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SYSTEMIC AND LOCAL IMMUNOMODULATORY EFFECTS OF INTRAVESICAL BCG THERAPY IN PATIENTS WITH SUPERFICIAL URINARY BLADDER CARCINOMAS
0022-5347/00/1631-0296/0 THE JOURNAL OF UROLOGY® Copyright © 2000 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 163, 296 –299, January 2000 Printed in U.S.A.
SYSTEMIC AND LOCAL IMMUNOMODULATORY EFFECTS OF INTRAVESICAL BCG THERAPY IN PATIENTS WITH SUPERFICIAL URINARY BLADDER CARCINOMAS ¨ SSER-BEILE,* O. GUTZEIT, S. BAUER, A. KATZENWADEL, W. SCHULTZE-SEEMANN U. ELSA U. WETTERAUER
AND
From the Department of Urology, University of Freiburg, Freiburg, Germany
ABSTRACT
Purpose: The aim of the present study was to elucidate whether only local, or also systemic immunomodulatory effects may be induced by intravesical BCG therapy of superficial urinary bladder cancer. Materials and Methods: A total of 37 patients with stages Ta to T1b superficial transitional cell bladder carcinomas received 6 weekly BCG instillations after transurethral resection of the tumor. In a first group of 19 patients blood was taken before each BCG instillation and 6 weeks after the last one. In a second group of 18 patients blood and urine was taken before and 2, 6, and 24 hours after each BCG instillation. In the mitogen-stimulated whole blood cell cultures and in the urine samples the levels of the cytokines IL-1b, IL-2, IL-10, TNF-a and IFN-g were determined by enzymoimmunological tests. Additionally, in all plasma and urine samples the levels of TNF-p75-receptor (TNF-p75-R) were measured. Results: Comparison of ex vivo leukocyte cytokine production in the blood cell cultures of the patients of group I revealed no significant change in the levels of the cytokines. In contrast, TNF-p75-R plasma levels increased significantly during the experimental time of 12 weeks (p #0.01). In the blood cell cultures of the group II patients a different daytime variation of cytokine production was seen, compared to the 19 healthy controls. After BCG instillation the normal peak cytokine production in the evening was suppressed. A significant rise in plasma TNF-p-75-R levels was measured 24 hours after BCG instillation (p #0.05). In the urine of these patients significantly higher levels of all measured cytokines and TNF-p75-R were observed 6 to 24 hours after the instillation. Conclusions: The results suggest that besides the well known local immune activation, BCG instillation also leads to a modulation of peripheral immune mechanisms. KEY WORDS: bladder cancer, BCG, leukocyte cytokine, plasma TNF
Since its introduction in 1976, intravesical Bacillus Calmette-Gue´rin (BCG) therapy has been shown to be effective in the treatment of carcinoma in situ and can reduce tumor recurrence of transitional cell carcinoma of the bladder.1, 2 The antitumor effects of BCG seem to be related to local immunological mechanisms which are reflected by a transient increase of several cytokines3– 6 and the presence of activated immunocompetent leukocytes7, 8 in the urine within 24 hours after instillation. However, the cellular mechanism of its activity after interaction with the bladder wall remains largely unknown and until now it has not been proven whether this local reaction is accompanied by a systemic immune modulation. In an attempt to address this question and to monitor the systemic immunological response of patients following BCG treatment, we have performed a prospective study. We have measured the ex vivo cytokine production of peripheral leukocytes as well as plasma TNF-p75-receptor (TNF-p75-R) levels as independent immune parameters. MATERIALS AND METHODS
Patients. A total of 37 patients (22 men and 16 women) with superficial urothelial bladder carcinomas of the stages
pTa and pT1 and grades 1 to 3 without any other concurrent malignancies were included in the study. The ages of these patients were between 47 and 78 years (mean 65 years). Informed consent was obtained from all patients. About three weeks after transurethral resection (TUR) each patient received 6 instillations at weekly intervals with 1.8 3 108 colony-forming units of BCG strain Connaught, diluted in 50 ml. of normal saline. After instillation into the bladder it was retained for two hours. Urine cultures were obtained from all patients before each instillation to exclude the presence of bacterial infection. The clinical followup consisted of examination with cystoscopy, cytology and random biopsies. In the first group of 19 patients blood was taken before each BCG instillation and 6 weeks after the last one. As controls for this group 14 patients were chosen with superficial urinary bladder carcinomas who were not treated with BCG and from whom blood was taken at the same intervals. In the second group of 18 patients blood and urine was taken before (10 AM) and 2 (1 PM), 6 (5 PM) and 24 hours (10 AM) after each BCG instillation. As controls for this group 19 healthy persons were included from whom blood was taken at the same intervals and times. Blood samples. Ten ml. of heparinized venous blood were taken from each donor. The samples were kept at room temperature and used within 3 hours. A 2 ml. aliquot was
Accepted for publication June 14, 1999. * Requests for reprints: Department of Urology, Experimental Research Group, Stefan-Meier-Str. 8, D-79104 Freiburg, Germany. 296
