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Preliminary study on urinary cytokine levels in interstitial cystitis: does intravesical bacille Calmette-Guérin treat interstitial cystitis by altering the immune profile in the bladder?
ADULT UROLOGY
PRELIMINARY STUDY ON URINARY CYTOKINE LEVELS IN INTERSTITIAL CYSTITIS: DOES INTRAVESICAL BACILLE CALMETTE-GUE´RIN TREAT INTERSTITIAL CYSTITIS BY ALTERING THE IMMUNE PROFILE IN THE BLADDER? KENNETH M. PETERS, ANANIAS C. DIOKNO,
AND
BRUCE W. STEINERT
ABSTRACT Objectives. To obtain preliminary urinary cytokine data on subjects with active interstitial cystitis (IC), subjects with IC in remission after bacille Calmette-Gue´rin (BCG), and control (non-IC) subjects. IC is a severe, debilitating bladder disease of unknown etiology and no cure. In controlled clinical trials, intravesical BCG has been shown to be an effective and durable treatment for IC. The durability of this treatment led us to speculate on the mechanism by which intravesical BCG may treat IC. Evidence exists that IC may be mediated by an abnormal immune profile within the bladder. Intravesical BCG is known to stimulate the immune system of the bladder. Methods. Fresh voided urine was collected from 15 subjects with active IC, 9 subjects with IC who received intravesical BCG and had been in remission for an average of 2.6 years, and 11 non-IC subjects. The urine was immediately centrifuged, aliquoted, and frozen in liquid nitrogen. At the time of urine collection, a validated IC questionnaire was completed. The enzyme-linked immunosorbent assay technique was used to determine levels of urinary cytokines interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF), human granulocyte-macrophage colony stimulating factor (hGM-CSF), IL-1beta, and interferongamma (IFN-gamma). Results. Cytokines IL-4, IL-10, IL-12, TNF, hGM-CSF, IL-1beta, and IFN-gamma were not detected. Significant elevations in symptom scores and IL-2, IL-6, and IL-8 were found in the urine of subjects with active IC compared with subjects with IC in remission and control subjects. The urinary cytokine levels and symptom scores were identical in the IC group who had received BCG and the control group. Conclusions. Elevations in symptom scores and urinary cytokine levels were seen in subjects with active IC, suggesting an abnormal immune profile in this disease. Subjects with IC in remission after receiving BCG had identical cytokine levels and symptom scores as non-IC control subjects. Intravesical BCG may be effective in treating IC by correcting an aberrant immune imbalance in the bladder, leading to long-term symptomatic improvement. A prospective study is ongoing to further investigate the role of the immune system in IC. UROLOGY 54: 450–453, 1999. © 1999, Elsevier Science Inc.
I
nterstitial cystitis (IC) is an inflammatory disease of the bladder characterized by urinary urgency, frequency, and suprapubic pain. A recent study suggests the prevalence of IC is 60 in 100,000 individuals in the United States.1 The number of undiagnosed individuals affected may
From the Department of Urology, William Beaumont Hospital, Royal Oak, Michigan Reprint requests: Kenneth M. Peters, M.D., Department of Urology, William Beaumont Hospital, 3535 West 13 Mile Road, Suite 438, Royal Oak, MI 48073 Submitted: February 15, 1999, accepted (with revisions): March 10, 1999
450
© 1999, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
be much higher. The cause of IC is unknown despite a century of study. Intravesical bacille Calmette-Gue´rin (BCG) is a novel therapy for the treatment of IC. The efficacy, durability, and safety of intravesical BCG in the treatment of IC was demonstrated recently in a controlled clinical trial.2,3 This raises the question of why BCG would be effective in the treatment of IC. Although this therapy has been used for years in the treatment of bladder cancer, its exact mechanism of action is unknown. Pellegrini and coworkers4 hypothesize that the disregulation of the T-helper cell type 1 (Th-1)/T-helper cell type 2 0090-4295/99/$20.00 PII S0090-4295(99)00162-4
(Th-2) response correlates with tumor stage and may be related to the tumor progression and metastasis observed in advanced cases. BCG is a strong stimulus of the immune system in the bladder, and there is evidence that in some patients IC may be an autoimmune disease.5–10 Aberrant HLA-DR expression is found in the bladder of patients with IC and in other autoimmune diseases, including thyroid disease, inflammatory bowel disease, and psoriasis and in pancreatic cells in diabetes. HLA-DR is known to function in the immune system by presenting processed antigen to activate lymphocytes and serves to trigger local immune responses. An abnormal expression of HLA-DR on the urothelium of IC bladders may indicate a specific type of inflammation involving an unusual pattern of cytokine networks. It is known that specific cytokines stimulate either a Th-1 or Th-2 response.11 In general, a Th-2 response is thought to be associated with disease states, favoring antibody formation and inappropriate tissue necrosis. The maturation of T-lymphocytes concentrates along