0022-534 7/91/1455-1078$03.00/0 THE JOURNAL OF UROLOGY Copyright© 1991 by AMERICAN UROLOGICAL ASSOCIATION, INC.
Vol. 145, 1078-1081, May 1991
Printed in U.S.A.
THE EFFECTS OF RECOMBINANT HUMAN INTERERON-GAMMA ON A PANEL OF HUMAN BLADDER CANCER CELL LINES S. HAWKYARD,* K. JAMES, S. PRESCOTT, A. M. JACKSON, A. W. S. RITCHIE, J. F. SMYTH G. D. CHISHOLM
AND
From the Departments of Surgery/Urology and the Imperial Cancer Research Fund, Medical Oncology Unit, Western General Hospital, Edinburgh, United Kingdom
ABSTRACT
We have examined the Major Histocompatibility Complex class II antigen inducing capabilities of recombinant human interferon-gamma, on a panel of human transitional cell carcinoma lines which have been raised from original tumours of varying histological grades: RT4 (grade 1), RT112 (grade 2) and MGH-Ul (grade 3). Cells were examined for class II antigens using an indirect immunofluorescent staining method and analysed on a fluorescence activated cell sorter. Twenty percent of RT4 cells constitutively expressed class II antigen. Both RT112 and MGH-Ul were repeatedly found to be negative for this antigen prior to treatment with interferon-gamma. Following treatment with interferon-gamma all three lines showed an increase in class II antigen expression, which was consistently dependent on both the length of incubation and concentration of interferon-gamma. A differential susceptibility was found amongst the three cell lines which may relate to the histological grade of the parent tumor. KEY
WORDS: bladder cancer, interferon-gamma, cell culture
Interferon-gamma is a peptide cytokine produced by antigen specific T cells during an immune response, and also Natural Killer (NK) cells stimulated by interleukin-2 1. Interferongamma has been shown to have several actions of relevance to tumour immunology. Firstly, it appears to be directly cytostatic and possibly cytotoxic to tumour cells. 2 Secondly, and perhaps more importantly, it fulfills a number of immunomodulatory functions, notably activation of macrophages and NK cells and proliferation and activation of T and B cells. 1 Also of importance in this regard is the stimulation of both normal and malignant cells to express the Major Histocompatibility Complex (MHC) class I and class II antigens by interferongamma.1·3 These molecules are crucial to the control of T cell interactions and thus of specific immune responses. 4 Class II MHC antigens, commonly referred to as HLA-DR antigens, are not normally expressed on epithelial cells, including urothelium. 5 We have previously shown, that malignant urothelium converts from HLA-DR negative to HLA-DR positive after BCG treatment for superficial transitional cell carcinoma,6 at a time when interferon-gamma is present in the urine. 7 This suggested that in these circumstances interferon-gamma might be the HLA-DR inducing agent. In order to investigate this further, we have examined the effects of purified recombinant interferon-gamma on the HLA-DR expression of a panel of transitional cell tumour lines corresponding to low, intermediate and high grade malignancy. MATERIAL AND METHODS
Cell culture. Cell lines RT4, RT112 and MGH-Ul were kindly given to us by Dr. J. Masters of the Institute of Urology, London (table 1). All were mycoplasma free as tested by the Public Health Laboratory Service, Porton Down, England. Culture was performed in RPMI 1640 with Hepes (Gibco Ltd., Uxbridge, England.) supplemented with 5% heat inactivated fetal calf serum (FCS) (Sera-Lab, Crawley Down, England.), L-glutamine (two mM), sodium pyruvate (5 mM), penicillin (50 Uml.- 1), streptamycin (50 µ,g. ml.- 1) and amphotericin B (2.5 Accepted for publication November 28, 1990. * Requests for reprints: Department of Surgery, Wilkie Laboratory, Teviot Place, Edinburgh EH8 9AG, Scotland. Funded by the Imperial Cancer Research Fund.