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removed for total leukocyte count, FACS analysis, and for preparation of plasma samples. Whole blood cell cultures. Cultures were performed as described previously with a system for which optimal conditions and kinetics of cytokine production were established.9 In brief, heparinized venous blood was diluted 1/10 with RPMI 1640 medium (Seromed, Berlin, Germany), which was supplemented with 50 U/ml. penicillin (Seromed) and 50 mg./ml. streptomycin (Seromed) and distributed in 0.5 ml. aliquots in 12 mm. polystyrol tubes. For stimulation, 50 ml. phytohemagglutinin (PHA; Wellcome, Burgwedel, Germany) at a final concentration of 10 mg./ml. or 50 ml. pokeweed mitogen at 5 mg./ml. (PWM; Sigma, Deisenhofen, Germany) were added. Cultures were incubated at 37C in a humidified atmosphere of 5% CO2. After 2 (for IL-1-b, IL-2 and TNF-a) or 4 days (for IL-10 and IFN-g) of culture without change of medium, 320 ml. of supernatant were removed from each tube and frozen at 220C until they were assayed for cytokine levels. Urine samples. Samples were kept cool and processed immediately. After centrifugation for 10 minutes at 3000 g to remove cells and cellular debris the samples were adjusted to pH 7.2 with sodium hydroxide and filtered through a 0.45 mm. filter (Milipore). Aliquots were stored at 270C. Prior to cytokine determination the samples were thawed to room temperature and mixed gently. Determination of cytokines. Enzyme-linked immunoassays (ELISA) were applied for qualitative and quantitative determinations of the cytokines as described previously.9 These tests were based on the sandwich principle and performed in one step. Recombinant human cytokines were used as standards. In the IL-1b test the first immobilized antibody was a polyclonal goat anti-hu-IL-1b antibody. For the detection of bound IL-1b, the pod-linked Fab-fragment of a polyclonal goat anti-hu-IL-1b antibody was used (assay range: 100 – 1,000 pg. IL-1b/ml.). For the IL-2 ELISA, microtiter plates were coated with two monoclonal mouse anti-hu-IL-2 antibodies (clone 3D5 and clone 7B1) and a third peroxidase(pod)-linked monoclonal mouse anti-hu-IL-2 antibody (clone 13A6) was used for detection of bound IL-2 (assay range: 50 –1,000 pg. IL-2/ml.). For the IL-10 ELISA, microtiter plates were coated with the monoclonal mouse anti-hu-IL-10 antibody (clone ilt-3) and a second pod-linked monoclonal mouse anti-hu-IL-10 antibody (clone ilt-9) was used for detection of bound IL-10, (assay range: 50 –500 pg. IL-10/ml.). For determination of the dimeric IFN-g, the coating antibody and detection antibody was a monoclonal mouseanti-hu-IFN-g antibody (clone 69). This ELISA had an assay range of 50 –1,000 pg. IFN-g/ml. In the TNF-a ELISA the first immobilized antibody was a monoclonal mouse anti-hu-TNF-a-antibody (clone 6b) and the second a pod-coupled polyclonal rabbit anti-hu-TNF-aantibody. The assay range was 20 –500 pg. TNF-a/ml. For the TNF-p75-receptor test, microtiter plates were coated with a monoclonal mouse anti-hu-TNF-p75-R antibody (clone utr-4). For the detection of bound TNF-p75-R peroxidase-bound recombinant human TNF-a was used (assay range: 100 –1,000 pg. TNF-p75-R/ml.). Determination of T cell subsets by flow cytometry. Cells were stained with conjugated monoclonal antibodies of the Simultest™ reagents (Becton Dickinson, Heidelberg, Germany) and measured by flow cytometry using the SimulSET™ software (Becton Dickinson). In brief, 100 ml. of heparinized blood were mixed and incubated at 20C with the appropriate amount of each Simultest™ reagent in separate tubes. Contaminating erythrocytes were then removed by addition of FACSR Lysing Solution (Becton Dickinson) to the tubes. Samples were subsequently fixed with 1% formaldehyde.
297
Statistical analysis. The results in the patient and control groups were statistically evaluated using the analysis of variance, SASR procedure GLM (SAS Institute, Inc, Cary, NC). Due to the skewed distribution, all values were logarithmically transformed in preparation for the variance analysis. RESULTS
Comparison of ex vivo cytokine production and plasma TNF-p75 receptor levels during the 6 weekly BCG instillations (patient group I). From the 19 patients in group 1 blood was taken before each weekly BCG-instillation and 6 weeks after the last instillation. Since blood was always taken in the morning, no alterations resulting from day time variations could be expected. In the blood cell cultures a rather wide range of cytokine levels was found after mitogenic stimulation. The values followed a skewed distribution, therefore data are given as “box plots” and not as a mean with standard deviation. There was no significant change in the production of any of the measured cytokines in the leukocyte cultures from blood taken before the first and every following BCG instillation as well as 6 weeks after the last one. In contrast, the levels of soluble TNF-p75 receptor increased significantly during the experimental time of 12 weeks (p #0.01). Box plots of the data before the first, before the sixth and 6 weeks after the sixth BCG instillation are shown in fig. 1. During this time no major alterations in absolute peripheral blood mononuclear cell counts nor the lymphocyte subset ratios for the CD31 T cells, the CD191 B cells the CD161/ CD561 NK cells and the CD41, CD81 and HLA-DR1 T cells were seen. Comparison of ex vivo cytokine production and plasma
FIG. 1. Box plots of plasma TNF-p75-R levels before first, before sixth and 6 weeks after sixth BCG instillation (19 patients of group I).