two distinct pathways, either Th-1 or Th-2 as defined by the prevalent cytokines. The pathways can have a common stimulus such as infection or tissue damage, but the response pathway determines whether the condition will resolve or develop into a chronic, ultimately debilitating condition. Intravesical BCG is a known stimulus of the Th-1 cytokine pathway.12,13 A study by Ochs et al.14 has shown a common autoantigen in serum from patients with IC and those with atopic dermatitis. Atopic dermatitis is a Th-2-mediated skin disease that is chronic and relapsing and associated with allergic activity to foods and other common antigens.15 This finding lends evidence that IC may be a Th-2-mediated disease. In addition, urine of patients with IC was found to have a fivefold increase in interleukin (IL)-6 production compared with urine of controls.16 This elevation was found only in urine collected from the bladder and not urine collected from the ureter. IL-6 is a known stimulus of the Th-2 response, and the degree of elevation of IL-6 correlated with the patient’s symptoms. Furthermore, inhibitors to IL-2, which stimulates a Th-1 response, were found in urine from patients with IC.17 The above studies suggest that IC may be driven by a Th-2 cytokine network leading to an immunopathologic response. Of interest, studies of college students before final examinations have demonstrated a shift from a Th-1 to a Th-2 response, presumably due to stress.18 An almost universal finding in IC is that symptoms worsen during times of stress. This may be secondary to a further shifting of immune response from Th-1 to Th-2. UROLOGY 54 (3), 1999
A Th-1 response favors protection and is thought to be beneficial. A Th-1 response downregulates the inappropriate Th-2-driven responses that can cause immunopathologic features. It recognizes stressed cells, leading to cytolysis of these cells, reduces Th-2-mediated allergic responses, and has the potential to treat chronic infections and autoimmune diseases. Interestingly, BCG is a strong stimulus of the Th-1 response by causing marked elevation of IL-1, IL-2, interferon-gamma (IFNgamma), and tumor necrosis factor (TNF), which are stimulants of this response.12,13,19,20 Thus, the efficacy of BCG in treating IC may be due to stimulation of a Th-1 cytokine network, allowing for destruction of stressed immunogenic cells and promoting reparative conditions. Correcting the underlying abnormal immunologic event may be the reason why intravesical BCG is effective in treating IC and may explain the long-term improvement in symptoms. The purpose of this study was to obtain preliminary information regarding our hypothesis that there is a difference in the urinary cytokine levels of subjects with active IC compared with subjects in remission after BCG and control patients. MATERIAL AND METHODS A total of 35 urine samples were collected comprising 15 subjects (14 women, 1 man) with active IC, 9 women with IC but in remission 2.6 years after receiving intravesical BCG, and 11 female control (non-IC) subjects. Urinalysis was performed, and no pyuria or bacteria suggestive of urinary tract infection was identified. In addition, urine cultures were performed on all samples from the subjects with active IC, and all were sterile. The mean age was 51 years in the active IC group, 50 years in the remission group, and 34 years in the control group. The number of years with IC was 6.4 in the active IC group and 8.8 in the remission group. The control group had no active urologic disease or symptoms at the time of urine collection. The diagnosis of IC was based strictly on the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criteria,21 and no subjects had had a urinary tract infection, hydrodistension, or any intravesical treatment for at least 12 weeks before the urine collection. Fresh voided urine was immediately centrifuged, aliquoted, and frozen in liquid nitrogen. Subjects completed the validated University of Wisconsin IC questionnaire at the time of urine collection.22,23 The enzyme-linked immunosorbent assay technique was used to measure urinary cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNF, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), IL-1beta, and IFN-gamma. Each sample was analyzed in triplicate and compared to a standard curve. To correct for hydration status, cytokine concentrations are expressed as picograms per milligram creatinine. Statistical differences between groups were determined by the nonparametric Tukey test.
RESULTS IL-4, IL-10, IL-12, TNF, hGM-CSF, IL-1beta, and IFN-gamma were undetectable in all the urine samples. IL-2, IL-6, and IL-8 were measur451
TABLE I. Symptom scores and urinary cytokine levels* in subjects with active interstitial cystitis, interstitial cystitis in remission after BCG, and control subjects
†
Active IC BCG/IC Control
n
Symptom Score
IL-2
IL-6
IL-8
15 9 11
32.8 3.6 4.0
70 26 11.4
7.0 0.8 2.6
44.1 5.2 13.6
KEY: BCG ⫽ bacille Calmette-Gue´rin; IL ⫽ interleukin; IC ⫽ interstitial cystitis. * In picograms per milligram creatinine. † P ⬍0.05 compared with BCG/IC and control; no difference between BCG/IC group and control.
