Evidence for Urothelial Cell Activation in Interstitial Cystitis

0022-534 7/93/1493-04 70$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1993 by AMERICAN UROLOGICAL ASSOCIATION, INC.

Vol. 149, 470-475, March 1993

Printed in U.S.A.

EVIDENCE FOR UROTHELIAL CELL ACTIVATION IN INTERSTITIAL CYSTITIS MONICA LIEBERT,* GARY WEDEMEYER, JUDITH A. STEIN, RAYMOND WASHINGTON, JR., GARY FAERBER, ANDREW FLINT AND H. BARTON GROSSMAN From the Section of Urology and Department of Pathology, University of Michigan, Ann Arbor, Michigan

ABSTRACT

Bladder biopsy samples from 17 interstitial cystitis patients and 20 controls were evaluated for urothelial cell activation using a panel of monoclonal antibodies to HLA-DR, intercellular adhesion molecule 1, interleukin 1 a and tumor necrosis factor a. Urothelial cells in the majority (13 of 16, 81 %) of the biopsies from patients with interstitial cystitis showed increased expression of HLADR, while fewer samples were positive for intercellular adhesion molecule 1 (3 of 16, 19%), interleukin 1 a (2 of 17, 12%) or tumor necrosis factor a (1 of 15, 7%). No urothelial cell expression of intercellular adhesion molecule 1, interleukin 1 a or tumor necrosis factor a was detected in the controls, and only 1 of 20 control samples contained HLA-DR positive urothelial cells. These results suggest that an unusual type of cellular activation is present in interstitial cystitis. In vitro studies with cultured normal urothelial cells indicated that cells activated with 'Y interferon and tumor necrosis factor a expressed intercellular adhesion molecule 1 and HLA-DR, although increases in intercellular adhesion molecule 1 expression occurred earlier. Urothelial cells in interstitial cystitis patients may be defective in ability to express intercellular adhesion molecule 1. Alternatively, the differential expression of HLA-DR and intercellular adhesion molecule 1 in interstitial cystitis specimens may represent a functional subset of interstitial cystitis or reflect different stages of the disease. Urothelial cell activation in interstitial cystitis may result in aberrant immune responses and immune activation within the bladder. Because HLA-DR can be detected in paraffin-embedded tissues, evaluation of urothelial cell HLA-DR expression, although not specific for interstitial cystitis, may become a useful tool in the pathological evaluation of biopsy tissues from patients with this disease. KEY WORDS: cystitis, HLA-DR antigens, bladder

Interstitial cystitis is a poorly understood bladder condition causing pain and increasing frequency of urination, and resulting in some cases in fibrosis that can limit bladder capacity. 1 The histopathological appearance of this disease is submucosal inflammation, largely of lymphocytes, along with thinned or ulcerated urothelial layers. 2 The cause and pathophysiology of interstitial cystitis are unknown. Immune cells, when activated, show increased expression of several cell surface antigens, including HLA-DR and intercellular adhesion molecule 1.3 These activated immune cells also may produce cytokines, such as interleukin 1 a and tumor necrosis factor a. Recent reports indicate that epithelial cells also can respond to immune stimulation by the production of cytokines and expression of an activated cell surface phenotype. 4 - 8 Previous results from our laboratory have shown that cultured normal urothelial cells activated in vitro express increased cellular HLA-DR and intercellular adhesion molecule 1, and produce interleukin 1 a and interleukin 8, another cytokine. 9 These results suggest that urothelial cells can participate in inflammation such as occurs in interstitial cystitis. To determine if urothelial cell activation is present in interstitial cystitis, we investigated the cell surface phenotype and cytokine expression by urothelial cells from biopsies of patients with interstitial cystitis. Accepted for publication August 14, 1992. Supported by a grant from the Interstitial Cystitis Association and Grant 1-R01-DK44804 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health and National Health and Human Services. Read at annual meeting of American Urological Association, Washington, D. C., May 10-14, 1992. * Requests for reprints: Section of Urology, University of Michigan, Box 0330, Room 2916 TC, Ann Arbor, Michigan 48109. 470