298
IMMUNOMODULATORY EFFECTS OF INTRAVESICAL BCG THERAPY
TNF-p75 receptor levels before and after each BCG instillation (patient group II). From the 18 patients of group 2 blood was taken before (10 AM) and 2 (1 PM), 6 (5 PM) and 24 hours (10 AM) after each weekly BCG instillation. Additionally, from 19 healthy persons blood was taken at the same times. In the whole blood cell cultures of the healthy controls clear day time variations of cytokine production were observed. Peak production of all measured cytokines was seen at 5 PM. In the patients treated with BCG a different day time variation was found, in as far as at 5 PM ex vivo leukocyte cytokine production was lower compared to the controls and this was seen after each of the six BCG instillations. The difference was most evident for IFN-g (p #0.001). Median IFN-g values of the patients and controls are given in fig. 2. Comparison of ex vivo cytokine production in the blood cell cultures taken before and 24 hours after BCG instillation (identical day time) revealed no significant difference in the production of any of the cytokines measured. In contrast, TNF-p75-R plasma levels showed a significant increase during this time (p #0.05). Box plots are given in fig. 3. Also in this patient group, no significant alterations in absolute peripheral blood mononuclear cell counts or lymphocyte subset ratios were found before and after BCG instillation. Measurement of urine cytokine levels before and after BCG instillation. From the patients of group II urine was taken before and 2, 6 and 24 hours after BCG instillation. After BCG instillation a significant increase in the urine levels of all measured cytokines (p #0.001) was found. For IL-1b, IL-2, and TNF-a the highest values were measured 6 hours after BCG instillation, for IFN-g 24 hours after instillation. Urine levels of TNF-p75-R decreased 2 and 6 hours after instillation and then increased after 24 hours. DISCUSSION
In this study we could not only show a local secretion of cytokines into the urine but also systemic effects of the BCG instillation therapy. A local effect has been demonstrated by several authors showing increased cytokine levels in the urine after BCG instillation which were not due to normal daytime variations. Some reflect an inflammatory response, such as IL-1, IL-6, IL-8 and TNF-a,10 –12 while others, such as IL-2 and IFN-g, indicate specific T cell activation.8, 13, 14 Also increased urine TNF receptor levels have been reported.15 A local lymphocytic infiltration16 and cytokine production was assessed in the bladder wall of most patients receiving intra-
FIG. 2. Median IFN-g levels in whole blood cell cultures before and 2, 6 and 24 hours after BCG instillation (18 patients of group II, 6 BCG cycles) compared to daytime variations of 19 healthy controls.
FIG. 3. Box-plots of plasma TNF-p75-R levels before and 2, 6 and 24 hours after BCG instillation (18 patients of group II, 6 BCG cycles).
vesical BCG17 and by intracellular cytokine staining it was demonstrated that this local response was highly complex.18 In vitro studies showed that BCG triggers leukocytes not only for cytokine production but also for antitumor activity.19, 20 There are very few publications about the systemic immunomodulatory effects of the BCG instillation therapy. Studying lymphocyte subsets revealed an increase of suppressor T cells and a decrease of helper T cells 3 to 6 months after beginning of treatment;21, 22 however, in another study, T cell subset distribution was not found to be influenced by the BCG treatment.23 To our knowledge the present study is the first that systematically investigates effects of the BCG instillation therapy on the ex vivo leukocyte cytokine production in a statistical relevant number of patients. Except for the effect on the day time variations we found no change of the mitogeninduced cytokine production after the BCG instillations. These data are in accordance with those of other studies showing an unchanged lectin-induced production of IL-224 and an unchanged reactivity of peripheral blood lymphocytes to mitogens23 before and after BCG therapy. An interesting phenomenon in our study was the depression of the late afternoon peak cytokine production in the BCG-treated patients compared to the controls. This daytime variation of human cytokine production has been associated to plasma cortisol levels.25, 26 Therefore, it may be assumed that the lower cytokine levels of the patients at 5 PM result