TABLE
1. Cell line characteristics
Histological Doubling Limiting Time Dilution Originator Grade of Reference Original Tumour (Hours) (Cells/Well) RT4 RT112 MGHUl
Rigby Rigby Prout
Gl G2 G3
51 42 20
5 5 1
9 10 11
µ,g. ml.- 1) which shall be referred to hereafter as complete
medium. For routine culture and interferon incubations, cells were grown in 25 cm. 2 plastic flasks (J. Bibby Science Products Ltd., Stone, Staffordshire, England.). Preliminary growth curves were determined for each line, by seeding one ml. aliquots at the appropriate cell concentration in 24 well plates (Sterilin Ltd., Feltham, England.). Every 24 to 48 hours cells were recovered from three wells by trypsinisation (trypsin/ ethylenediaminetetraacetic acid [EDTA] solution, 0.5 gl.- 1 trypsin and 0.2 gl.- 1 of sodium EDTA dissolved in magnesium and calcium-free phosphate buffered saline) and counted using an improved Neubauer hemocytometer. The remaining wells were re-fed with complete medium. The lowest cell density which resulted in single colonies being formed was also determined. Ninety-six wells, flat bottomed plates (Sterilin) were seeded at between 100 and 0.5 cells per well, in 200 µ,l. of complete medium. Seven days later the colonies in each well were counted. Interferon treatment. Each line was grown until in log phase. At this stage the supernatant was replaced with seven ml. of complete medium supplemented with several different concentrations (O, 10, 100, 500 and 1000 U ml.- 1) of recombinant human interferon-gamma (Boehringer Mannheim, Lewes, Sussex, England). After 48 hours the supernatant was discarded and a single cell suspension made from the monolayer using trypsin/EDT A solution. The suspensions were then stained immediately using an indirect immunofluorescent technique. A time course study was set up, in which the 3 lines were incubated as above with 500 U ml.- 1 of interferon-gamma for 48, 72 and 120 hours. At 48 and 96 hours the supernatants were removed and replaced with fresh medium supplemented with 500 U ml.- 1 of interferon-gamma. Indirect immunofluorescent staining. Aliquots of 5 X 105 cells
1078
1079
EFFECTS OF INTERFEROl·~-GAivHvIA ON BLADDER TU"I:v10R CELLS Ii~ VITRO
under test, were dispensed in triplicate into 500 µl. microtitre tubes (Sarstedt, West Germany). The cells were pelleted by centrifuging at 140 g for five minutes, the supernatant removed, and the cells dispersed again by vortexing. One hundred microlitres of mouse antihuman-HLA DR monoclonal antibody (DA6.231, Dr. K Guy, Human Genetics Unit, Medical Research Council, Edinburgh) was added to the cells at the optimal dilution and incubated at 4C for 45 minutes. The cells were then washed twice with phosphate buffered saline containing 1 % (vol:vol) FCS and 0.01 % sodium azide. After the second wash, 100 µL of fluorescein isothiocyanate (FITC) conjugated sheep anti mouse-IgG antibody (Sigma Chemical Company Ltd., Dorset, England.) was added to the dispersed cell pellet and incubated for a further 45 minutes at 4C. Finally the cells were washed twice with the wash solution and fixed in 1% formaldehyde. Negative controls were set up for each cell line in which the primary antibody was replaced by 100 µL of an irrelevant monoclonal antibody, anti-CD4 (Scottish Antibody Production Unit, Carluke, Scotland). An immortalised human B-cell line which is designated HBl was used as a positive controL This cell line was raised in the department of Surgery. It is a spontaneously transformed human B cell line derived from a peripheral blood lymphocyte preparation. The cell suspensions were analysed 12 hours later using a Coulter EPICS C fluorescence activated cell sorter (FACS), for both the percentage of cells staining positively for HLA-DR antigen and the mean intensity of those stained (the mean intensity relates to the antigen density). The gain and photomultiplier tube voltage setting were kept constant throughout and the background fluorescence was set at 1 % in the negative control samples. Intensity of staining was given as channel numbers on a linear scale to facilitate comparisons between different dose and time point values. Statistical analysis. This was performed using the Wilcoxon's Rank Sum Test. 8 RESULTS
Although the growth characteristics of the three cell lines examined, RT4 (Grade 1), RT112 (Grade 2) and MGH-Ul (Grade 3), have already been investigated, 9 "100 11 we repeated growth studies for each in our laboratory. The results are shown in figure 1 and table 1, and are in keeping with those previously reported and appropriate for the grades of the cell lines. Figure 2 shows baseline expression of HLA-DR antigens by 120 ,I. ,I.