able (Table I). Subjects with active IC had significant elevations in urinary cytokines IL-2, IL-6, and IL-8 and in symptom scores compared with control subjects or subjects with IC who had received BCG and were in remission (mean 2.6 years from BCG). Subjects with IC who had received intravesical BCG had cytokine levels and symptom scores statistically identical to those without IC. COMMENT BCG is an attenuated strain of the bovine tuberculosis organism Mycobacterium bovis. This strain was originally developed as a vaccine against Mycobacterium tuberculosis, the cause of tuberculosis in humans. Reports of activity against various tumors by BCG appeared as early as 1935.24 However, Morales and coworkers25 first reported the intravesical use of BCG to treat superficial bladder cancer in 1976, and this has continued to be the most efficacious treatment for superficial bladder cancer. Bladder cancer and IC share common symptoms, including urinary frequency and urgency. Intravesical BCG is a novel therapy for the treatment of IC. Our own double-blind, placebocontrolled, randomized trial of 30 patients2 demonstrated that instilling the tuberculosis vaccine BCG into the bladder is beneficial as a treatment for IC symptoms. Patients with IC treated with BCG achieved remission and remained free of IC-related symptoms for more than 2 years, suggesting durability of its effect.3 Intravesical BCG instillation is a Food and Drug Administration approved treatment for superficial cancer of the bladder and likely counteracts cancer by stimulating the host immune system. IC may be caused by an aberrant immune system. The present study was designed to determine preliminary cytokine data on subjects with active IC, subjects diagnosed with IC and in remission after intravesical BCG, and control (non-IC) subjects. The subjects who had received BCG were on average 2.6 years from completing a single 6-week course of intravesical BCG. Those who responded to BCG required no further treatment for their IC. 452
The significant elevation in IL-2, IL-6, and IL-8 in the subjects with active IC is in line with our hypothesis that IC may be an immune-modulated disease. This group also had elevated symptom scores compared with subjects with IC treated with BCG or controls. Elevations in urine IL-6 and IL-8 have been demonstrated in subjects with active bacterial cystitis.26 The elevation of IL-6 and IL-8 in addition to IL-2 in sterile urine of the patients with active IC may be a result of chronic inflammation or may be secondary to an aberrant immune profile in the bladder, leading to the inflammation. Intravesical BCG is known to alter the immune profile in the bladder and may counteract IC by correcting this immune imbalance. The major limitations of this study were that the cytokine profile of the BCG-treated group was not known before treatment and the control group was younger than the IC group. It is noteworthy, however, that subjects with IC treated with BCG had cytokine levels and symptom scores identical to control subjects, despite the age difference. Intravesical BCG is currently being tested in Phase III, prospective, randomized, placebo-controlled trials for IC. A study is concurrently being undertaken to collect urine from all subjects with IC on study entry, while undergoing intravesical instillation (with BCG or placebo), and during their 6-month follow-up. Symptoms will also be assessed and response to treatment determined. These urine samples are being stored in liquid nitrogen for future determination of urinary cytokines. These data will determine the urine cytokine profile of subjects with IC entering the study. In addition, the peaks in cytokine production during bladder instillation will be identified and compared between those who received BCG or placebo. Finally, the cytokine levels will be determined 6 months after patients complete the intravesical treatment. The follow-up cytokine levels will be compared with the initial levels to see whether the immune profile changed. Additionally, correlations will be made between changes in cytokine levels and clinical response. Furthermore, data will UROLOGY 54 (3), 1999
be analyzed to determine whether stimulation of certain cytokines during the 6-week instillation period resulted in a good clinical response, as suggested in bladder cancer.27 Finally, we may be able to predict who may respond to intravesical BCG on the basis of their initial cytokine profile and limit the use of BCG to this specific population. If future studies demonstrate that IC is associated with an immune imbalance and that BCG is effective in treating this disease by correcting this imbalance, this would open the door to a new avenue of research and potentially more effective and less toxic treatments for IC. CONCLUSIONS These preliminary data demonstrate that subjects with active IC have elevations in urinary cytokine levels compared with controls, suggesting an abnormal immune profile in IC. Subjects with IC in remission 2.6 years after BCG had cytokine profiles identical to controls, suggesting that BCG may alter the immune profile in IC and lead to long-term improvement in symptoms. Future studies are ongoing to definitively determine the bladder urine immune profile in IC and to determine the impact BCG has on this profile and ultimately on clinical response. REFERENCES 1. Curhan GC, Speizer FE, Hunter DJ, et al: Epidemiology of interstitial cystitis: a population based study. J Urol 161: 549 –552, 1999. 2. Peters K, Diokno A, Steinert B, et al: The efficacy of intravesical Tice威 bacillus Calmette-Gue´rin (BCG) in the treatment of interstitial cystitis (IC): a double-blind, prospective, placebo controlled trial. J Urol 157: 2090 –2094, 1996. 3. Peters KM, Diokno AC, Steinert BW, et al: The efficacy of intravesical TICE威 bacillus Calmette-Gue´rin (BCG) in the treatment of interstitial cystitis (IC): long-term follow-up. J Urol 159: 1483–1487, 1998. 4. Pellegrini P, Berghella AM, Del Beato T, et al: Disregulation in Th1 and Th2 subsets of CD4⫹ T cells in peripheral blood of colorectal cancer patients and involvement in cancer establishment and progression. Cancer Immunol Immunother 42: 1– 8, 1996. 5. Liebert M, Wedemeyer G, Stein JA, et al: Evidence for urothelial cell activation in interstitial cystitis. J Urol 149: 470 – 475, 1993. 6. Christmas TJ, and Bottazzo GF: Abnormal urothelial HLA-DR expression in interstitial cystitis. Clin Exp Immunol 87: 450 – 454, 1992. 7. Silk MR: Bladder antibodies in interstitial cystitis. J Urol 103: 307–309, 1970. 8. Keay S, Zhang C-O, Trifillis AL, et al: Urine autoantibodies in interstitial cystitis. J Urol 157: 1083–1087, 1997. 9. Ochs RL, Stein TW Jr, Peebles CL, et al: Autoantibodies in interstitial cystitis. J Urol 151: 587–592, 1994.