from relatively high cortisol levels due to the instillation stress. In contrast to the unchanged ex vivo leukocyte cytokine production, we found significantly higher plasma TNF-p75-R levels 24 hours after each BCG instillation and also a gradual increase of this parameter during the whole experimental time of about 12 weeks. It has been shown that the soluble TNF-p75 receptors originate mainly from activated T cells, B cells or neutrophils and increased plasma levels have been determined as a marker of immune activation.27 Since we have not found a polyclonal unspecific systemic immune activation after BCG instillation, we suggest that the increased TNF-p75-R plasma levels may be the result of a specific T cell activation by BCG antigens, possibly after attachment to the bladder wall, which was demonstrated by other authors.28 –30 In conclusion, not only a local but also a systemic immunomodulatory effect could be shown after BCG instillation
IMMUNOMODULATORY EFFECTS OF INTRAVESICAL BCG THERAPY
therapy. Further studies might show if TNF-R-p75 plasma levels can be correlated to a specific peripheral T cell reaction against BCG antigens and if this parameter has prognostic value for the outcome of this immunotherapy. Acknowledgments. The authors wish to thank B. Ho¨fflin for excellent technical assistance and F. Jehle for his help with preparing the manuscript. REFERENCES
1. Morales, A., Eidinger, D. and Bruce, A. W.: Intracavitary Bacillus Calmette-Gue´rin in the treatment of superficial bladder tumors. J Urol, 116: 180, 1976. 2. Lamm, D. L., Thor, D. E., Winters, W. D., Stogdill, V. D. and Radwin, H. M.: BCG immunotherapy of bladder cancer: inhibition of tumor recurrence and associated immune responses. Cancer, 48: 82, 1981. 3. Haaff, E. O., Catalona, W. J. and Ratliff, T. L.: Detection of interleukin 2 in the urine of patients with superficial bladder tumors after treatment with intravesical BCG. J Urol, 136: 970, 1986. 4. Bo¨hle, A., Nowc, C. H., Ulmer, A. J., Musehold, J., Gerdes, J., Hofstetter, A. G. and Flad, H.-D.: Elevations of cytokines interleukin-1, interleukin-2 and tumor necrosis factor in the urine of patients after intravesical Bacillus Calmette-Gue´rin immunotherapy. J Urol, 144: 59, 1990. 5. Bettex-Galland, M., Studer, U. E., Walz, A., Dewald, B. and Baggiolini, M.: Neutrophil-activating peptide-1/interleukin-8 detection in human urine during acute bladder inflammation caused by transurethral resection of superficial cancer and Bacillus Calmette-Gue´rin administration. Eur Urol, 19: 171, 1991. 6. De Boer, E. C., De Jong, W. H., Steerenberg, P. A., Aarden, L. A., Tetteroo, E., De Groot, E. R., Van der Meijden, A. P. M., Vegt, P. D. J., Debruyne, F. M. J. and Ruitenberg, E. J.: Induction of urinary interleukin-1 (IL-1), IL-2, IL-6, and tumor necrosis factor during intravesical immunotherapy with Bacillus Calmette-Gue´rin in superficial bladder cancer. Cancer Immunol Immunother, 34: 306, 1992. 7. De Boer, E. C., De Jong, W. H. D., Van der Meijden, A. P. M., Steerenberg, P. A., Witjes, J. A., Vegt, P. D. J., Debruyne, F. M. J. and Ruitenberg, E. J.: Presence of activated lymphocytes in the urine of patients with superficial bladder cancer after intravesical immunotherapy with Bacillus CalmetteGue´rin. Cancer Immunol Immunother, 33: 411, 1991. 8. Patard, J. J., Muscatelli-Groux, B., Saint, F., Popov, Z., Maille, P., Abbou, C. and Chopin, D.: Evaluation of local immune response after intravesical Bacillus Calmette-Gue´rin treatment for superficial bladder cancer. Br J Urol, 78: 709, 1996. 9. Elsa¨sser-Beile, U., von Kleist, S. and Gallati, H.: Evaluation of a test system for measuring cytokine production in human whole blood cell cultures. J Immunol Meth, 139: 191, 1991. 10. De Boer, E. C., De Jong, W. H., Steerenberg, P. A., Van der Meijden, A. P., Aarden, L. A., Debruyne, F. M. and Ruitenberg, E. J.: Leukocytes and cytokines in the urine of superficial bladder cancer patients after intravesical immunotherapy with Bacillus Calmette-Gue´rin. In Vivo, 5: 671, 1991. 11. Thalmann, G. N., Dewald, B., Baggiolini, M. and Studer, U. E.: Interleukin-8 expression in the urine after Bacillus CalmetteGue´rin therapy: a potential prognostic factor of tumor recurrence and progression. J Urol, 158: 1340, 1997. 12. DeBoer, E. C., Somogyi, L., De Ruiter, G. J., De Reijke, T. M., Kurth, K. H. and Schamhart, D. H.: Role of interleukin-8 in onset of the immune response in intravesical BCG therapy for superficial bladder cancer. Urol Res, 25: 31, 1997. 13. Ratliff, T. L., Haaff, E. O. and Catalona, W. J.: Interleukin-2 production during intravesical Bacillus Calmette-Gue´rin therapy for bladder cancer. Clin Immunol Immunopathol, 40: 375, 1986. 14. Prescott, S., James, K., Hargreave, T. B., Chisholm, G. D. and Smyth, J. F.: Radio-immunoassay detection of interferongamma in urine after intravesical Evans BCG therapy. J Urol, 144: 1248, 1990. 15. Jackson, A. M., Alexandrov, A. B., Prescott, S. and James, K.:
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Production of urinary tumour necrosis factors and soluble tumour necrosis factor receptors in bladder cancer patients after Bacillus Calmette-Gue´rin immunotherapy. Cancer Immunol Immunother, 40: 119, 1995. Boccafoschi, C., Montefiore, F., Pavesi, M., Pastormerlo, M. and Betta, P. G.: Late effects of intravesical Bacillus CalmetteGue´rin immunotherapy on bladder mucosa infiltrating lymphocytes: an immunohistochemical study. Eur Urol, 27: 334, 1995. Bo¨hle, A., Busemann, E., Gerdes, J., Ulmer, A. J., Flad, H. D. and Jocham, D.: Long-term immunobiological effects of intravesical Bacillus Calmette-Gue´rin against bladder carcinoma recurrences. Dev Biol Stand, 77: 199, 1992. Sander, B., Damm, O., Gustafsson, B., Andersson, U. and Hakansson, L.: Localization of IL-1, IL-2, IL-4, IL-8 and TNF in superficial bladder tumors treated with intravesical Bacillus Calmette-Gue´rin. J Urol, 156: 536, 1996. Kurisu, H., Matsuyama, H., Ohmoto, Y., Shimabukuro, T. and Naito, K.: Cytokine-mediated antitumor effect of Bacillus Calmette-Gue´rin on tumor cells in vitro. Cancer Immunol Immunother, 39: 249, 1994. Thanha¨user, A., Bo¨hle, A., Schneider, B., Reiling, N., Mattern, T., Ernst, M., Flad, H. D. and Ulmer, A. J.: The induction of Bacillus Calmette-Gue´rin-activated killer cells requires the presence of monocytes and T-helper type-1 cells. Cancer Immunol Immunother, 40: 103, 1995. Sarica, K., Baltaci, S., Beduk, Y., Suzer, O., Kilic, S., Muftuglu, Y. Z. and Gogus, O.: Evaluation of cellular immunity following Bacillus Calmette-Gue´rin therapy in patients with superficial bladder cancer. Urol Int, 54: 137, 1995. Yoshimura, K. and Yamauchi, T.: Influence of intravesical Bacillus Calmette-Gue´rin therapy on systemic immunological status. Ont J Urol, 4: 456, 1997. van der Meijden, A. P., Steerenberg, P. A., van Hoogstraaten, I. M., Kerckhaert, J. A., Schreinemachers, L. M., Harthoorn-Lasthuizen, E. J., Hagenaars, A. M., de Jong, W. H., Debruijne, F. M. and Ruitenberg, E. J.: Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with Bacillus CalmetteGue´rin. Cancer Immunol Immunother, 28: 287, 1989. Hermann, G. G., Petersen, K. R., Zeuthen, J. and Steven, K.: Intravesical BCG therapy in bladder carcinoma. Effect on cytotoxicity, IL-2 production and phenotype of peripheral blood mononuclear cells. Scand J Urol Nephrol, 25: 269, 1991. Born, J., Lange, T., Hansen, K., Molle, M. and Fehm, H. L.: Effects of sleep and circadian rhythm on human circulating immune cells. J Immunol, 158: 4454, 1997. Petrovsky, N. and Harrison, L. C.: Diurnal rhythmicity of human cytokine production: a dynamic disequilibrium in T helper cell type 1/T helper cell type 2 balance? J Immunol, 158: 5163, 1997. Aderka, D.: The potential biological and clinical significance of the soluble tumor necrosis factor receptors. Cytokine Growth Factor Rev, 7: 231, 1996. Zlotta, A., Drowart, A., van Vooren, J. P., Simon, J., Schulman, C. C. and Huygen, K.: Evolution of cellular and humoral response against Tuberculin and antigen 85 complex during intravesical treatment with BCG of superficial bladder cancer. Acta Urol Belg, 62: 63, 1994. Zlotta, A. R., Drowart, A., Huygen, K., de Bruyn, J., Shekarsarai, H., Decock, M., Pirson, M., Jurion, F., Palfliet, K., Denis, O., Mascart, F., Simon, J., Schulman, C. C. and van Vooren, J. P.: Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with Bacillus Calmette-Gue´rin (BCG) in patients with superficial bladder cancer. Clin Exp Immunol, 109: 157, 1997. Zlotta, A. R., Drowart, A., van Vooren, J. P., de Cock, M., Pirson, M., Palfliet, K., Jurion, F., Vanonckelen, A., Simon, J., Schulman, C. C. and Huygen, K.: Evolution and clinical significance of the T cell proliferative and cytokine response directed against the fibronectin binding antigen 85 complex of Bacillus Calmette-Gue´rin during intravesical treatment of superficial bladder cancer. J Urol, 157: 492, 1997.
Vol. 163, 296 –299, January 2000 Printed in U.S.A.