MATERIALS AND METHODS

Cell culture. Normal urothelial cells were propagated from ureteral segments obtained from living-related kidney donors as previously described. 10 Briefly, urothelial cells were scraped from the ureteral lumen into serum-free medium. The cultures were fed twice a week with fresh medium. After 10 to 14 days small colonies of urothelial cells appear in the culture; these cells were passed by trypsinization into tissue culture flasks. Cultures were used for experiments at passages 2 to 6. In some experiments the cells were stimulated by the addition of 'Y interferon at 1,000 units per ml. and tumor necrosis factor a at 500 units per ml. added to the serum-free medium. Antibodies. All primary antibodies were murine monoclonal antibodies, including MOPC-21, a murine IgGl myeloma protein; W6/32, a monoclonal antibody to the HLA class I framework antigen; antiHLA-DR antibody IVA 12; antiHLA-DR antibody (HB145); anti-intercellular adhesion molecule 1; BQ16, a monoclonal antibody prepared in our laboratory directed against the a 6 integrin subunit; antitumor necrosis factor a, and anti-interleukin 1 a. Enzyme-linked immunosorbent assay (ELISA). Antigen expression was evaluated using ELISA as previously described.11· 12 Briefly, cultured normal urothelial cells were plated into a sterile 96-well tissue culture dish and allowed to adhere for 24 hours in serum-free medium. Fresh medium was added, including in some cases 'Y interferon (1,000 units per ml.) and/ or tumor necrosis factor a (500 units per ml.). The cells were cultured under these conditions for various periods. Cultures were terminated by fixing the cells for 10 minutes with formalin (10% in phosphate buffered saline). After fixation, the formalin was removed and the fixed cells were washed and stored in phosphate buffered saline at 4C until tested. For ELISA the

E'lIDE:NCE FOR URO'I'H:ELIAL CELL ACTIVATION IN 1t:.JTERSTITIAL CYSTITIS

cells were initially blocked for 30 minutes with phosphate buffered saline containing 5% 'Y globulin-free fetal bovine serum, then rinsed in phosphate buffered saline and incubated for 1 hour with primary antibodies, including MOPC-21, a murine myeloma protein as negative control used at 10 µg./ml.; antiHLA-DR (HB145), used as tissue cultured supernatant, and anti-intercellular adhesion molecule 1, used at 10 µg./ml. Antibodies that required dilution were diluted in fetal bovine serum/phosphate buffered saline. After this incubation the plates were washed 5 times with phosphate buffered saline, and then incubated for 1 hour with peroxidase conjugated goat antimouse immunoglobulin diluted 1/500 in fetal bovine serum/ phosphate buffered saline. The plates were washed 5 times with phosphate buffered salin€ and developed with indicator (0.5% 2,2' -azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] diluted 1/10 in 0.1 M. citrate buffer, pH 4) containing hydrogen peroxide for 30 minutes. The resulting blue-green color was read at 600 nm. on a plate reader. The experiment was duplicated using cells from different donors. Flow cytometry. Cultured urothelial cells were stimulated for 3 days in serum-free medium supplemented with 500 units per ml. tumor necrosis factor a and 1,000 units per ml. 'Y interferon, and control cells were cultured in unsupplemented serum-free medium. The cells were trypsinized to obtain a single cell suspension and washed once with fetal bovine serum/phosphate buffered saline. Aliquots of the cells were incubated for 1 hour on ice with MOPC-21 (negative control at 10 µg./ml.), W6/32 (antiHLA class I framework antibody, hybridoma tissue culture supernatant), anti-intercellular adhesion molecule 1 (purified antibody diluted to 4 µg./ml.) or antiHLA-DR (HB145, hybridoma tissue culture supernatant). All antibody dilutions were prepared in fetal bovine serum/phosphate buffered saline. After this incubation the cells were washed in fetal bovine serum/ phosphate buffered saline, and fluorescein-isothiocyanate conjugated goat anti-mouse IgG,A,M diluted 1/40 in fetal bovine serum/phosphate buffered saline was added to each aliquot and the cells were incubated in the dark for 1 hour on ice. The cells were washed twice and resuspended in fetal bovine serum/ phosphate buffered saline, and evaluated on a Coulter EPICS C flow cytometer. The same experiment was performed using cells from 3 different donors with similar results. Tissue staining. Informed consent was obtained from patients with the diagnosis of interstitial cystitis as defined by National Institutes of Health criteria. 1 Bladder biopsies were obtained immediately after hydrodistention to confirm the presence of glomerulations. Most tissues were obtained from new patients at initial evaluation. None of the patients had Hunner's ulcers. Biopsies were either taken randomly or from areas showing glomerulations. Control tissues included normal ureter from transplant donors or patients with renal or bladder cancer, grossly normal-appearing areas of bladder obtained from cystectomy specimens from patients with bladder cancer and a bladder biopsy from 1 patient undergoing transurethral resection of the prostate for benign prostatic hypertrophy. All tissues were snap frozen in liquid nitrogen, embedded in frozen tissue mounting medium and sectioned on a cryostat. Tissue sections were fixed for 5 minutes in ice-cold acetone, then incubated for 30 minutes with normal horse serum diluted 1/20 in phosphate buffered saline to block nonspecific antibody binding. The sections were blotted and covered with primary antibody. The primary antibodies included MOPC-21 (negative control at 10 µg./ml.), antiHLA-DR (diluted 1/50), anti-intercellular adhesion molecule 1 (4 /.lg./ml.), W6/32 (antiHLA class I framework antibody, hybridoma tissue culture supernatant), anti-interleukin 1 a (1/500), antitumor necrosis factor a (1/500) and BQ16 (hybridoma tissue culture supernatant). W6/32 and BQ16 served as positive controls. The sections were incubated with primary antibody for 1 hour, washed 3 times in phosphate buffered saline, then incubated for 1 hour in the dark with fluorescein isothiocyanate