UROLOGY 54 (3), 1999
10. Joustra B, Karrenbeld A, and Mensink H: Specific autoantibodies in interstitial cystitis patients suggest an auto-immune etiology (abstract). J Urol 155: 431A, 1996. 11. Mosmann TR, and Moore KW: The role of IL-10 in crossregulation of Th1 and Th2 responses. Immunol Today 12: A49 –A53, 1997. 12. Jackson AM, Alexandroff AB, Kelley RW, et al: Changes in urinary cytokines and soluble intercellular adhesion molecule-1 (ICAM-1) in bladder cancer patients after bacillus Calmette-Gue´rin (BCG) immunotherapy. Clin Exp Immunol 99: 369 –375, 1995. 13. Jackson AM, and James K: Understanding the most successful immunotherapy for cancer. Immunologist 2: 208 –215, 1994. 14. Ochs RL, Muro Y, Chan EKL, et al: Autoantibodies to DSF70 in patients with interstitial cystitis or atopic dermatitis. Presented at the 1997 International Research Symposium on Interstitial Cystitis, Washington, DC. 15. Fujimura T, Yamanashi R, Masuzawa M, et al: Conversion of the CD⫹ T cell Th2-dominant type to Th1-dominant type after varicella-zoster virus infection in atopic dermatitis. J Allergy Clin Immunol 100: 274 –282, 1997. 16. Lotz M, Villiger P, Hugli T, et al: Interleukin-6 and interstitial cystitis. J Urol 152: 869 – 873, 1994. 17. Shingleton WB, and Fleischmann J: Urinary interleukin-2 inhibitor and the voiding symptoms in women patients with interstitial cystitis. Semin Urol 9: 120 –123, 1991. 18. Rook GAW, and Stanford JL: Adjuvants, endocrines and conserved epitopes: factors to consider when designing “therapeutic vaccines.” Int J Pharm 17: 91–102, 1995. 19. Bo¨hle A, Nowc C, Ulmer AJ, et al: Elevations of cytokines interleukin-1, interleukin-2 and tumor necrosis factor in the urine of patients after intravesical bacillus CalmetteGue´rin immunotherapy. J Urol 144: 59 – 64, 1990. 20. Kaempfer R, Gerez L, Farbstein H, et al: Prediction of response to treatment in superficial bladder carcinoma through pattern of interleukin-2 gene expression. J Clin Oncol 14: 1778 –1786, 1996. 21. Gillenwater JY, and Wein AJ: Summary of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases workshop on interstitial cystitis, National Institutes of Health, Bethesda, Maryland, August 28 –29, 1987. J Urol 140: 203– 206, 1988. 22. Goin JE, Olaleye D, Peters KM, et al: Psychometric analysis of the university of Wisconsin interstitial cystitis scale: implications for use in randomized clinical trials. J Urol 159: 1085–1090, 1998. 23. Keller ML, McCarthy DO, and Neider RS: Measurement of symptoms of interstitial cystitis. A pilot study. Urol Clin North Am 21: 67–71, 1994. 24. Holmgren I: La tuberculine et le BCG chez les cancereux. Schweiz Med Wochenschr 65: 1203–1206, 1935. 25. Morales A, Eidinger D, and Bruce AW: Intracavitary bacillus Calmette-Gue´rin in the treatment of superficial bladder tumors. J Urol 116: 180 –183, 1976. 26. Benson M, Jodal U, Agace W, et al: Interleukin (IL)-6 and IL-8 in children with febrile urinary tract infection and asymptomatic bacteriuria. J Infect Dis 174: 1080 –1084, 1996. 27. Thalman GN, Dewald B, Baggiolini M, et al: Interleukin-8 expression in the urine after bacillus Calmette-Gue´rin therapy: a potential prognostic factor of tumor recurrence and progression. J Urol 158: 1340 –1344, 1997.