100
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Vol. 145, 1078-1081, May 1991
Printed in U.S.A.
THE EFFECTS OF RECOMBINANT HUMAN INTERERON-GAMMA ON A PANEL OF HUMAN BLADDER CANCER CELL LINES S. HAWKYARD,* K. JAMES, S. PRESCOTT, A. M. JACKSON, A. W. S. RITCHIE, J. F. SMYTH G. D. CHISHOLM
AND
From the Departments of Surgery/Urology and the Imperial Cancer Research Fund, Medical Oncology Unit, Western General Hospital, Edinburgh, United Kingdom
ABSTRACT
We have examined the Major Histocompatibility Complex class II antigen inducing capabilities of recombinant human interferon-gamma, on a panel of human transitional cell carcinoma lines which have been raised from original tumours of varying histological grades: RT4 (grade 1), RT112 (grade 2) and MGH-Ul (grade 3). Cells were examined for class II antigens using an indirect immunofluorescent staining method and analysed on a fluorescence activated cell sorter. Twenty percent of RT4 cells constitutively expressed class II antigen. Both RT112 and MGH-Ul were repeatedly found to be negative for this antigen prior to treatment with interferon-gamma. Following treatment with interferon-gamma all three lines showed an increase in class II antigen expression, which was consistently dependent on both the length of incubation and concentration of interferon-gamma. A differential susceptibility was found amongst the three cell lines which may relate to the histological grade of the parent tumor. KEY
WORDS: bladder cancer, interferon-gamma, cell culture
Interferon-gamma is a peptide cytokine produced by antigen specific T cells during an immune response, and also Natural Killer (NK) cells stimulated by interleukin-2 1. Interferongamma has been shown to have several actions of relevance to tumour immunology. Firstly, it appears to be directly cytostatic and possibly cytotoxic to tumour cells. 2 Secondly, and perhaps more importantly, it fulfills a number of immunomodulatory functions, notably activation of macrophages and NK cells and proliferation and activation of T and B cells. 1 Also of importance in this regard is the stimulation of both normal and malignant cells to express the Major Histocompatibility Complex (MHC) class I and class II antigens by interferongamma.1·3 These molecules are crucial to the control of T cell interactions and thus of specific immune responses. 4 Class II MHC antigens, commonly referred to as HLA-DR antigens, are not normally expressed on epithelial cells, including urothelium. 5 We have previously shown, that malignant urothelium converts from HLA-DR negative to HLA-DR positive after BCG treatment for superficial transitional cell carcinoma,6 at a time when interferon-gamma is present in the urine. 7 This suggested that in these circumstances interferon-gamma might be the HLA-DR inducing agent. In order to investigate this further, we have examined the effects of purified recombinant interferon-gamma on the HLA-DR expression of a panel of transitional cell tumour lines corresponding to low, intermediate and high grade malignancy. MATERIAL AND METHODS
Cell culture. Cell lines RT4, RT112 and MGH-Ul were kindly given to us by Dr. J. Masters of the Institute of Urology, London (table 1). All were mycoplasma free as tested by the Public Health Laboratory Service, Porton Down, England. Culture was performed in RPMI 1640 with Hepes (Gibco Ltd., Uxbridge, England.) supplemented with 5% heat inactivated fetal calf serum (FCS) (Sera-Lab, Crawley Down, England.), L-glutamine (two mM), sodium pyruvate (5 mM), penicillin (50 Uml.- 1), streptamycin (50 µ,g. ml.- 1) and amphotericin B (2.5 Accepted for publication November 28, 1990. * Requests for reprints: Department of Surgery, Wilkie Laboratory, Teviot Place, Edinburgh EH8 9AG, Scotland. Funded by the Imperial Cancer Research Fund.