SYSTEMIC AND LOCAL IMMUNOMODULATORY EFFECTS OF INTRAVESICAL BCG THERAPY IN PATIENTS WITH SUPERFICIAL URINARY BLADDER CARCINOMAS ¨ SSER-BEILE,* O. GUTZEIT, S. BAUER, A. KATZENWADEL, W. SCHULTZE-SEEMANN U. ELSA U. WETTERAUER
AND
From the Department of Urology, University of Freiburg, Freiburg, Germany
ABSTRACT
Purpose: The aim of the present study was to elucidate whether only local, or also systemic immunomodulatory effects may be induced by intravesical BCG therapy of superficial urinary bladder cancer. Materials and Methods: A total of 37 patients with stages Ta to T1b superficial transitional cell bladder carcinomas received 6 weekly BCG instillations after transurethral resection of the tumor. In a first group of 19 patients blood was taken before each BCG instillation and 6 weeks after the last one. In a second group of 18 patients blood and urine was taken before and 2, 6, and 24 hours after each BCG instillation. In the mitogen-stimulated whole blood cell cultures and in the urine samples the levels of the cytokines IL-1b, IL-2, IL-10, TNF-a and IFN-g were determined by enzymoimmunological tests. Additionally, in all plasma and urine samples the levels of TNF-p75-receptor (TNF-p75-R) were measured. Results: Comparison of ex vivo leukocyte cytokine production in the blood cell cultures of the patients of group I revealed no significant change in the levels of the cytokines. In contrast, TNF-p75-R plasma levels increased significantly during the experimental time of 12 weeks (p #0.01). In the blood cell cultures of the group II patients a different daytime variation of cytokine production was seen, compared to the 19 healthy controls. After BCG instillation the normal peak cytokine production in the evening was suppressed. A significant rise in plasma TNF-p-75-R levels was measured 24 hours after BCG instillation (p #0.05). In the urine of these patients significantly higher levels of all measured cytokines and TNF-p75-R were observed 6 to 24 hours after the instillation. Conclusions: The results suggest that besides the well known local immune activation, BCG instillation also leads to a modulation of peripheral immune mechanisms. KEY WORDS: bladder cancer, BCG, leukocyte cytokine, plasma TNF
Since its introduction in 1976, intravesical Bacillus Calmette-Gue´rin (BCG) therapy has been shown to be effective in the treatment of carcinoma in situ and can reduce tumor recurrence of transitional cell carcinoma of the bladder.1, 2 The antitumor effects of BCG seem to be related to local immunological mechanisms which are reflected by a transient increase of several cytokines3– 6 and the presence of activated immunocompetent leukocytes7, 8 in the urine within 24 hours after instillation. However, the cellular mechanism of its activity after interaction with the bladder wall remains largely unknown and until now it has not been proven whether this local reaction is accompanied by a systemic immune modulation. In an attempt to address this question and to monitor the systemic immunological response of patients following BCG treatment, we have performed a prospective study. We have measured the ex vivo cytokine production of peripheral leukocytes as well as plasma TNF-p75-receptor (TNF-p75-R) levels as independent immune parameters. MATERIALS AND METHODS
Patients. A total of 37 patients (22 men and 16 women) with superficial urothelial bladder carcinomas of the stages
pTa and pT1 and grades 1 to 3 without any other concurrent malignancies were included in the study. The ages of these patients were between 47 and 78 years (mean 65 years). Informed consent was obtained from all patients. About three weeks after transurethral resection (TUR) each patient received 6 instillations at weekly intervals with 1.8 3 108 colony-forming units of BCG strain Connaught, diluted in 50 ml. of normal saline. After instillation into the bladder it was retained for two hours. Urine cultures were obtained from all patients before each instillation to exclude the presence of bacterial infection. The clinical followup consisted of examination with cystoscopy, cytology and random biopsies. In the first group of 19 patients blood was taken before each BCG instillation and 6 weeks after the last one. As controls for this group 14 patients were chosen with superficial urinary bladder carcinomas who were not treated with BCG and from whom blood was taken at the same intervals. In the second group of 18 patients blood and urine was taken before (10 AM) and 2 (1 PM), 6 (5 PM) and 24 hours (10 AM) after each BCG instillation. As controls for this group 19 healthy persons were included from whom blood was taken at the same intervals and times. Blood samples. Ten ml. of heparinized venous blood were taken from each donor. The samples were kept at room temperature and used within 3 hours. A 2 ml. aliquot was
Accepted for publication June 14, 1999. * Requests for reprints: Department of Urology, Experimental Research Group, Stefan-Meier-Str. 8, D-79104 Freiburg, Germany. 296