453
PRELIMINARY STUDY ON URINARY CYTOKINE LEVELS IN INTERSTITIAL CYSTITIS: DOES INTRAVESICAL BACILLE CALMETTE-GUE´RIN TREAT INTERSTITIAL CYSTITIS BY ALTERING THE IMMUNE PROFILE IN THE BLADDER? KENNETH M. PETERS, ANANIAS C. DIOKNO,
AND
BRUCE W. STEINERT
ABSTRACT Objectives. To obtain preliminary urinary cytokine data on subjects with active interstitial cystitis (IC), subjects with IC in remission after bacille Calmette-Gue´rin (BCG), and control (non-IC) subjects. IC is a severe, debilitating bladder disease of unknown etiology and no cure. In controlled clinical trials, intravesical BCG has been shown to be an effective and durable treatment for IC. The durability of this treatment led us to speculate on the mechanism by which intravesical BCG may treat IC. Evidence exists that IC may be mediated by an abnormal immune profile within the bladder. Intravesical BCG is known to stimulate the immune system of the bladder. Methods. Fresh voided urine was collected from 15 subjects with active IC, 9 subjects with IC who received intravesical BCG and had been in remission for an average of 2.6 years, and 11 non-IC subjects. The urine was immediately centrifuged, aliquoted, and frozen in liquid nitrogen. At the time of urine collection, a validated IC questionnaire was completed. The enzyme-linked immunosorbent assay technique was used to determine levels of urinary cytokines interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF), human granulocyte-macrophage colony stimulating factor (hGM-CSF), IL-1beta, and interferongamma (IFN-gamma). Results. Cytokines IL-4, IL-10, IL-12, TNF, hGM-CSF, IL-1beta, and IFN-gamma were not detected. Significant elevations in symptom scores and IL-2, IL-6, and IL-8 were found in the urine of subjects with active IC compared with subjects with IC in remission and control subjects. The urinary cytokine levels and symptom scores were identical in the IC group who had received BCG and the control group. Conclusions. Elevations in symptom scores and urinary cytokine levels were seen in subjects with active IC, suggesting an abnormal immune profile in this disease. Subjects with IC in remission after receiving BCG had identical cytokine levels and symptom scores as non-IC control subjects. Intravesical BCG may be effective in treating IC by correcting an aberrant immune imbalance in the bladder, leading to long-term symptomatic improvement. A prospective study is ongoing to further investigate the role of the immune system in IC. UROLOGY 54: 450–453, 1999. © 1999, Elsevier Science Inc.
I
nterstitial cystitis (IC) is an inflammatory disease of the bladder characterized by urinary urgency, frequency, and suprapubic pain. A recent study suggests the prevalence of IC is 60 in 100,000 individuals in the United States.1 The number of undiagnosed individuals affected may
From the Department of Urology, William Beaumont Hospital, Royal Oak, Michigan Reprint requests: Kenneth M. Peters, M.D., Department of Urology, William Beaumont Hospital, 3535 West 13 Mile Road, Suite 438, Royal Oak, MI 48073 Submitted: February 15, 1999, accepted (with revisions): March 10, 1999
450
© 1999, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
be much higher. The cause of IC is unknown despite a century of study. Intravesical bacille Calmette-Gue´rin (BCG) is a novel therapy for the treatment of IC. The efficacy, durability, and safety of intravesical BCG in the treatment of IC was demonstrated recently in a controlled clinical trial.2,3 This raises the question of why BCG would be effective in the treatment of IC. Although this therapy has been used for years in the treatment of bladder cancer, its exact mechanism of action is unknown. Pellegrini and coworkers4 hypothesize that the disregulation of the T-helper cell type 1 (Th-1)/T-helper cell type 2 0090-4295/99/$20.00 PII S0090-4295(99)00162-4
(Th-2) response correlates with tumor stage and may be related to the tumor progression and metastasis observed in advanced cases. BCG is a strong stimulus of the immune system in the bladder, and there is evidence that in some patients IC may be an autoimmune disease.5–10 Aberrant HLA-DR expression is found in the bladder of patients with IC and in other autoimmune diseases, including thyroid disease, inflammatory bowel disease, and psoriasis and in pancreatic cells in diabetes. HLA-DR is known to function in the immune system by presenting processed antigen to activate lymphocytes and serves to trigger local immune responses. An abnormal expression of HLA-DR on the urothelium of IC bladders may indicate a specific type of inflammation involving an unusual pattern of cytokine networks. It is known that specific cytokines stimulate either a Th-1 or Th-2 response.11 In general, a Th-2 response is thought to be associated with disease states, favoring antibody formation and inappropriate tissue necrosis. The maturation of T-lymphocytes concentrates along two distinct pathways, either Th-1 or Th-2 as defined by the prevalent cytokines. The pathways can have a common stimulus