Cancer Immunol Immunother(1990) 31: 182- 186
~
ancer mmunology mmunotherapy
© Springer-Verlag 1990
Presence of interleukin-2 in urine of superficial bladder cancer patients after intravesical treatment with bacillus Calmette-Guérin W. H. De Jongl, E. C. De Boer~, A. P. M. Van Der Meijden2, 3, p. Vegt4, P. A. Steerenberg 1, F. M. J. Debruyne z, and E. J. Ruitenberg I 1Laboratoryfor Pathology,National Institute of Public Health and EnvironmentalProtection,P. O. Box 1, 3720 BA Bilthoven,The Netherlands 2 Departmentof Urology,UniversityHospitalNijmegen, Nijmegen 3Departmentof Urology,Grootziekengasthuis,'s-Hertogenbosch 4 Departmentof Urology,St. ElisabethHospital, Leiderdorp Received 11 July 1989/Accepted20 December 1989
Summary. Urine samples were obtained from patients with superficial bladder cancer after immunotherapy with bacillus Calmette-Guérin (BCG). The patients were repeatedly (once a week for 6 consecutive weeks) treated with intravesical administration of approximately 5 x 10s culturable particles of BCG. Some patients received more than six BCG instillations. The urine samples were investigated for the presence of interleukin-2 (IL-2) in an in vitro bioassay using a murine cytotoxic T cell line (CTTL-16) that shows IL-2-dependent growth. Preliminary experiments indicated the presence of inhibitory factors in the urine. This inhibitory activity was abolished after 24 h dialysis. In a neutralization assay with both polyvalent and monoclonal anti-(human IL-2) antibody it was demonstrated that there was indeed IL-2 in the urine samples. In 8 of 11 patients the presence of IL-2 in the urine was demonstrated. The IL-2 production was directly related to the BCG administration as samples obtained just before the BCG instillation were always negative. In IL-2-positive samples a maximum level of IL-2 was observed between 2 h and 6 h after the BCG instillation. In urine samples obtained 24 h after the BCG IL-2 was not detected. In most patients the urine became positive after the third or fourth BCG instillation
Introduction Since the report of Morales et al. [17] in 1976 it has been demonstrated by several independent research groups that local administration of bacillus Calmette-Guérin (BCG) prevents recurrences in superficial bladder cancer, and can resolve carcinoma of the bladder in situ [3, 8, 15]. The BCG was applied by repeated intravesical administration
• O f f p r i n t r e q u e s t s to."
W. H. de Jong
once a week for 6 consecutive weeks. In an experimental murine bladder tumor system (MBT-2) similar BCG treatment caused a reduction in tumor take [20, 22, 25]. Although it is known that BCG can exert various effects on different parts of the immune system, and has shown antitumor activity in various animal tumour systems, the mechanism by which BCG exerts its antitumor activity remains unclear [6, 16]. After local administration at the tumor site BCG evokes a granulomatous inflammatory reaction in which mononuclear cells from the macrophage lineage are predominantly present as epithelioid cells [5, 10]. How the immune system is able to recognise a previously non-immunogenic tumor after BCG administration is unknown. BCG may be acting as an adjuvant inducing recognition of tumor antigens. Recently it was found that in the draining lymph node the expression of major histocompatibility complex (MHC) class Il antigens is enhanced after local BCG administration. This was observed not only after intradermal injection, but also after intravesical administration of BCG [26] (unpublished data). MHC class II antigens play a major role in the recognition and the processing of antigens in the immune response. In man also, the reason why BCG after intravesical administration has antitumor activity for superficial bladder cancer is not known. Both in man and in animals BCG induces scattered foci of inflammatory cells in the bladder wall, part of which show granulomatous characteristics [7, 14, 27]. The repeated BCG administration may be considered as secondary antigen challenges resulting in delayed-type hypersensitivity reactions in the bladder wall. Recently, Ratliff et al. demonstrated the presence of interleukin 2 (IL-2) in the urine of BCG-treated bladder cancer patients [9, 21]. Apart from its role as a T-cell growth factor, where IL-2 may be an intermediate in a secondary immune response such as a delayed-type hypersensitivity reaction, the induction of so-called lymphokine-activated killer cells by IL-2 was observed more recently [24]. The latter function of IL-2 especially is of interest, as these lymphokine-activated killer cells are able to kill a variety of tumor cells.
