Cystitis Cystica: An Electron and Immunofluorescence Microscopic Study

0022-5347 /87 /1374-0764$02.00/0 THE JOURNAL OF UROLOGY

Vol. 137, April Printed in U.S.A.

Copyright © 1987 by The Williams & Wilkins Co.

CYSTITIS CYSTICA: AN ELECTRON AND IMMUNOFLUORESCENCE MICROSCOPIC STUDY McCLELLAN M. WALTHER,* WALLACE G. CAMPBELL, JR., DAVID P. O'BRIEN, III, JOSEPH K. WHEATLEY AND SAM D. GRAHAM, JR. From the Departments of Urology and Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia

ABSTRACT

Cystitis cystica was studied with the aid of electron and immunofluorescence microscopy. By electron microscopy, the epithelium demonstrated morphologic features suggestive of an active metabolism. Secretory-type granules were seen in the cytoplasm just beneath the luminal membrane of surface columnar cells. Microvilli of the plasma membrane also were seen at the luminal surface. Rough endoplasmic reticulum and Golgi apparatuses were present. The cells were rich in mitochon dria. By immunofluorescence microscopy, IgA, secretory piece and IgM were localized in the epithelial cells, especially at the luminal surfaces. IgG was occasionally found. These findings contrast markedly with the transitional cells and their relatively scanty content of secretory-type organelles. In addition, they may explain the large amounts of IgA in the urine of patients with cystitis cystica. The association of cystitis cystica with chronic infections of the urinary tract and with chronic irritation by foreign bodies in the urinary tract has long been recognized. Increasingly, evidence suggests that clinically symptomatic lesions are a manifestation of a recently appreciated immunological mechanism by which the urinary tract attempts to react to an infection or irritation within the bladder. In support of this, Hill1 was able to produce the lesions of pyeloureteritis cystica and glandularis by introducing E. coli intravenously in rabbits after unilateral ureteral ligation. Further data suggest the local secretion and/or production of IgA in patients with cystitis cystica. IgA and secretory IgA are normally present in the urine. Elevated IgA and secretory IgA in the urine has been demonstrated in children with urinary tract infections. 2 IgA and secretory IgA excretion in the urine of children with the cystoscopic findings of cystitis cystica has been shown to be significantly elevated over that of normal children.'1 Bladder washings of patients with defunctionalized urinary bladders were found to contain an IgA:IgG ratio eight times greater than that found in serum. 4 Since this elevation in ratio was seen in both men and women, a non-prostatic origin of the secretory IgA was suggested. Unfortunately, in that study the presence of cystitis cystica was not documented. With these considerations in mind, we studied a series of patients with cystitis cystica by electron and immunofluorescence microscopy. MATERIALS AND METHODS

Cold cup biopsies from 24 patients with the cystoscopic findings of cystitis cystica and glandularis were studied by light microscopy. The World Health Organization nomenclature, cystitis cystica, is used henceforth, since no justification has been put forth for histologic differentiation between cystitis cystica and cystitis glandularis. 5 • 6 Fourteen patients had cystitis cystica with tall columnar cells lining the lumens of the cysts. Five patients had cystitis cystica with flat cells lining the lumens. One patient had von Brunn's nests only, and four had no evidence of cystitis cystica. No mucin-producing cells or evidences of the colonic variant of cystitis cystica were seen in any of the biopsies, implying that these features are unusual in cystitis cystica. Biopsy proven Accepted for publication October 15, 1986. *Requests for reprints: Emory Clinic, Dept. of Urology, 1365 Clifton Road N.E., Atlanta, GA 30322.

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specimens were then examined further using electron and immunofluorescence microscopy. The study was ultimately confined to seven males and four females. The patients ranged in age from 21 to 82 years (mean age = 57 yrs., median age = 61 yrs.). Five were white and six were black. The patients' diagnosis was pelvic lipomatosis in three, recurrent urinary tract infection in three, irritative voiding symptoms in two, carcinoma of the prostate in two, and benign prostatic hypertrophy in one. There was no evidence of reflux in this group. All patients had normal submocosal tunnels at cystoscopy. Only one patient had a cystogram performed, which showed no reflux. One patient had a history of pyelonephritis. Urine cultures grew Pseudomonas aeruginosa in one, Serratia marcescens in one, yielded no growth in four, and were contaminated or were not done in five. RESULTS