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removed for total leukocyte count, FACS analysis, and for preparation of plasma samples. Whole blood cell cultures. Cultures were performed as described previously with a system for which optimal conditions and kinetics of cytokine production were established.9 In brief, heparinized venous blood was diluted 1/10 with RPMI 1640 medium (Seromed, Berlin, Germany), which was supplemented with 50 U/ml. penicillin (Seromed) and 50 mg./ml. streptomycin (Seromed) and distributed in 0.5 ml. aliquots in 12 mm. polystyrol tubes. For stimulation, 50 ml. phytohemagglutinin (PHA; Wellcome, Burgwedel, Germany) at a final concentration of 10 mg./ml. or 50 ml. pokeweed mitogen at 5 mg./ml. (PWM; Sigma, Deisenhofen, Germany) were added. Cultures were incubated at 37C in a humidified atmosphere of 5% CO2. After 2 (for IL-1-b, IL-2 and TNF-a) or 4 days (for IL-10 and IFN-g) of culture without change of medium, 320 ml. of supernatant were removed from each tube and frozen at 220C until they were assayed for cytokine levels. Urine samples. Samples were kept cool and processed immediately. After centrifugation for 10 minutes at 3000 g to remove cells and cellular debris the samples were adjusted to pH 7.2 with sodium hydroxide and filtered through a 0.45 mm. filter (Milipore). Aliquots were stored at 270C. Prior to cytokine determination the samples were thawed to room temperature and mixed gently. Determination of cytokines. Enzyme-linked immunoassays (ELISA) were applied for qualitative and quantitative determinations of the cytokines as described previously.9 These tests were based on the sandwich principle and performed in one step. Recombinant human cytokines were used as standards. In the IL-1b test the first immobilized antibody was a polyclonal goat anti-hu-IL-1b antibody. For the detection of bound IL-1b, the pod-linked Fab-fragment of a polyclonal goat anti-hu-IL-1b antibody was used (assay range: 100 – 1,000 pg. IL-1b/ml.). For the IL-2 ELISA, microtiter plates were coated with two monoclonal mouse anti-hu-IL-2 antibodies (clone 3D5 and clone 7B1) and a third peroxidase(pod)-linked monoclonal mouse anti-hu-IL-2 antibody (clone 13A6) was used for detection of bound IL-2 (assay range: 50 –1,000 pg. IL-2/ml.). For the IL-10 ELISA, microtiter plates were coated with the monoclonal mouse anti-hu-IL-10 antibody (clone ilt-3) and a second pod-linked monoclonal mouse anti-hu-IL-10 antibody (clone ilt-9) was used for detection of bound IL-10, (assay range: 50 –500 pg. IL-10/ml.). For determination of the dimeric IFN-g, the coating antibody and detection antibody was a monoclonal mouseanti-hu-IFN-g antibody (clone 69). This ELISA had an assay range of 50 –1,000 pg. IFN-g/ml. In the TNF-a ELISA the first immobilized antibody was a monoclonal mouse anti-hu-TNF-a-antibody (clone 6b) and the second a pod-coupled polyclonal rabbit anti-hu-TNF-aantibody. The assay range was 20 –500 pg. TNF-a/ml. For the TNF-p75-receptor test, microtiter plates were coated with a monoclonal mouse anti-hu-TNF-p75-R antibody (clone utr-4). For the detection of bound TNF-p75-R peroxidase-bound recombinant human TNF-a was used (assay range: 100 –1,000 pg. TNF-p75-R/ml.). Determination of T cell subsets by flow cytometry. Cells were stained with conjugated monoclonal antibodies of the Simultest™ reagents (Becton Dickinson, Heidelberg, Germany) and measured by flow cytometry using the SimulSET™ software (Becton Dickinson). In brief, 100 ml. of heparinized blood were mixed and incubated at 20C with the appropriate amount of each Simultest™ reagent in separate tubes. Contaminating erythrocytes were then removed by addition of FACSR Lysing Solution (Becton Dickinson) to the tubes. Samples were subsequently fixed with 1% formaldehyde.
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Statistical analysis. The results in the patient and control groups were statistically evaluated using the analysis of variance, SASR procedure GLM (SAS Institute, Inc, Cary, NC). Due to the skewed distribution, all values were logarithmically transformed in preparation for the variance analysis. RESULTS
Comparison of ex vivo cytokine production and plasma TNF-p75 receptor levels during the 6 weekly BCG instillations (patient group I). From the 19 patients in group 1 blood was taken before each weekly BCG-instillation and 6 weeks after the last instillation. Since blood was always taken in the morning, no alterations resulting from day time variations could be expected. In the blood cell cultures a rather wide range of cytokine levels was found after mitogenic stimulation. The values followed a skewed distribution, therefore data are given as “box plots” and not as a mean with standard deviation. There was no significant change in the production of any of the measured cytokines in the leukocyte cultures from blood taken before the first and every following BCG instillation as well as 6 weeks after the last one. In contrast, the levels of soluble TNF-p75 receptor increased significantly during the experimental time of 12 weeks (p #0.01). Box plots of the data before the first, before the sixth and 6 weeks after the sixth BCG instillation are shown in fig. 1. During this time no major alterations in absolute peripheral blood mononuclear cell counts nor the lymphocyte subset ratios for the CD31 T cells, the CD191 B cells the CD161/ CD561 NK cells and the CD41, CD81 and HLA-DR1 T cells were seen. Comparison of ex vivo cytokine production and plasma
FIG. 1. Box plots of plasma TNF-p75-R levels before first, before sixth and 6 weeks after sixth BCG instillation (19 patients of group I).