183 In this study w e i n v e s t i g a t e d the p r e s e n c e o f I L - 2 in the urine o f b l a d d e r c a n c e r patients treated intravesically with B C G - R I V M or B C G - T I C E . M o s t patients were treated with B C G - R I V M , w h i c h has b e e n d e m o n s t r a t e d to have antitumor p o t e n c y both in l a b o r a t o r y a n i m a l t u m o r systems and in s p o n t a n e o u s l y occurring animal tumors [4, 12, 13]. In a r a n d o m i z e d phase III study for the treatment o f superficial b l a d d e r cancer in man, B C G - R I V M h a d equal antit u m o r activity w h e n c o m p a r e d to m i t o m y c i n C [3].
Materials and methods
Results
Patients. Urine specimens were obtained from patients with a stage pTa
or pT1 papillary transitional cell carcinoma of the bladder. These patients received intravesical immunotherapy with bacillus Calmette-Guérin within 2 weeks after transurethral resection of the tumor(s). BCG-RIVM (eight patients) or BCG-TICE (three patients), approximately 5 x 108 culturable particles, was administered in 50 ml 0.9% saline once a week for 6 consecutive weeks. Some patients were treated with more than six intravesical BCG instillations. Urine samples. After collection the urine was kept on melting ice and cenlrifuged (10 min, 500 g) to remove cells and debris. The supematant was collected and stored at -20 ° C. After thawing, the urine was dialyzed against 50 times the volume in phosphate-buffered saline using a dialysis membrane, molecular mass cut-off 6 - 8 kDa (Spectmm Medic Ind., Los Angeles, Calif, USA). The phosphate-buffered saline was refreshed four times, the last time with RPMI-1640 (Gibco Europe, Breda, The Netherlands). Initially the urine samples were dialyzed for 2 days at 4 ° C. Urine specimens obtained later were dialyzed for 24 h (see results Fig. 1). Specimens were sterilized by fillration through a 0.22-gm filter, and stored at -20 ° C until use in the IL-2 bioassay. IL-2 bioassay. For the detection of IL-2 the IL-2-dependent murine
cytotoxic T cell line CTLL- 16 (kindly provided by Dr. L. Aarden, CLB, Amsterdam) was used. CTLL-16 cells were cultured in RPMI-1640 tissue-culture medium supplemented with 10% (v/v) crude recombinant IL-2 (Sanofi, Toulouse, France), 10% (v/v) heat-inactivated fetal calf serum, penicillin (100 U/tal), streptomycin (100 gg/ml), fungizone (0.26 gg/ml) and 50 gM 2-mercaptoethanol. CTLL-16 cells were used in the IL-2 bioassay 3 days after change of the culture medium. For determination of the presence of IL-2 in the urine specimens, the IL-2 and fungizone were omitted from the tissue-culture medium. The bioassay was performed in flat-bottomed microtiter wells (Costar, Cambridge, Mass, USA). In each well 5 x 103 CTLL-16 cells in 100 gl were seeded, and 100 gl test sample or standard recombinant IL-2
m-" o
(Biogen, Geneva, Switzerland) was added. Cells were cultured for 24 h at 37 ° C, 5% CO2 in a humidified atmosphere. For determinations of the CTLL-16 protiferation [methyl-3H]thymidine (5 gCi/ml, 185 kBq/ml; Radiochemical Centre, Amersham, Bucks., UK) was added during the last 4 h of culture. Cellular DNA was harvested on glass-fiber filters using a multiple cell culture harvester (Flow Laboratories, Irvine, Ayreshire, Scotland). [3H]Thymidine incorporation was measured in an ISO cap/300 liquid scintillation counter (Nuclear Inc., Des Plaines, Ill, USA). The results are expressed as units IL-2 based on a standard titration curve of recombinant IL-2, which was included in each separate assay.