Three specimens were suitable for both electron microscopy (EM) and immunofluorescence microscopy (IM). Four specimens were suitable only for EM anrl fou_r only for IM, Previously described techniques were employed. 7 The findings are summarized in table 1. Because of the small size of the biopsies, both EM and IM often could not be done on the same specimen. Electron microscopy revealed that the epithelium of most cystic structures was characterized by stratification of rather elongate cells with nuclei often basally placed (fig. lA ). The cytoplasm of these cells usually contained abundant mitochondria. Numerous secondary lysosomes were present. Golgi apparatuses and rough endoplasmic reticulum (RER) were present. The cells of the urothelial surface were characterized by tight junctions at their margins. The cytoplasm beneath the plasma membranes of the surface cells contained small secretory-type granules with electron dense centers (SG) and a peripheral single limiting membrane (fig. lB). The plasma membrane on the surface sometimes had areas of disruption and duplication where sloughing of the cells apparently had occurred. Varying numbers of microvilli were seen in areas where sloughing had not occurred. The surface microvilli (MV) were true microvilli, with core filaments and a terminal web (fig. 2). Complex lateral spaces were sporadically present. Lipid droplets and residual bodies were occasionally seen. At the base of the lesions (fig. lC) the cells were flatter and more characteristic of transitional epithielium. The cytoplasm was rela-

f'J; 7Ov

CYSTITIS CYSTIC.A TABLE

Pt# Sex Age

Ja

M

1. Results of eleven patients studied by electron and immunofluorescence microscopy Light Microscopy

2

Columnar epithelium Dark and light cells Tight junctions Sparse microvilli with occasional rootlets Surface granules Mitochondria Golgi Rer

Not adequate

Cystitis cystica Tall superficial cells

Stratified columnar epithelium Dark and light cells Tight junctions Surface granules Few golgi Rer Moderate mitochondria

Not adequate

Granulation tissue with cystitis cystica Few glands with tall epithelium

Not adequate

Pas: lgA, + Neg: C and fibrinogen

Chronic cystitis cystica and focal squamous metaplasia One gland lined hy tall epithelium

Stratified epithelium Light cells Tight junctions Surface sloughing with few microvilli Surface granules Rer Many mitochondria

Not adequate

Mild chronic cystitis cystica Few glands Focal squamous metaplasia

Not adequate

Pos: lgA, + lgM,+ Neg: C and fibrinogen

Chronic cystitis cystica

Columnar epithelium Predominantly light cells Tight junctions Surface sloughing of membrane and granules Irregular microvilli in areas of no recent sloughing Surface granules Many mitochondria Many golgi Rer Much ser Complex lateral spaces Paucity of organelles in basilar cells

Not adequate

Chronic cystitis cystica Tall superficial cells

Stratified columnar epithelium Dark and light cells Tight junctions Few granules Primitive microvilli Mitochondria Golgi Much rer Much ser Complex lateral spaces with lateral microvilli

Pas: lgA, +++TO++++ SC,++++ lgG, + lgM, ++TO+++ Neg: C and fibrinogen

Chronic cystitis cystica Tall superficial cells

Stratified epithelium Predominantly light cells Tight junctions Surface granules Microvilli with rootlets Mitochondria Many golgi Rer Few lateral spaces

Pos: lgA, ++TO+++ SC, ++TO+++ lgG, + lgM, +TO++ Neg: C and fibrinogen

64

3

M 82

4b

F 53

5

F 64

6"

M 28

7c,d

M 60

8"·d

M 61

Fluorescence Microscopy

Cystitis cystica Tall superficial cells

21

M

Electron Microscopy

766 TABLE 1.

WALTHER AND ASSOCIATES

Continued Pt# Sex Age gd

F 36

10

F

Light Microscopy

Electron Microscopy

Chronic cystitis Brunn's nests Focal squamous metaplasia

Not adequate

Pos: IgA, + SC,+ IgG, + IgM, + Neg: C and fibrinogen

Cystitis cystica Tall superficial cells

Not adequate

Pos: IgA, +++ SC,++++ IgG, +++ IgM, +++ Neg: C and fibrinogen

Cystitis cystica with calcifications Tall superficial cells

Columnar epithelium Dark and light cells Tight junctions Surface granules Microvilli with rootlets Mitochondria Golgi Much rer Ser Lipid droplets Lateral spaces with lateral microvilli Many residual bodies

Pos: IgA, + SC,+ lgG, + IgM, + Neg: C and fibrinogen

75

11

M 80

Fluorescence Microscopy

M = male. F = female. Age in years. 'Pelvic lipomatosis. hSerratia urine infection. 'Pseudomonas urine infection. dRecurrent urine infection. RER Endoplasmic Reticulum. SER = Smooth Endoplasmic Reticulum. SC = Secretory component. C = Complement.