such as infection or tissue damage, but the response pathway determines whether the condition will resolve or develop into a chronic, ultimately debilitating condition. Intravesical BCG is a known stimulus of the Th-1 cytokine pathway.12,13 A study by Ochs et al.14 has shown a common autoantigen in serum from patients with IC and those with atopic dermatitis. Atopic dermatitis is a Th-2-mediated skin disease that is chronic and relapsing and associated with allergic activity to foods and other common antigens.15 This finding lends evidence that IC may be a Th-2-mediated disease. In addition, urine of patients with IC was found to have a fivefold increase in interleukin (IL)-6 production compared with urine of controls.16 This elevation was found only in urine collected from the bladder and not urine collected from the ureter. IL-6 is a known stimulus of the Th-2 response, and the degree of elevation of IL-6 correlated with the patient’s symptoms. Furthermore, inhibitors to IL-2, which stimulates a Th-1 response, were found in urine from patients with IC.17 The above studies suggest that IC may be driven by a Th-2 cytokine network leading to an immunopathologic response. Of interest, studies of college students before final examinations have demonstrated a shift from a Th-1 to a Th-2 response, presumably due to stress.18 An almost universal finding in IC is that symptoms worsen during times of stress. This may be secondary to a further shifting of immune response from Th-1 to Th-2. UROLOGY 54 (3), 1999
A Th-1 response favors protection and is thought to be beneficial. A Th-1 response downregulates the inappropriate Th-2-driven responses that can cause immunopathologic features. It recognizes stressed cells, leading to cytolysis of these cells, reduces Th-2-mediated allergic responses, and has the potential to treat chronic infections and autoimmune diseases. Interestingly, BCG is a strong stimulus of the Th-1 response by causing marked elevation of IL-1, IL-2, interferon-gamma (IFNgamma), and tumor necrosis factor (TNF), which are stimulants of this response.12,13,19,20 Thus, the efficacy of BCG in treating IC may be due to stimulation of a Th-1 cytokine network, allowing for destruction of stressed immunogenic cells and promoting reparative conditions. Correcting the underlying abnormal immunologic event may be the reason why intravesical BCG is effective in treating IC and may explain the long-term improvement in symptoms. The purpose of this study was to obtain preliminary information regarding our hypothesis that there is a difference in the urinary cytokine levels of subjects with active IC compared with subjects in remission after BCG and control patients. MATERIAL AND METHODS A total of 35 urine samples were collected comprising 15 subjects (14 women, 1 man) with active IC, 9 women with IC but in remission 2.6 years after receiving intravesical BCG, and 11 female control (non-IC) subjects. Urinalysis was performed, and no pyuria or bacteria suggestive of urinary tract infection was identified. In addition, urine cultures were performed on all samples from the subjects with active IC, and all were sterile. The mean age was 51 years in the active IC group, 50 years in the remission group, and 34 years in the control group. The number of years with IC was 6.4 in the active IC group and 8.8 in the remission group. The control group had no active urologic disease or symptoms at the time of urine collection. The diagnosis of IC was based strictly on the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criteria,21 and no subjects had had a urinary tract infection, hydrodistension, or any intravesical treatment for at least 12 weeks before the urine collection. Fresh voided urine was immediately centrifuged, aliquoted, and frozen in liquid nitrogen. Subjects completed the validated University of Wisconsin IC questionnaire at the time of urine collection.22,23 The enzyme-linked immunosorbent assay technique was used to measure urinary cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNF, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), IL-1beta, and IFN-gamma. Each sample was analyzed in triplicate and compared to a standard curve. To correct for hydration status, cytokine concentrations are expressed as picograms per milligram creatinine. Statistical differences between groups were determined by the nonparametric Tukey test.
RESULTS IL-4, IL-10, IL-12, TNF, hGM-CSF, IL-1beta, and IFN-gamma were undetectable in all the urine samples. IL-2, IL-6, and IL-8 were measur451
TABLE I. Symptom scores and urinary cytokine levels* in subjects with active interstitial cystitis, interstitial cystitis in remission after BCG, and control subjects
†
Active IC BCG/IC Control
n
Symptom Score
IL-2
IL-6
IL-8
15 9 11
32.8 3.6 4.0
70 26 11.4
7.0 0.8 2.6
44.1 5.2 13.6
KEY: BCG ⫽ bacille Calmette-Gue´rin; IL ⫽ interleukin; IC ⫽ interstitial cystitis. * In picograms per milligram creatinine. † P ⬍0.05 compared with BCG/IC and control; no difference between BCG/IC group and control.