471

conjugated goat anti-rnouse IgG diluted in fetal bovine serum/phosphate buffered saline and filtered before use with a 0.45 µm. low protein-binding filter for 1 hour in the dark. The sections were washed 3 times with phosphate buffered saline and mounted in glycerine containing 5% 0.1 M. sodium bicarbonate, pH 9.6, to reduce quenching. The slides were stored at 4C in the dark until they were evaluated on a fluorescence microscope. Reactivity with BQ16 identified the junction of the urothelium with the basement membrane-like area above the lamina propria for orientation and confirmed that urothelium was present in the biopsy specimens. Reactions were scored as negative, weakly positive, 1+ positive or 2+ positive based on the intensity of the reaction. Weakly positive reactions are scored as negative in the table. If fewer than 50% of the urothelial cells were positive with 1+ or 2+ intensity, the reaction was noted to be patchy. Positive reactions were documented by photography. RESULTS

Tissue staining. Frozen tissue specimens from 19 patients were stained with a panel of monoclonal antibodies to evaluate the expression of activation antigens. Two specimens failed to stain with the anti-integrin a 6 antibody and, therefore, the presence of urothelial cells in the specimens could not be positively identified. These samples were not included in further analysis. As shown in the table 13 of 16 specimens (81 %) from patients with interstitial cystitis showed expression of HLA-DR, while only 3 of 16 (19%) and 2 of 17 (12%) showed expression of intercellular adhesion molecule 1 and interleukin 1 a, respectively. Samples positive for intercellular adhesion molecule 1 or interleukin 1 a were also positive for HLA-DR expression. Little expression of any of these antigens was observed in the normal bladder samples, as shown in the table. Examples of tissue staining for HLA-DR and interleukin 1 a are shown in figure 1. In figure 1, A all urothelial cells present show strong staining for HLA-DR. In figure 1, D patchy (although strong) HLA-DR staining of urothelial cells is seen throughout the various urothelial cell layers. Expression of activation antigens by cultured normal urothelial cells. Cultured normal urothelial cells were activated with 'Y interferon and tumor necrosis factor a (1,000 and 500 units per ml., respectively) for 3 days, harvested and evaluated by flow cytometry for expression of the activation antigens HLA-DR and intercellular adhesion molecule 1. As indicated in figure 2, urothelial cells showed no binding of antiHLA-DR or antiintercellular adhesion molecule 1 before stimulation, while after stimulation increased binding of HLA-DR and intercellular adhesion molecule 1 antibodies was detected. These results confirm our previous observations with ureteral pieces stimulated in situ9 and indicate that urothelial cells, like keratinocytes, show little or no expression of HLA-DR or intercellular adhesion molecule 1 before stimulation but are capable of responding to cytokine stimulation by the expression of intercellular adhesion molecule 1 and HLA-DR. 7, 3, 13 The time-course of this response was evaluated by ELISA, as shown in figure 3. Cells were stimulated with 'Y interferon and tumor necrosis factor a (1,000 and 500 units per ml., respectively) for 1 hour and then fresh culture medium was placed in each well. The cultures were continued for various times after stimulation, As shown in figure 3, cultured normal urothelial cells express significant levels of intercellular adhesion molecule 1 within 6 hours after stimulation with 'Y interferon and tumor necrosis factor a. However, the levels of HLADR remained low until 1 to 2 days after culture, and they continued to increase until reaching a plateau at 3 days after stimulation. DISCUSSION