TABLE
1. Cell line characteristics
Histological Doubling Limiting Time Dilution Originator Grade of Reference Original Tumour (Hours) (Cells/Well) RT4 RT112 MGHUl
Rigby Rigby Prout
Gl G2 G3
51 42 20
5 5 1
9 10 11
µ,g. ml.- 1) which shall be referred to hereafter as complete
medium. For routine culture and interferon incubations, cells were grown in 25 cm. 2 plastic flasks (J. Bibby Science Products Ltd., Stone, Staffordshire, England.). Preliminary growth curves were determined for each line, by seeding one ml. aliquots at the appropriate cell concentration in 24 well plates (Sterilin Ltd., Feltham, England.). Every 24 to 48 hours cells were recovered from three wells by trypsinisation (trypsin/ ethylenediaminetetraacetic acid [EDTA] solution, 0.5 gl.- 1 trypsin and 0.2 gl.- 1 of sodium EDTA dissolved in magnesium and calcium-free phosphate buffered saline) and counted using an improved Neubauer hemocytometer. The remaining wells were re-fed with complete medium. The lowest cell density which resulted in single colonies being formed was also determined. Ninety-six wells, flat bottomed plates (Sterilin) were seeded at between 100 and 0.5 cells per well, in 200 µ,l. of complete medium. Seven days later the colonies in each well were counted. Interferon treatment. Each line was grown until in log phase. At this stage the supernatant was replaced with seven ml. of complete medium supplemented with several different concentrations (O, 10, 100, 500 and 1000 U ml.- 1) of recombinant human interferon-gamma (Boehringer Mannheim, Lewes, Sussex, England). After 48 hours the supernatant was discarded and a single cell suspension made from the monolayer using trypsin/EDT A solution. The suspensions were then stained immediately using an indirect immunofluorescent technique. A time course study was set up, in which the 3 lines were incubated as above with 500 U ml.- 1 of interferon-gamma for 48, 72 and 120 hours. At 48 and 96 hours the supernatants were removed and replaced with fresh medium supplemented with 500 U ml.- 1 of interferon-gamma. Indirect immunofluorescent staining. Aliquots of 5 X 105 cells
1078
1079
EFFECTS OF INTERFEROl·~-GAivHvIA ON BLADDER TU"I:v10R CELLS Ii~ VITRO
under test, were dispensed in triplicate into 500 µl. microtitre tubes (Sarstedt, West Germany). The cells were pelleted by centrifuging at 140 g for five minutes, the supernatant removed, and the cells dispersed again by vortexing. One hundred microlitres of mouse antihuman-HLA DR monoclonal antibody (DA6.231, Dr. K Guy, Human Genetics Unit, Medical Research Council, Edinburgh) was added to the cells at the optimal dilution and incubated at 4C for 45 minutes. The cells were then washed twice with phosphate buffered saline containing 1 % (vol:vol) FCS and 0.01 % sodium azide. After the second wash, 100 µL of fluorescein isothiocyanate (FITC) conjugated sheep anti mouse-IgG antibody (Sigma Chemical Company Ltd., Dorset, England.) was added to the dispersed cell pellet and incubated for a further 45 minutes at 4C. Finally the cells were washed twice with the wash solution and fixed in 1% formaldehyde. Negative controls were set up for each cell line in which the primary antibody was replaced by 100 µL of an irrelevant monoclonal antibody, anti-CD4 (Scottish Antibody Production Unit, Carluke, Scotland). An immortalised human B-cell line which is designated HBl was used as a positive controL This cell line was raised in the department of Surgery. It is a spontaneously transformed human B cell line derived from a peripheral blood lymphocyte preparation. The cell suspensions were analysed 12 hours later using a Coulter EPICS C fluorescence activated cell sorter (FACS), for both the percentage of cells staining positively for HLA-DR antigen and the mean intensity of those stained (the mean intensity relates to the antigen density). The gain and photomultiplier tube voltage setting were kept constant throughout and the background fluorescence was set at 1 % in the negative control samples. Intensity of staining was given as channel numbers on a linear scale to facilitate comparisons between different dose and time point values. Statistical analysis. This was performed using the Wilcoxon's Rank Sum Test. 8 RESULTS
Although the growth characteristics of the three cell lines examined, RT4 (Grade 1), RT112 (Grade 2) and MGH-Ul (Grade 3), have already been investigated, 9 "100 11 we repeated growth studies for each in our laboratory. The results are shown in figure 1 and table 1, and are in keeping with those previously reported and appropriate for the grades of the cell lines. Figure 2 shows baseline expression of HLA-DR antigens by 120 ,I. ,I.
100
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'
;:;"'
""
~
•o
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~
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A MGIUJl(GJ)