able (Table I). Subjects with active IC had significant elevations in urinary cytokines IL-2, IL-6, and IL-8 and in symptom scores compared with control subjects or subjects with IC who had received BCG and were in remission (mean 2.6 years from BCG). Subjects with IC who had received intravesical BCG had cytokine levels and symptom scores statistically identical to those without IC. COMMENT BCG is an attenuated strain of the bovine tuberculosis organism Mycobacterium bovis. This strain was originally developed as a vaccine against Mycobacterium tuberculosis, the cause of tuberculosis in humans. Reports of activity against various tumors by BCG appeared as early as 1935.24 However, Morales and coworkers25 first reported the intravesical use of BCG to treat superficial bladder cancer in 1976, and this has continued to be the most efficacious treatment for superficial bladder cancer. Bladder cancer and IC share common symptoms, including urinary frequency and urgency. Intravesical BCG is a novel therapy for the treatment of IC. Our own double-blind, placebocontrolled, randomized trial of 30 patients2 demonstrated that instilling the tuberculosis vaccine BCG into the bladder is beneficial as a treatment for IC symptoms. Patients with IC treated with BCG achieved remission and remained free of IC-related symptoms for more than 2 years, suggesting durability of its effect.3 Intravesical BCG instillation is a Food and Drug Administration approved treatment for superficial cancer of the bladder and likely counteracts cancer by stimulating the host immune system. IC may be caused by an aberrant immune system. The present study was designed to determine preliminary cytokine data on subjects with active IC, subjects diagnosed with IC and in remission after intravesical BCG, and control (non-IC) subjects. The subjects who had received BCG were on average 2.6 years from completing a single 6-week course of intravesical BCG. Those who responded to BCG required no further treatment for their IC. 452
The significant elevation in IL-2, IL-6, and IL-8 in the subjects with active IC is in line with our hypothesis that IC may be an immune-modulated disease. This group also had elevated symptom scores compared with subjects with IC treated with BCG or controls. Elevations in urine IL-6 and IL-8 have been demonstrated in subjects with active bacterial cystitis.26 The elevation of IL-6 and IL-8 in addition to IL-2 in sterile urine of the patients with active IC may be a result of chronic inflammation or may be secondary to an aberrant immune profile in the bladder, leading to the inflammation. Intravesical BCG is known to alter the immune profile in the bladder and may counteract IC by correcting this immune imbalance. The major limitations of this study were that the cytokine profile of the BCG-treated group was not known before treatment and the control group was younger than the IC group. It is noteworthy, however, that subjects with IC treated with BCG had cytokine levels and symptom scores identical to control subjects, despite the age difference. Intravesical BCG is currently being tested in Phase III, prospective, randomized, placebo-controlled trials for IC. A study is concurrently being undertaken to collect urine from all subjects with IC on study entry, while undergoing intravesical instillation (with BCG or placebo), and during their 6-month follow-up. Symptoms will also be assessed and response to treatment determined. These urine samples are being stored in liquid nitrogen for future determination of urinary cytokines. These data will determine the urine cytokine profile of subjects with IC entering the study. In addition, the peaks in cytokine production during bladder instillation will be identified and compared between those who received BCG or placebo. Finally, the cytokine levels will be determined 6 months after patients complete the intravesical treatment. The follow-up cytokine levels will be compared with the initial levels to see whether the immune profile changed. Additionally, correlations will be made between changes in cytokine levels and clinical response. Furthermore, data will UROLOGY 54 (3), 1999
be analyzed to determine whether stimulation of certain cytokines during the 6-week instillation period resulted in a good clinical response, as suggested in bladder cancer.27 Finally, we may be able to predict who may respond to intravesical BCG on the basis of their initial cytokine profile and limit the use of BCG to this specific population. If future studies demonstrate that IC is associated with an immune imbalance and that BCG is effective in treating this disease by correcting this imbalance, this would open the door to a new avenue of research and potentially more effective and less toxic treatments for IC. CONCLUSIONS These preliminary data demonstrate that subjects with active IC have elevations in urinary cytokine levels compared with controls, suggesting an abnormal immune profile in IC. Subjects with IC in remission 2.6 years after BCG had cytokine profiles identical to controls, suggesting that BCG may alter the immune profile in IC and lead to long-term improvement in symptoms. Future studies are ongoing to definitively determine the bladder urine immune profile in IC and to determine the impact BCG has on this profile and ultimately on clinical response. REFERENCES 1. Curhan GC, Speizer FE, Hunter DJ, et al: Epidemiology of interstitial cystitis: a population based study. J Urol 161: 549 –552, 1999. 2. Peters K, Diokno A, Steinert B, et al: The efficacy of intravesical Tice威 bacillus Calmette-Gue´rin (BCG) in the treatment of interstitial cystitis (IC): a double-blind, prospective, placebo controlled trial. J Urol 157: 2090 –2094, 1996. 3. Peters KM, Diokno AC, Steinert BW, et al: The efficacy of intravesical TICE威 bacillus Calmette-Gue´rin (BCG) in the treatment of interstitial cystitis (IC): long-term follow-up. J Urol 159: 1483–1487, 1998. 4. Pellegrini P, Berghella AM, Del Beato T, et al: Disregulation in Th1 and Th2 subsets of CD4⫹ T cells in peripheral blood of colorectal cancer patients and involvement in cancer establishment and progression. Cancer Immunol Immunother 42: 1– 8, 1996. 5. Liebert M, Wedemeyer G, Stein JA, et al: Evidence for urothelial cell activation in interstitial cystitis. J Urol 149: 470 – 475, 1993. 6. Christmas TJ, and Bottazzo GF: Abnormal urothelial HLA-DR expression in interstitial cystitis. Clin Exp Immunol 87: 450 – 454, 1992. 7. Silk MR: Bladder antibodies in interstitial cystitis. J Urol 103: 307–309, 1970. 8. Keay S, Zhang C-O, Trifillis AL, et al: Urine autoantibodies in interstitial cystitis. J Urol 157: 1083–1087, 1997. 9. Ochs RL, Stein TW Jr, Peebles CL, et al: Autoantibodies in interstitial cystitis. J Urol 151: 587–592, 1994.