Our results have shown that cultured urothelial cells respond to stimulation in vitro with 'Y interferon and tumor necrosis

472

LIEBERT AND ASSOCIATES

Tissue staining Pt. No.-Age*-Sex

Tissue Staining Bladder Conditiont

Intracellular Adhesion Molecule 1

HLA-DR

Interleukin 1 "

Tumor Necrosis Factor "

Interstitial cystitis group 1- 49 -F

13- 36

-F

Small

Pos. Pos. Pos. Pos. Pos. Pos. Pos. Patchy Patchy Neg. Neg. Not done or tissue was lost Neg.

14- 39 15- 69

-F -F

Small Normal

Patchy Pos.

16- 21 -F 17- 57 -F Total No. pos./total No.

Normal Normal

Pos. Patchy 13/16

23456789101112-

36 -F 30 -F 52 -F 27 -F 39 -F 23 -F 45 -F 36 -F 38 -F 87 -F 49 -F

Small Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal

Neg. Neg. Neg. Neg. Neg. Neg. Pos. Neg. Neg. Neg. Neg. Neg.

Neg. Neg. Neg. Neg. Neg. Neg. Pos. Patchy Neg. Neg. Neg. Neg.

Neg.

Neg.

Patchy Not done or tissue was lost Pos. Neg. 3/16

Neg. Neg. Neg. Neg. 2/17

0/9 0/4 0/1

0/9 0/4 0/1

0/6 0/20 Not significant

0/5 0/19 Not significant

Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Not done or tissue was lost Not done or tissue was lost Patchy Not done or tissue was lost Neg. Neg. 1/15

Control group

18-26-47-82-M 27-30-53-70-F 31- 67-M 32-37-38-64 Total No. pos./total No.

Bladder Ca:j: Bladder Ca Benign prostatic hypertrophy Normal ureter

1/9 0/4 0/1 0/6 1/20 p <0.005§

0/9 0/4 Not done or tissue was lost 0/5 0/18 Not significant

* Age at time specimen was obtained.

t Bladder capacity was defined as small if less than 350 ml. or normal if greater than 350 ml. with the patient under anesthesia. :j: Samples taken from grossly normal-appearing areas in cystectomy specimens for bladder cancer. § Significance of difference between interstitial cystitis and control samples as determined by chi-square analysis.

FIG. 1. A to C, fluorescence microscopy of sections from interstitial cystitis biopsy. A, strong HLA-DR stain of all urothelial cells below white arrow. B, MOPC-21 (negative control antibody) contrasts lack of urothelial cell staining below white arrow. C, strong interleukin 1 a stain of all urothelial cells. A to C, reduced from Xl60. D and E, sections from another patient. D, patchy HLA-DR stain of urothelial cells at several cell layers below white arrowhead. E, MOPC-21 (negative control antibody) contrasts lack of urothelial cell staining below white arrowhead. D and E, reduced from X400. F, section from grossly normal area from patient with bladder cancer stained with HLA-DR shows slight staining of urothelial cells below white arrows. Reduced from Xl60.

factor a by expression of HLA-DR and intercellular adhesion molecule 1, antigens that were not detected before stimulation. This response occurred quickly in vitro with intercellular adhesion molecule 1 expression detected within 6 hours of stimulation and HLA-DR within 24 to 48 hours of stimulation. This expression persisted in vitro until 7 days after stimulation and for intercellular adhesion molecule 1 it occurred as soon as 30 minutes after stimulation (data not shown). In biopsies from patients with interstitial cystitis increased expression of HLA-DR was observed in 13 of 16 specimens

(81 %), while increased expression of intercellular adhesion molecule 1 was detected in 3 of 16 (19%) and interleukin 1 a in 2 of 17 (12%). These specimens were obtained immediately following hydrodistention, which potentially could contribute to an activation process. However, the in vitro time-course study indicated that HLA-DR expression on urothelial cells was not detected until 24 to 48 hours after activation, and that increased urothelial cell intercellular adhesion molecule 1 expression was detected first. These results would suggest that the interval was too short to produce activation, and that if

EVIDENCE FOR URDTHELJ:AL CELL ACTIVATION i:N INTERSTITIAL CYSTITIS

A.