= Rough

FIG. 1. A, 30-year-old black male with pelvic lipomatosis and previously biopsied cystitis glandularis had recurrent irritative voiding symptoms without urinary tract infection. Biopsy revealed stratified type of cystitis cystica. EM was performed. Secretory-type granules are seen just below luminal surface. L = lumen, B = basal portion. Lead citrate and uranyl acetate. X6585. B, detail of luminal surface of cystitis cystica. SG = secretory granule, T J = tight junction, G = golgi apparatus. Lead citrate and uranyl acetate. X13,675. C, detail of base of cystitis cystica. Cells rest on basal lamina (arrows). Lead citrate and uranyl citrate, X 8450. (table 1, pt. no. 6)

tively devoid of organelles and electron dense granules were not seen. The epithelium rested on a basal lamina. Some cystic structures within the superficial lamina propria were lined by a columnar epithelium that showed a tendency to be pseudostratified (fig. 3). Light and dark cells were present, which is not uncommon in epithelial surfaces. The lighter cells were more commonly located along the base of the lesion. The darker cells became more common at the luminal surface and were characterized by an increased density of organelles, particularly rough endoplasmic reticulum, smooth endoplasmic reticulum, and Golgi apparatuses, in comparison with the light cells. The increased density of organelles suggests that the dark cells are the metabolically more active cells. Sparse microvilli with rootlets lined the luminal surface. Secretory granules were

present in both type of cells. Complex lateral spaces with lateral microvilli were sometimes present. Immunofluorescence studies revealed that the surface epithelium of cystitis cystica stained positively for the presence of IgA, IgM, and secretory piece. A representative positive stain is shown in fig. 4A. Background staining of the interstitial tissue was present and is normal. A negative control using antiC1q was negative (fig. 4B). Squamous metaplasia seen in one case did not stain for IgA, IgM, secretory piece, IgG or complement. DISCUSSION

Cystitis cystica is a commonly seen, but poorly understood lesion of the urinary tract. Both children8 and adults 9 may be

CYSTITIS CYSTICA

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epithelium. The cells are more polygonal with centrally placed nuclei. They have fewer mitochondria and rare secretory-type granules. These studies demonstrate the similarity between luminal lining cells in both the pseudostratified and stratified types of cystitis cystica at the electron microscopic level. The development of cystitis cystica of the mucin producing type, as shown by our biopsies, is not a common occurrence. This study contained no cases of this type and therefore adds no information regarding the morphologic features of this variant. An active metabolism in the epithelial cell is suggested by the presence of secretory-type granules just below the luminal surface. There appears to be a progression from a resting phase of light cells at the base to more mature dark cells which eventually slough at the surface. Immunofluorescence studies localize IgA, secretory piece, and IgM to the surface portion of the epithelium, where the secretory type granules were located. It would thus appear that cystitis cystica and glandularis may be part of a humoral immunological surveillance mechanism for the urinary tract. In the GI tract, the mechanism of delivery of lgA has been clearly defined. 12 · 13 lgA produced by the tissues is taken up by the columnar cells of intestinal epithelium and then bound to secretory piece produced by the epithelial cell. The secretory piece-IgA complex is then transported to the luminal surface by cytoplasmic vesicles and excreted. Further work must be done to define the precise mode of secretion of IgA in the urinary tract, but our studies suggest that a similar mechanism may be at work.

Fm. 2. 80-year-old man was evaluated for obstructive symptoms. Cystoscopy revealed prostatic enlargement and cystitis cystica. This was confirmed by biopsy. EM was performed. Typical secretory granules (SG) and true microvilli (MV) with core filaments and terminal bar are present. T J = tight junction. Lead citrate and uranyl citrate. X20,070. (table 1, pt. no. 11)