In an initial series o f e x p e r i m e n t s the time for dialysis o f the urine was determined. F o r that p u r p o s e r e c o c m b i n a n t h u m a n I L - 2 (4.0 units/ml) was a d d e d to d i a l y z e d urine obtained f r o m healthy volunteers. A s shown in Fig. 1, the presence o f IL-2 c o u l d not be detected in the in vitro b i o a s s a y in undiluted n o n - d i a l y z e d urine. T h e urine therefore c o n t a i n e d c o m p o n e n t s inhibiting the proliferative activity i n d u c e d b y IL-2. This is also d e m o n s t r a t e d b y the fact that in t w o f o l d dilutions o f the urine I L - 2 could be detected. D i a l y s i s o f the urine a b o l i s h e d the inhibition of the I L - 2 activity. F r o m 24 h onwards a similar result was o b t a i n e d both with I L - 2 a d d e d to urine and with I L - 2 a d d e d as control to tissue-culture m e d i u m . T h e urine samples tested were d i a l y z e d for 24 h and the m e d i u m was refreshed once. Urine s a m p l e s were collected before and at different time points after the intravesical B C G administration. In pretreatment s a m p l e s I L - 2 could not be detected (Table 1). I L - 2 c o u l d be detected in urine o b t a i n e d in the p e r i o d 2 - 6 h after B C G instillation. A t 24 h after B C G instillation IL-2 could no l o n g e r be detected (see Fig. 2). In six patients a c o m p l e t e B C G treatment course o f six intravesical B C G administrations was f o l l o w e d for the production o f I L - 2 in the urine. T w o patients did not p r o d u c e I L - 2 during the six B C G administrations. A f t e r the first three B C G instillations I L - 2 could be detected in the urine o f two patients. A f t e r the sixth B C G instillation I L - 2 was present in the urine o f four of the six patients investigated (Fig. 3). O n the basis o f a control titration curve with
60
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~
ancer mmunology mmunotherapy
© Springer-Verlag 1990
Presence of interleukin-2 in urine of superficial bladder cancer patients after intravesical treatment with bacillus Calmette-Guérin W. H. De Jongl, E. C. De Boer~, A. P. M. Van Der Meijden2, 3, p. Vegt4, P. A. Steerenberg 1, F. M. J. Debruyne z, and E. J. Ruitenberg I 1Laboratoryfor Pathology,National Institute of Public Health and EnvironmentalProtection,P. O. Box 1, 3720 BA Bilthoven,The Netherlands 2 Departmentof Urology,UniversityHospitalNijmegen, Nijmegen 3Departmentof Urology,Grootziekengasthuis,'s-Hertogenbosch 4 Departmentof Urology,St. ElisabethHospital, Leiderdorp Received 11 July 1989/Accepted20 December 1989
Summary. Urine samples were obtained from patients with superficial bladder cancer after immunotherapy with bacillus Calmette-Guérin (BCG). The patients were repeatedly (once a week for 6 consecutive weeks) treated with intravesical administration of approximately 5 x 10s culturable particles of BCG. Some patients received more than six BCG instillations. The urine samples were investigated for the presence of interleukin-2 (IL-2) in an in vitro bioassay using a murine cytotoxic T cell line (CTTL-16) that shows IL-2-dependent growth. Preliminary experiments indicated the presence of inhibitory factors in the urine. This inhibitory activity was abolished after 24 h dialysis. In a neutralization assay with both polyvalent and monoclonal anti-(human IL-2) antibody it was demonstrated that there was indeed IL-2 in the urine samples. In 8 of 11 patients the presence of IL-2 in the urine was demonstrated. The IL-2 production was directly related to the BCG administration as samples obtained just before the BCG instillation were always negative. In IL-2-positive samples a maximum level of IL-2 was observed between 2 h and 6 h after the BCG instillation. In urine samples obtained 24 h after the BCG IL-2 was not detected. In most patients the urine became positive after the third or fourth BCG instillation
Introduction Since the report of Morales et al. [17] in 1976 it has been demonstrated by several independent research groups that local administration of bacillus Calmette-Guérin (BCG) prevents recurrences in superficial bladder cancer, and can resolve carcinoma of the bladder in situ [3, 8, 15]. The BCG was applied by repeated intravesical administration
• O f f p r i n t r e q u e s t s to."