Unstimulated Cell$ HlA-DR

300

R!J El

B.

4

Stimulated Cells

HLA DR 0

200

II

0

DR MOPC

DA MOPC

200

C.

Unstimulated Cells ICAM·1

D. 200

ICAM-1

~ ICAM-1

200

0

Stimulated Cells !221 ICAM I\/IOPC

MOPC

0 100

100

0

O 0

100

200

o

100

:wo

FIG. 2. Flow cytometric evaluation of HLA-DR (A and B) and intercellular adhesion molecule 1 (ICAM-1, C and D) expression on cultured urothelial cells before (A and C) and after (B and D) stimulation with 'Y interferon and tumor necrosis factor a. MOPC, MOPC-21 (negative control antibody). X axis is mean channel number, which is relative fluorescence intensity on logarithmic scale. Y axis is number of cells. Results are from single donor. Similar results were obtained with 2 other different donors. ICAM-1 AND DR EXPRESSION ON CUL TU RED NORMAL UROTHELIAL CELLS AFTER STIMULATION

1.0

ICAM-1 0.8

u"' z

0.6

"'"'a: 0

0.4

"'"' "'

0.2

0.0 0

20

40

60

80

100

120

TIME AFTER STIMULATION (HOURS)

FIG. 3. HLA-DR and intercellular adhesion molecule 1 (ICAM-1) expression on cultured urothelial cells after 'Y interferon and tumor necrosis factor a stimulation with time as evaluated by ELISA. Error bars indicate ±1 standard deviation.

activation was secondary to the hydrodistention and if the response was more rapid in vivo than detected in vitro, more frequent intercellular adhesion molecule 1 expression should be detected. Our results showed more frequent expression of HLA-DR than intercellular adhesion molecule 1 in interstitial cystitis biopsies, which suggests that hydrodistention was not the cause of activation in these tissues. Urothelial cells do not normally express HLA-DR. 14' 15 However, HLA-DR expression on apparently normal urothelial cells has been found after bacillus Calmette-Guerin treatment for bladder cancer. 16-2° Furthermore, urothelial cells in other control tissues in these studies, including bladder biopsies from patients receiving mitomycin C for bladder cancer and patients with nonspecific cystitis, did not express HLA-DR. 16 ' 18' 19 Of 16 biopsy specimens from our patients with interstitial cystitis 13 (81 %) demonstrated increased expression of HLA-DR. In a previous study Zhou et al also reported increased urothelial cell expression of HLA-DR in interstitial cystitis. 21 However, they thought that the expression of HLA-DR on urothelial cells was secondary to inflammation present in the interstitial cystitis tissues. Additionally, these investigators reported increased HLA-DR expression on the urothelial cells in biopsies from patients with nonspecific cystitis. We compared the urothelial

HLA-DR expression in biopsies from patients with interstitial cystitis to grossly normal bladder specimens obtained from patients with bladder cancer. These control specimens also have significant inflammation in the majority of cases (as evaluated by pathologist A. F.). However, increased urothelial cell HLA-DR was detected in only 1 of 13 of these cases (8%), and no other activation antigens were detected in these controls. These findings suggest that the expression of HLA-DR antigens on urothelial cells detected in the interstitial cystitis specimens is not merely due to the presence of inflammatory cells. Christmas and Bottazzo recently reported increased expression of HLA-DR on urothelial cells in interstitial cystitis biopsies and not in control tissues (bladder specimens from normal women and from patients with chronic cystitis). 22 Induction of HLA-DR expression on epithelial cells that normally do not express this antigen has been noted in several pathological conditions. HLA-DR is found in autoimmune diseases, including thyroid epithelium in autoimmune thyroid diseases, 23 ' 24 colonic epithelium in inflammatory bowel diseases,25'26 keratinocytes in psoriasis 8 ' 27·28 and pancreatic (3 cells in diabetes. 24 HLA-DR expression has been shown to be included on epithelial cells in some infectious diseases as well, including on corneal epithelial cells in trachoma, 29 colonic epithelium in infections of the bowel, 25 gastric epithelial cells in H. pylori gastritis 30 and cervical epithelium in viral infection of the cervix. 31 HLA-DR is also induced on keratinocytes in contact dermatitis and allergic patch tests. 4 ' 6' 26 In many cases induction of HLA-D R is spatially associated with inflammatory infiltrates. 4' 8 • 23 ' 26 ' 27·31 · 32 HLA-DR is known to function in the immune system by presenting processed antigen to activate lymphocytes, 3 The expression of HLA-DR antigens may serve to trigger or to increase local immune responses. 24,33 Induction of intercellular adhesion molecule 1 expression has also been identified on epithelial cells in pathological states. Intercellular adhesion molecule 1 is induced in vitro on renal proximal tubular cells and keratinocytes after cytokine stimulation.7"8'13'34 In vivo intercellular adhesion molecule 1 expression is increased on keratinocytes after allergic patch testing and in several disease states. 4'6'8' 27 Increased expression of intercellular adhesion molecule 1 is associated with leukocyte trafficking in the skin,4· 6 and it has a role in antigen presentation to a cloned T cell line by activated cultured renal proximal tubular cells. 34