affected. Infection or obstruction may or may not coexist. The presence of these lesions almost invariably portends a prolonged clinical course. It is therefore interesting that in grossly normal bladders examined at autopsy, the microscopic presence of Brunn's nests, cystitis cystica, and cystitis glandularis is the norm rather than the exception. Weiner and coworkers 10 found Brunn's nests in 89% and cystitis cystica in 60% of 100 grossly normal urinary bladders. Only one of 100 bladders had neither Brunn's nests nor cystitis cystica. Ito and coworkersn reported Brunn's nests in 93.6% and cystitis glandularis in 71.2% of 125 grossly normal urinary bladders. Six of 125 bladders had neither Brunn's nests nor cystitis glandularis. These data suggest that Brunn's nests are a normal anatomic finding, and may be present in all bladders. It is suggested that the Brunn's nests lie dormant, and, under the stimulus of infection or irritation, proliferate and mature in a local attempt to rid the bladder of the offensive body. Normal transitional epithelium consists of polygonal cells without organelles associated with secretory activity. In contrast, cystitis cystica consists of polygonal to columnar cells with basally placed nuclei. The cells are stratified or pseudostratified and surround a central lumen. True microvilli occur along the surface of the plasma membranes. These cells are rich in mitochondria, Golgi apparatuses, rough endoplasmic reticulum, and electron dense secetory-type granules located below the plasma membrane at the luminal surface. Stratified below this layer are cells more characteristic of transitional

FIG. 3. 21-year-old black male with pelvic lipomatosis and previously biopsied cystitis glandularis had irritative voiding symptoms with no urinary tract infection. Biopsy revealed pseudostratified type of cystitis cystica and EM was performed. This section shows large perinuclear Golgi, secretory granules, and true microvilli, typical of cystitis cystica. SG = secretory granule, T J = tight junction, G = Golgi apparatus. Lead citrate and uranyl citrate. X9010. (table 1, pt. no. 1)

768

WALTHER AND ASSOCIATES

Fm. 4. lmmunofluorescence study of cystitis cystica. A, this cross section of nodule of cystitis cystica demonstrates strong surface uptake of anti-lgM (arrows). X250. B, minimal staining is seen with negative control, anti-C1q. X250. (table 1, pt. no. 7) In summary, the characteristic gland-like structures of cystitis cystica are lined by epithelium with features of secretory cells including abundant mitochondria, Golgi apparatuses, and rough endoplasmic reticulum. Secretory-type granules are consistently seen just beneath the plasma membranes of the surface cells. lmmunofluorescence localizes lgA, lgM and secretory pieGe-in -the -epitae1ium-0f-these-Gell-s. -'I'-aese-f-indi-ngs may account, in part, for the large amounts of lgA and secretory piece found in the urine of some patients with these conditions. Acknowledgments. Special thanks to Robert A. Santoianni and Willy B. Thomas for their technical expertise in performing the electron microscopic and immunofluorescence microscopic studies.

5. Koss, L. G.: Atlas of Tumor Pathology, Tumors of the Urinary Bladder. AFIP. Washington. Fascicle 11, Second Series, p. 5,

1975. 6. Mostofi, F. K., Sobin, L. and Torloni, H.: International Histological

7. 8. 9. 10.

REFERENCES

11. 1. Hill, G. S.: Experimental production of pyeloureteritis cystica and glandularis. Invest. Urol., 9: 1, 1971. 2. Uehling, D. T. and Steihm, E. R.: Elevated urinary secretory lgA in children with urinary tract infection. Pediatrics, 47: 40, 1971. 3. Uehling, D. T. and King, L. R.: Secretory immunoglobulin-A excretion in cystitis cystica. Urology, 1: 305, 1973. 4. Feldman, B. H., Herdman, R. and Hong, R.: Local immunomechanism of the urinary tract. Invest. Urol., 8: 575, 1971.

12. 13.

Classification of Tumours. Geneva: World Health Organization, No. 10, p. 33, 1973. Nolting, S. F. and Campbell, W. G. Jr.: Subepithelial argyrophilic spicular structures- in -renal amyloidosi:s=an·aid in di:agno-sis. Human Pathol., 12: 724, 1981. Belman, A. B.: The clinical significance of cystitis cystica in girls: results of a prospective study. J. Urol., 119: 661, 1978. Parker, C.: Cystitis cystica and glandularis: a study of 40 cases. Proc. Royal Society Med., 63: 239, 1970. Wiener, D. P., Koss, L. G., Sablay, B. and Freed, S. Z.: The prevalence and significance of Brunn's nests, cystitis cystica, and squamous metaplasia in normal bladders. J. Urol., 122: 317, 1979. Ito, N., Hirose, M., Shirai, T., Tsuda, H., Nakanishi, K. and Fukushima, S.: Lesions of the urinary bladder epithelium in 125 autopsy cases. Acta Pathol. Japan, 31: 545, 1981. Brown, W.R., Isobe, Y. and Nakane, P. K.: Studies on translocaton of immunoglobulins across intestinal epithelium. Gastroenterology, 71: 985, 1976. Poger, M. and Lamm, M.: Localization of free and bound secretory component in human intestinal epithelial cells. J. Exp. Med., 139: 629, 1974.