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TNF-p75 receptor levels before and after each BCG instillation (patient group II). From the 18 patients of group 2 blood was taken before (10 AM) and 2 (1 PM), 6 (5 PM) and 24 hours (10 AM) after each weekly BCG instillation. Additionally, from 19 healthy persons blood was taken at the same times. In the whole blood cell cultures of the healthy controls clear day time variations of cytokine production were observed. Peak production of all measured cytokines was seen at 5 PM. In the patients treated with BCG a different day time variation was found, in as far as at 5 PM ex vivo leukocyte cytokine production was lower compared to the controls and this was seen after each of the six BCG instillations. The difference was most evident for IFN-g (p #0.001). Median IFN-g values of the patients and controls are given in fig. 2. Comparison of ex vivo cytokine production in the blood cell cultures taken before and 24 hours after BCG instillation (identical day time) revealed no significant difference in the production of any of the cytokines measured. In contrast, TNF-p75-R plasma levels showed a significant increase during this time (p #0.05). Box plots are given in fig. 3. Also in this patient group, no significant alterations in absolute peripheral blood mononuclear cell counts or lymphocyte subset ratios were found before and after BCG instillation. Measurement of urine cytokine levels before and after BCG instillation. From the patients of group II urine was taken before and 2, 6 and 24 hours after BCG instillation. After BCG instillation a significant increase in the urine levels of all measured cytokines (p #0.001) was found. For IL-1b, IL-2, and TNF-a the highest values were measured 6 hours after BCG instillation, for IFN-g 24 hours after instillation. Urine levels of TNF-p75-R decreased 2 and 6 hours after instillation and then increased after 24 hours. DISCUSSION
In this study we could not only show a local secretion of cytokines into the urine but also systemic effects of the BCG instillation therapy. A local effect has been demonstrated by several authors showing increased cytokine levels in the urine after BCG instillation which were not due to normal daytime variations. Some reflect an inflammatory response, such as IL-1, IL-6, IL-8 and TNF-a,10 –12 while others, such as IL-2 and IFN-g, indicate specific T cell activation.8, 13, 14 Also increased urine TNF receptor levels have been reported.15 A local lymphocytic infiltration16 and cytokine production was assessed in the bladder wall of most patients receiving intra-
FIG. 2. Median IFN-g levels in whole blood cell cultures before and 2, 6 and 24 hours after BCG instillation (18 patients of group II, 6 BCG cycles) compared to daytime variations of 19 healthy controls.
FIG. 3. Box-plots of plasma TNF-p75-R levels before and 2, 6 and 24 hours after BCG instillation (18 patients of group II, 6 BCG cycles).
vesical BCG17 and by intracellular cytokine staining it was demonstrated that this local response was highly complex.18 In vitro studies showed that BCG triggers leukocytes not only for cytokine production but also for antitumor activity.19, 20 There are very few publications about the systemic immunomodulatory effects of the BCG instillation therapy. Studying lymphocyte subsets revealed an increase of suppressor T cells and a decrease of helper T cells 3 to 6 months after beginning of treatment;21, 22 however, in another study, T cell subset distribution was not found to be influenced by the BCG treatment.23 To our knowledge the present study is the first that systematically investigates effects of the BCG instillation therapy on the ex vivo leukocyte cytokine production in a statistical relevant number of patients. Except for the effect on the day time variations we found no change of the mitogeninduced cytokine production after the BCG instillations. These data are in accordance with those of other studies showing an unchanged lectin-induced production of IL-224 and an unchanged reactivity of peripheral blood lymphocytes to mitogens23 before and after BCG therapy. An interesting phenomenon in our study was the depression of the late afternoon peak cytokine production in the BCG-treated patients compared to the controls. This daytime variation of human cytokine production has been associated to plasma cortisol levels.25, 26 Therefore, it may be assumed that the lower cytokine levels of the patients at 5 PM result from relatively high cortisol levels due to the instillation stress. In contrast to the unchanged ex vivo leukocyte cytokine production, we found significantly higher plasma TNF-p75-R levels 24 hours after each BCG instillation and also a gradual increase of this parameter during the whole experimental time of about 12 weeks. It has been shown that the soluble TNF-p75 receptors originate mainly from activated T cells, B cells or neutrophils and increased plasma levels have been determined as a marker of immune activation.27 Since we have not found a polyclonal unspecific systemic immune activation after BCG instillation, we suggest that the increased TNF-p75-R plasma levels may be the result of a specific T cell activation by BCG antigens, possibly after attachment to the bladder wall, which was demonstrated by other authors.28 –30 In conclusion, not only a local but also a systemic immunomodulatory effect could be shown after BCG instillation
IMMUNOMODULATORY EFFECTS OF INTRAVESICAL BCG THERAPY
therapy. Further studies might show if TNF-R-p75 plasma levels can be correlated to a specific peripheral T cell reaction against BCG antigens and if this parameter has prognostic value for the outcome of this immunotherapy. Acknowledgments. The authors wish to thank B. Ho¨fflin for excellent technical assistance and F. Jehle for his help with preparing the manuscript. REFERENCES
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