474

LIEBERT AND ASSOCIATES

Thus, the typical response of activated or inflamed epithelial cells is the expression of intercellular adhesion molecule 1 and HLA·DR. However, induction of HLA-DR without increased epithelial cell intercellular adhesion molecule 1 expression has been reported on keratinocytes in Sezary's syndrome," 5 in cutaneous T cell lymphomas 27 and on gastric epithelial cells in H. pylori gastritis. 30 These responses may be the result of stimulation by a specific cytokine or set of cytokines that induce or cause persistence of HLA-DR expression without increasing intercellular adhesion molecule 1 expression. The biological significance of this expression is not known. In this study and our previous studies 9 we have shown that in short-term and long-term culture urothelial cells respond to cytokine stimulation with the expression of intercellular adhesion molecule 1 and HLA-DR. In contrast, urothelial cells in the majority of interstitial cystitis samples shov, only HLA-DR and not intercellular adhesion molecule 1 expression. Intercellular adhesion molecule 1 was found in only a few of the interstitial cystitis specimens. These observations may indicate that a specific type of inflammation (possibly an unusual cytokine or set of cytokines) is present in interstitial cystitis, or that urothelial cells from such patients may be defective in ability to express intercellular adhesion molecule 1. Alternatively, the differential expression of HLA-DR and intercellular adhesion molecule 1 in interstitial cystitis specimens may represent a functional subset of cystitis or reflect different stages of the disease. This type of urothelial cell activation in interstitial cystitis may result in aberrant immune responses and immune activation within the bladder. This activation could be the result of urothelial cell participation in cytokine networks (as proposed for keratinocytes 5 •6 ). We have previously shown that urothelial cells can produce and respond to cytokine signals in vitro. 9 Interstitial cystitis may involve a vicious circle in a cytokine network similar to the immune cycle originally proposed by Gordon et al for interstitial cystitis. 36 Furthermore, urothelial HLA-DR expression, although not specific for interstitial cystitis, may become a useful tool in the pathological evaluation of biopsy tissues from patients with this disease, especially because HLA-DR can be detected in paraffinembedded tissues. Dr. Suyu Shu provided comments and suggestions, and Margaret Brozovich assisted with the flow cytometry experiments.

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REFERENCES 1. Hanno, P., Levin, R. M., Monson, F. C., Teuscher, C., Zhou, Z. Z., Ruggieri, M., Whitmore, K. and Wein, A. J.: Diagnosis of interstitial cystitis. J. Urol., 143: 278, 1990. 2. Lynes, W. L., Flynn, S. D., Shortliffe, L. D. and Stamey, T. A.: The histology of interstitial cystitis. Amer. J. Surg. Path., 14: 969, 1990. 3. Male, D., Champion, B., Cooke, A. and Owen, M.: Advanced Immunology, 2nd ed. Philadelphia: J. B. Lippincott Co., 1991. 4. Griffiths, C. E. M. and Nickoloff, B. J.: Keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression precedes dermal T lymphocytic infiltration in allergic contact dermatitis (Rhus dermatitis). Amer. J. Path., 135: 1045, 1989. 5. Kupper, T. S.: The activated keratinocyte: a model for inducible cytokine production by non-bone marrow-derived cells in cutaneous inflammatory and immune responses. J. Invest. Dermatol., suppl. 6, 94: 1468, 1990. 6. Nickoloff, B. J.: Role of interferon-gamma in cutaneous trafficking of lymphocytes with emphasis on molecular and cellular adhesion events. Arch. Dermatol., 124: 1835, 1988. 7. Griffiths, C. E. M., Esmann, J., Fisher, G. J., Voorhees, J. J. and Nickoloff, B. J.: Differential modulation of keratinocyte intercellular adhesion molecule-I expression by gamma interferon and phorbol ester: evidence for involvement of protein kinase C signal transduction. Brit. J. Dermatol., 122: 333, 1990. 8. Griffiths, C. E. M., Voorhees, J. J. and Nickoloff, B. J.: Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal and inflamed skin: modulation by recom-

21.