W. H. de Jong
once a week for 6 consecutive weeks. In an experimental murine bladder tumor system (MBT-2) similar BCG treatment caused a reduction in tumor take [20, 22, 25]. Although it is known that BCG can exert various effects on different parts of the immune system, and has shown antitumor activity in various animal tumour systems, the mechanism by which BCG exerts its antitumor activity remains unclear [6, 16]. After local administration at the tumor site BCG evokes a granulomatous inflammatory reaction in which mononuclear cells from the macrophage lineage are predominantly present as epithelioid cells [5, 10]. How the immune system is able to recognise a previously non-immunogenic tumor after BCG administration is unknown. BCG may be acting as an adjuvant inducing recognition of tumor antigens. Recently it was found that in the draining lymph node the expression of major histocompatibility complex (MHC) class Il antigens is enhanced after local BCG administration. This was observed not only after intradermal injection, but also after intravesical administration of BCG [26] (unpublished data). MHC class II antigens play a major role in the recognition and the processing of antigens in the immune response. In man also, the reason why BCG after intravesical administration has antitumor activity for superficial bladder cancer is not known. Both in man and in animals BCG induces scattered foci of inflammatory cells in the bladder wall, part of which show granulomatous characteristics [7, 14, 27]. The repeated BCG administration may be considered as secondary antigen challenges resulting in delayed-type hypersensitivity reactions in the bladder wall. Recently, Ratliff et al. demonstrated the presence of interleukin 2 (IL-2) in the urine of BCG-treated bladder cancer patients [9, 21]. Apart from its role as a T-cell growth factor, where IL-2 may be an intermediate in a secondary immune response such as a delayed-type hypersensitivity reaction, the induction of so-called lymphokine-activated killer cells by IL-2 was observed more recently [24]. The latter function of IL-2 especially is of interest, as these lymphokine-activated killer cells are able to kill a variety of tumor cells.
183 In this study w e i n v e s t i g a t e d the p r e s e n c e o f I L - 2 in the urine o f b l a d d e r c a n c e r patients treated intravesically with B C G - R I V M or B C G - T I C E . M o s t patients were treated with B C G - R I V M , w h i c h has b e e n d e m o n s t r a t e d to have antitumor p o t e n c y both in l a b o r a t o r y a n i m a l t u m o r systems and in s p o n t a n e o u s l y occurring animal tumors [4, 12, 13]. In a r a n d o m i z e d phase III study for the treatment o f superficial b l a d d e r cancer in man, B C G - R I V M h a d equal antit u m o r activity w h e n c o m p a r e d to m i t o m y c i n C [3].