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10. Joustra B, Karrenbeld A, and Mensink H: Specific autoantibodies in interstitial cystitis patients suggest an auto-immune etiology (abstract). J Urol 155: 431A, 1996. 11. Mosmann TR, and Moore KW: The role of IL-10 in crossregulation of Th1 and Th2 responses. Immunol Today 12: A49 –A53, 1997. 12. Jackson AM, Alexandroff AB, Kelley RW, et al: Changes in urinary cytokines and soluble intercellular adhesion molecule-1 (ICAM-1) in bladder cancer patients after bacillus Calmette-Gue´rin (BCG) immunotherapy. Clin Exp Immunol 99: 369 –375, 1995. 13. Jackson AM, and James K: Understanding the most successful immunotherapy for cancer. Immunologist 2: 208 –215, 1994. 14. Ochs RL, Muro Y, Chan EKL, et al: Autoantibodies to DSF70 in patients with interstitial cystitis or atopic dermatitis. Presented at the 1997 International Research Symposium on Interstitial Cystitis, Washington, DC. 15. Fujimura T, Yamanashi R, Masuzawa M, et al: Conversion of the CD⫹ T cell Th2-dominant type to Th1-dominant type after varicella-zoster virus infection in atopic dermatitis. J Allergy Clin Immunol 100: 274 –282, 1997. 16. Lotz M, Villiger P, Hugli T, et al: Interleukin-6 and interstitial cystitis. J Urol 152: 869 – 873, 1994. 17. Shingleton WB, and Fleischmann J: Urinary interleukin-2 inhibitor and the voiding symptoms in women patients with interstitial cystitis. Semin Urol 9: 120 –123, 1991. 18. Rook GAW, and Stanford JL: Adjuvants, endocrines and conserved epitopes: factors to consider when designing “therapeutic vaccines.” Int J Pharm 17: 91–102, 1995. 19. Bo¨hle A, Nowc C, Ulmer AJ, et al: Elevations of cytokines interleukin-1, interleukin-2 and tumor necrosis factor in the urine of patients after intravesical bacillus CalmetteGue´rin immunotherapy. J Urol 144: 59 – 64, 1990. 20. Kaempfer R, Gerez L, Farbstein H, et al: Prediction of response to treatment in superficial bladder carcinoma through pattern of interleukin-2 gene expression. J Clin Oncol 14: 1778 –1786, 1996. 21. Gillenwater JY, and Wein AJ: Summary of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases workshop on interstitial cystitis, National Institutes of Health, Bethesda, Maryland, August 28 –29, 1987. J Urol 140: 203– 206, 1988. 22. Goin JE, Olaleye D, Peters KM, et al: Psychometric analysis of the university of Wisconsin interstitial cystitis scale: implications for use in randomized clinical trials. J Urol 159: 1085–1090, 1998. 23. Keller ML, McCarthy DO, and Neider RS: Measurement of symptoms of interstitial cystitis. A pilot study. Urol Clin North Am 21: 67–71, 1994. 24. Holmgren I: La tuberculine et le BCG chez les cancereux. Schweiz Med Wochenschr 65: 1203–1206, 1935. 25. Morales A, Eidinger D, and Bruce AW: Intracavitary bacillus Calmette-Gue´rin in the treatment of superficial bladder tumors. J Urol 116: 180 –183, 1976. 26. Benson M, Jodal U, Agace W, et al: Interleukin (IL)-6 and IL-8 in children with febrile urinary tract infection and asymptomatic bacteriuria. J Infect Dis 174: 1080 –1084, 1996. 27. Thalman GN, Dewald B, Baggiolini M, et al: Interleukin-8 expression in the urine after bacillus Calmette-Gue´rin therapy: a potential prognostic factor of tumor recurrence and progression. J Urol 158: 1340 –1344, 1997.
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