22. 23.

24. 25. 26.

27.

binant gamma interferon and tumor necrosis factor. J. Amer. Acad. Dermatol., 20: 617, 1989. Liebert, M., Wedemeyer, G., Abruzzo, L. V., Kunkel, S. L., Hammerberg, C., Cooper, K. D. and Grossman, H.B.: Stimulated urothelial cells produce cytokines and express an activated cell surface antigenic phenotype. Sem. Urol., 9: 124, 1991. Liebert, M., Wedemeyer, G., Chang, J. H. C., Stein, J. A., McKeever, P. E., Carey, T. E., Flint, A., Steplewski, Z., Buchsbaum, D. J., Wahl, R. L. and Grossman, H.B.: Comparison of antigen expression on normal urothelial cells in tissue section and tissue culture. J. Urol., 144: 1288, 1990. Liebert, M., Ballou, B., Taylor, R. J., Reiland, J. M. and Hakala, T. R.: A method of membrane preparation for immunoassay. J. Immunol. Methods, 85: 97, 1985. Kennett, R. H.: Enzyme-linked antibody assay with cells attached to polyvinyl chloride plates. In: Monoclonal Antibodies: Hybridomas, A New Dimension in Biological Analysis. Edited by R.H. Kennett, T. J . .rv1cKearns and K. B. Bechtol. :t".Jew York: Plenuni Press, p. 376, 1980. Griffiths, C. E. M., Voorhees, J. J. and Nickoloff, B. J.: Gamma interferon induces different keratinocyte cellular patterns of expression of HLA-DR and DQ and intercellular adhesion molecule-I (ICAM-I) antigens. Brit. J. Dermatol., 120: 1, 1990. El-Demiry, M. I. M., Hargreave, T. B., Busuttil, A., James, K. and Chisholm, G. D.: Immunohistochemical identification of lymphocyte subsets and macrophages in normal human urothelium using monoclonal antibodies. Brit. J. Urol., 58: 436, 1986. Gardiner, R. A., Seymour, G. J., Lavin, M. F., Strutton, G. M., Gemmell, E. and Hazan, G.: Immunohistochemical analysis of the human bladder. Brit. J. Urol., 58: 19, 1986. el-Demiry, M. I. M., Smith, G., Ritchie, A. W. S., James, K., Cumming, J. A., Hargreave, T. B. and Chisholm, G. D.: Local immune responses after intravesical BCG treatment of carcinoma in situ. Brit. J. Urol., 60: 543, 1987. Stefanini, G. F., Bercovich, E., Mazzeo, V., Grigioni, W. F., Emili, E., D'Errico, A., Lo Cigno, M., Tamagnini, N. and Mazzetti, M.: Class I and class II HLA antigen expression by transitional cell carcinoma of the bladder: correlation with T-cell infiltration and BCG treatment. J. Urol., 141: 1449, 1989. Prescott, S., James, K., Busuttil, A., Hargreave, T. B., Chisholm, G. D. and Smyth, J. F.: HLA-DR expression by high grade superficial bladder cancer treated with BCG. Brit. J. Urol., 63: 264, 1989. Leong, A. 8.-Y., Wannakrairot, P., Jose, J. and Milios, J.: Bacillus Calmette-Guerin-treated superficial bladder cancer: correlation of morphology with immunophenotyping. J. Path., 162: 35, 1990. Bohle, A., Gerdes, J., Ulmer, A. J., Hofstetter, A. G. and Flad, H.-D.: Effects of local bacillus Calmette-Guerin therapy in patients with bladder carcinoma on immunocompetent cells of the bladder wall. J. Urol., 144: 53, 1990. Zhou, Z. Z., Zheng, Y., Monson, F. C., Wein, A. J., Ruggieri, M., Levin, R. M., Hanno, P., McKenna, B. A. W. and Teuscher, C.: Immunohistochemical analysis of the urinary bladder: a comparative study of the cellular immune response of patients diagnosed as having interstitial cystitis, noninterstitial cystitis voiding dysfunctions, and normal controls. Sem. Urol., 9: 115, 1991. Christmas, T. J. and Bottazzo, G. F.: Abnormal urothelial HLADR expression in interstitial cystitis. Clin. Exp. lmmunol., 87: 450, 1992. Hamilton, F., Black, M., Farquharson, M. S., Stewart, C. and Foulis, A. K.: Spatial correlation between thyroid epithelial cells expressing class II MHC molecules and interferon-gamma-containing lymphocytes in human thyroid autoimmune disease. Clin. Exp. Immunol., 83: 64, 1991. Foulis, A. K.: Class II major histocompatibility complex and organ specific autoimmunity in man. J. Path., 150: 5, 1986. McDonald, G. B. and Jewell, D. P.: Class II antigen (HLA-DR) expression by intestinal epithelial cells in inflammatory diseases of colon. J. Clin. Path., 40: 312, 1987. Fais, S., Pallone, F., Squarcia, 0., Biancone, L., Ricci, F., Paoluzi, P. and Boirivant, M.: HLA-DR antigens on colonic epithelial cells in inflammatory bowel disease: I. Relation to the state of activation of lamina propria lymphocytes and to the epithelial expression of other surface markers. Clin. Exp. Immunol., 68: 605, 1987. Vejlsgaard, G. L., Ralfkiaer, E., Avnstorp, C., Czajkowski, M.,