Materials and methods
Results
Patients. Urine specimens were obtained from patients with a stage pTa
or pT1 papillary transitional cell carcinoma of the bladder. These patients received intravesical immunotherapy with bacillus Calmette-Guérin within 2 weeks after transurethral resection of the tumor(s). BCG-RIVM (eight patients) or BCG-TICE (three patients), approximately 5 x 108 culturable particles, was administered in 50 ml 0.9% saline once a week for 6 consecutive weeks. Some patients were treated with more than six intravesical BCG instillations. Urine samples. After collection the urine was kept on melting ice and cenlrifuged (10 min, 500 g) to remove cells and debris. The supematant was collected and stored at -20 ° C. After thawing, the urine was dialyzed against 50 times the volume in phosphate-buffered saline using a dialysis membrane, molecular mass cut-off 6 - 8 kDa (Spectmm Medic Ind., Los Angeles, Calif, USA). The phosphate-buffered saline was refreshed four times, the last time with RPMI-1640 (Gibco Europe, Breda, The Netherlands). Initially the urine samples were dialyzed for 2 days at 4 ° C. Urine specimens obtained later were dialyzed for 24 h (see results Fig. 1). Specimens were sterilized by fillration through a 0.22-gm filter, and stored at -20 ° C until use in the IL-2 bioassay. IL-2 bioassay. For the detection of IL-2 the IL-2-dependent murine
cytotoxic T cell line CTLL- 16 (kindly provided by Dr. L. Aarden, CLB, Amsterdam) was used. CTLL-16 cells were cultured in RPMI-1640 tissue-culture medium supplemented with 10% (v/v) crude recombinant IL-2 (Sanofi, Toulouse, France), 10% (v/v) heat-inactivated fetal calf serum, penicillin (100 U/tal), streptomycin (100 gg/ml), fungizone (0.26 gg/ml) and 50 gM 2-mercaptoethanol. CTLL-16 cells were used in the IL-2 bioassay 3 days after change of the culture medium. For determination of the presence of IL-2 in the urine specimens, the IL-2 and fungizone were omitted from the tissue-culture medium. The bioassay was performed in flat-bottomed microtiter wells (Costar, Cambridge, Mass, USA). In each well 5 x 103 CTLL-16 cells in 100 gl were seeded, and 100 gl test sample or standard recombinant IL-2
m-" o
(Biogen, Geneva, Switzerland) was added. Cells were cultured for 24 h at 37 ° C, 5% CO2 in a humidified atmosphere. For determinations of the CTLL-16 protiferation [methyl-3H]thymidine (5 gCi/ml, 185 kBq/ml; Radiochemical Centre, Amersham, Bucks., UK) was added during the last 4 h of culture. Cellular DNA was harvested on glass-fiber filters using a multiple cell culture harvester (Flow Laboratories, Irvine, Ayreshire, Scotland). [3H]Thymidine incorporation was measured in an ISO cap/300 liquid scintillation counter (Nuclear Inc., Des Plaines, Ill, USA). The results are expressed as units IL-2 based on a standard titration curve of recombinant IL-2, which was included in each separate assay.
In an initial series o f e x p e r i m e n t s the time for dialysis o f the urine was determined. F o r that p u r p o s e r e c o c m b i n a n t h u m a n I L - 2 (4.0 units/ml) was a d d e d to d i a l y z e d urine obtained f r o m healthy volunteers. A s shown in Fig. 1, the presence o f IL-2 c o u l d not be detected in the in vitro b i o a s s a y in undiluted n o n - d i a l y z e d urine. T h e urine therefore c o n t a i n e d c o m p o n e n t s inhibiting the proliferative activity i n d u c e d b y IL-2. This is also d e m o n s t r a t e d b y the fact that in t w o f o l d dilutions o f the urine I L - 2 could be detected. D i a l y s i s o f the urine a b o l i s h e d the inhibition of the I L - 2 activity. F r o m 24 h onwards a similar result was o b t a i n e d both with I L - 2 a d d e d to urine and with I L - 2 a d d e d as control to tissue-culture m e d i u m . T h e urine samples tested were d i a l y z e d for 24 h and the m e d i u m was refreshed once. Urine s a m p l e s were collected before and at different time points after the intravesical B C G administration. In pretreatment s a m p l e s I L - 2 could not be detected (Table 1). I L - 2 c o u l d be detected in urine o b t a i n e d in the p e r i o d 2 - 6 h after B C G instillation. A t 24 h after B C G instillation IL-2 could no l o n g e r be detected (see Fig. 2). In six patients a c o m p l e t e B C G treatment course o f six intravesical B C G administrations was f o l l o w e d for the production o f I L - 2 in the urine. T w o patients did not p r o d u c e I L - 2 during the six B C G administrations. A f t e r the first three B C G instillations I L - 2 could be detected in the urine o f two patients. A f t e r the sixth B C G instillation I L - 2 was present in the urine o f four of the six patients investigated (Fig. 3). O n the basis o f a control titration curve with
60
x E ~J
30
g 4-13
o
20
ò u Œ r,-
jJ//
