet cell mucin contained predominantly sulphated and sialic acid by AB-PAS and HID-AB reactions, and 0-acetylated sialic acid by PB-KOH-PAS technique. The presence of 0-acetylated sialomutin is considered as a marker not only of the large intestines, but also of extraintestinal lesions and tumours derived from an intestinal type of epithelium (Lapertosa et al., 1985; Wells and Anderson, 1985). The mucin-staining pattern in the present cases, with various histochemical procedures, resembled that seen in cases of goblet cell metaplasia of the human urinary tract (Wells and Anderson, 1985). With the UEA-I and WGA reaction patterns, goblet cells throughout the crypt contained sugar residues of a-L-fucose, N-acetyl-o-glucosamine and sialic acid. The results of SBA and DBA staining patterns indicated that the goblet cells at the surface and in the upper half of the crypts contained large amounts of the residual sugar radicals N-acetyl-a-n-galactosamine, whereas the presence of a-n-mannose and a-n-glucose residues in the goblet cells in the lower half of crypts were indicated by the binding of Con A. Lectin-binding patterns of goblet ceils in cows differ from those of human intestinal metaplasia by the presence of both sugar residues of WGA and UEAI reactivities. The goblet cells in human intestinal metaplasia do not bind WGA and Lotus tetragonolobusagglutinin (LTA; specific for a+fucose) (Barresi and Marafioti, 1990). The colic goblet cells in man (Fischer, Klein, Vierbuthen, Skutta, Uhlenbruck and Fischer, 1984), and in the cows in the present study, bound UEA-I and WGA. Such differences between the goblet cells in cases of intestinal metaplasia of the urinary bladder and those in the normal large intestine in man cannot be explained by the results of this study. After digestion with neuraminidase, the reaction of AB pH 2.5 was somewhat reduced, whereas binding of PNA to goblet cells was greatly increased. The similarity of the lectin-staining pattern between WGA and PNA after enzyme digestion was also apparent. These results indicate the existence of terminal sialic acid residues. Moreover, PNA reaction after neuraminidase digestion revealed that goblet cells contained predominantly acidic glycoconjugates with sialic acid residues and penultimate galactose residues as sialic acid-galactose dimers. In intestinal metaplasia of the urinary bladder in man, PNA reactivity in the goblet cells can only be demonstrated after neuraminidase digestion, although under the cancerous conditions it is demonstrated with or without neuraminidase pretreatment (Barresi and Marafioti, 1990). This may indicate that the PNA-binding pattern could be useful for investigating whether a particular lesion is malignant or not. The glycoconjugates of goblet cells in bovine intestinal metaplasia of the urinary bladder as well as normal large intestine may contain penultimate residues to bind PNA, underlying terminal sialic acid residues (Stoward, Spicer and Miller, 1980; Karayannopoulou and Damjanov, 1987).

Histochemistry

of Bovine Polypoid

Cystitis

193

Histochemical and lectin histochemical reactions of goblet cells, in casesof goblet cell metaplasia of the urinary bladder, closely resemble the reactions of goblet cells in the normal bovine large intestine. Thus, these results indicate the homogeneity of glycoprotein components in both these goblet cell types. Since intestinal metaplasia has been observed frequently in tumours of the bovine urinary bladder (Yoshikawa and Oyamada, 1975), it would be worthwhile to investigate the relationship between goblet cells and such tumours of the urinary bladder in cattle.

References

Barresi, G. and Marafioti, T. (1990). Mucin histochemistry and lectin binding sitesin intestinal metaplasia of the urinary bladder. H&chemistry, 17, 219-223. Brobst, D. F., Cottrell, R. and Delez, A. (1971). Mutinous degeneration of the epithelium of the urinary tract of swine. Veterinary Pathology, 8, 485-489. Brobst, D. F. and Olson, C. (1963). Neoplastic and proliferative lesionsof the bovine urinary bladder. American Journal of Veterinary Research, 24, 105-l 10. Culling, C. F. A., Reid, P. E., Clay, M. G. and Dunn, W. L. (1974). The histochemical demonstration of 0-acetylated acid in gastrointestinal mucins. Their association with the potassium hydroxide-periodic acid-Schiff effect. Journal of Histochemistry and Cytochemistry, 22, 826-83

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Fischer, J., Klein, P.J., Vierbuchen, M., Skutta, B., Uhlenbruck, G. and Fischer, J. (1984). Characterization of glycoconjugates of human gastrointestinal mucosaby lectins. I. Histochemical distribution of lectin binding sitesin normal alimentary tract aswell asin benign and malignant gastric neoplasms.Journal of Histochemistry and Cytochemistry, 32, 681689.

Gordon, A. (1963). Intestinal metaplasia of the urinary tract epithelium. Journal of Pathology and Bacteriology, 85, 441-444. Karayannopoulou, G. and Damjanov, I. (1987). Lectin binding sites in the human gallbladder and cystic duct. Histochemistry, 88, 75-83. Kunze, E. (1986). Hyperplasia, Urinary Bladder, Rat. In Urinary Systems. T. C. Jones, V. Mohr and R. D. Hunt, Eds, Springer-Verlag, Berlin, pp. 291-310. Lapertosa, G., Baracchini, P., Fulcheri, E. and Tanzi, R. (1985). 0-acetylated sialic acid variants as markers of neoplasias which arise from the intestinal type epithelium. Verhandlungen der Deutschen Gesellschaft fir Pathologie, 69, 459. Lapertosa, G., Baracchini, P., Fulcheri, E. and Tanzi, R. (1986). 0-acetylated sialic acid variants in intestinal glandular metaplasia of the urinary tract. Histopathology, 10, 707-7 12.

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Skye, D. V. (1975). Hydronephrosis secondary to focal papillary hyperplasia of the urinary bladder of cattle. Journal of the American Veterinary Medical Association, 166, 596-598.

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Stoward, P. J., Spicer, S. S. and Miller, R. L. (1980). Histochemical reactivity of peanut lectin-horseradish peroxidase conjugate. Journal of Histochemistry and Cytochemistry, 28, 979-990.

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Yoshikawa, T. and Oyamada, T. (1975). Histopathology of papillary tumors in the bovine urinary bladder. Japanese Journal of Veterinary Science, 37, 469-479. Zachary, J. F. (1981). Cystitis cystica, cystitis glandularis, and Brunn’s nestsin a feline urinary bladder. Veterinary Pathology, 18, 113-l 16.

1

Received, March 13th, 1992 Accepted, May 2Oth, 1992