E'VIDEl'~CE FOR UROTJ-IELIAL CELL ACTIVATION lN II~TERSTI'TIAL- CYSTITIS

28.

29.

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Martin, S. D. and Rothlein, R.: Kinetics and characterization of intercellular adhesion molecule-I (ICAM-1) expression on keratinocytes in various inflammatory skin lesions and malignant cutaneous lymphomas. J. Amer. Acad. DermatoL, 20: 782, 1989. Gottlieb, A. B., Lifshitz, B., Fu, S. M., Staiano-Coico, L., Wang, C. Y. and Carter, D. M.: Expression of HLA-DR molecules by keratinocytes, and presence of Langerhans cells in the dermal infiltrate of active psoriatic plaques. J. Exp. Med., 164: 1013, 1986. Mabey, D. C. W., Bailey, R. L., Dunn, D., Jones, D., Willians, J. H. D., Whittle, H. C. and Ward, M. E.: Expression of MHC class II antigens by conjunctiva! epithelial cells in trachoma: implications concerning the pathogenesis of blinding disease. J. Clin. Path., 44: 285, 1991. Scheynius, A. and Engstrand, L.: Gastric epithelial cells in Helicobacter pylori-associated gastritis express HLA-DR but not ICAM-1. Scand. J. Immunol., 33: 237, 1991. Fais, S., Delle Fratte, F., Mancini, F., Cioni, V., Guadagno, M., Vetrano, G. and Pallone, F.: Human cervical epithelial cells that

32.

33.

34.

35.

36.

express HLA-DR associated with viral infection and activated mononuclear cell infiltmte. J. Clin. Path., 44: 290, 1991. Todd, I., Pujol-Borrell, R., Abdul-Karim, B. A. S., Hammond, L. J., Feldmann, M. and Bottazzo, G. F.: HLA-D subregion expression by thyroid epithelium in autoimmune thyroid diseases and induced in vitro. Clin. Exp. Immunol., 69: 532, 1987. Todd, I., Londei, 11/L, Pujol-Borrell, R., Mirakian, R., Feldmann, M. and Bottazzo, G, F.: HLA-D/DR expression on epithelial cells: the finger on the trigger? Ann. N. Y. Acad. Sci., 475: 241, 1986. Jevnikar, A. M., Wuthrich, R. P., Takei, F., Xu, H.-W., Brennan, D. C., Glimcher, L. H. and Rubin-Kelley, V. E.: Differing regulation and function of ICAM-1 and class II antigens on renal tubular cells. Kidney Int., 38: 417, 1990. Nickoloff, B. J., Griffiths, C. E. M., Baadsgaard, 0., Voorhees, J. J., Hanson, C. A. and Cooper, K. D.: Markedly diminished epidermal keratinocyte expression of intercellular adhesion molecule-1 (ICAM-1) In Sezary syndrome. J.A.M.A., 261: 2217, 1989. Gordon, H. L., Rossen, R. D., Hersh, E. M. and Yium, J. J.: Immunologic aspects of interstitial cystitis. J. Urol., 109: 228, 1973.