Role of Th1 and Th2 cytokines in BCG-induced IFN-γ production: cytokine promotion and simulation of BCG effect
Introduction
Intravesical BCG therapy has successfully been used in the treatment of superficial bladder cancer for over two decades [1], [2], [3]. Although the exact mechanism by which BCG mediates antitumor immunity remains unclear, a local nonspecific immunological reaction reflecting various activities of immune cells has been suggested [1], [2], [3]. One of the remarkable phenomena after BCG instillation is a transient secretion of several cytokines in patients' voided urine. Comparably, induction of an array of cytokines by BCG has also been observed in the immunocompetent cell cultures in vitro. Up to now, over a dozen cytokines have been reported to be induced by BCG stimulation, including IL-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, IL-18, IP-10, TNF-γ, GMCSF, and IFN-γ [1], [2], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. However, the specific role each of these cytokines plays in orchestrating the antitumor action of BCG is not clear. Despite this, in clinical practice a massive burst of urinary IFN-γ along with other T-helper-type 1 (Th1) cytokines (IL-12 and IL-2), after BCG treatment has been observed to be a common feature in BCG responders. In contrast, higher levels of Th2 cytokines IL-10 and/or IL-6 appear to be associated with BCG failure [14]. Correlatively, in mouse models IFN-γ and IL-12 but not IL-10 and IL-4 are suggested to be required for immunotherapeutic control of orthotopic bladder cancer [15], [16]. These observations support the idea that effective BCG therapy of bladder cancer requires proper activation of the Th1 immune pathway [17], [18].
Despite the success of current BCG therapy in superficial bladder cancer, 30–50% of patients either fail to respond initially or relapse within the first 5 years of treatment [19], [20], [21]. In addition, toxicity associated with BCG use is frequent and even occasionally life threatening [22], [23]. Apparently, conventional BCG therapy needs to be improved in both therapeutic efficacy and safety. Based on the current understanding of BCG's biological activities, several alternative approaches to improve immunotherapy of bladder cancer have been proposed, including those of concomitant administration of low-dose BCG plus costimulatory cytokines [24], [25], [26], [27], [28], and those of cytokine alone therapies [13], [24], [26], [29], [30]. Favorable clinical responses have been claimed in some of these studies [24], [28], [29], [30]. Since induction of Th1 cytokines is a major feature observed in clinical responders undergoing BCG therapy, this study was focused on the role of these cytokines in the regulation of BCG-mediated immune responses. The potential of these cytokines in augmenting BCG-mediated immune responses and the possibility of using these cytokines as substitutes for BCG to induce Th1 responses were also investigated. Since IFN-γ is a major Th1 restricted cytokine, easily detectable in urine and culture, and its urinary occurrence correlates most with that of IL-2 (a surrogate marker with prognostic value) during BCG therapy [5], [31], its expression was evaluated in this study to define the acquisition of a Th1 immune response.
Section snippets
Induction of IFN-γ by BCG requires involvement of various endogenously expressed Th1 and Th2 cytokines
In kinetic studies with mouse splenocyte cultures, BCG was found to induce expression of an array of cytokines, including IL-2, IL-6, IL-10, IL-12, GMCSF, TNF-α, and IFN-γ (Fig. 1A). This was best revealed with BCG-primed splenocytes since the levels induced by naı̈ve cells were much lower (data not shown). Both IL-4 and IL-18 were either undetected or negligible under the same conditions. It should be noted that the major Th1 cytokine IFN-γ is a late responsive cytokine, for which expression
Discussion
Due to its limitations in bladder cancer treatment, efforts to improve clinical efficacy of the conventional BCG regimen have been pursued for years. However, without a clearer understanding of the mechanism of BCG action, those improvements have largely been empirical. Since a proper induction of Th1 immune response appears necessary for BCG-mediated antitumor immunity, we focused on regulatory cytokines that favor the production of the Th1 restricted cytokine IFN-γ. We have identified a panel
BCG
A Pasteur strain of live BCG that had been previously transfected with the kanamycin resistance plasmid MV261 was used in all the experiments [46]. This BCG strain was routinely kept at 37 °C in 7H9 Middlebrook broth (Difco, Detroit, MI) supplemented with 10% ADC (5% BSA, 2% dextrose, and 0.85% NaCl), 0.05% Tween 80 (Sigma, St. Louis, MO), and 30 μg of kanamycin per ml under the condition of continuous shaking. Log phase cultures of viable BCG were quantified using the absorbance at 600 nm (
Acknowledgements
We are grateful to Dr Haruki Okamura (Hayashibara Biochemical Laboratories, Inc., Hyogo, Japan) for supplying mIL-18 ELISA reagents. This work was supported by USPHS Grant R29 CA64230 (National Cancer Institute, NIH) to M.A.O.
References (46)
- et al.
BCG immunotherapy of bladder cancer: 20 years on
Lancet
(1999) - et al.
Bacillus Calmette-Guérin immunotherapy for superficial bladder cancer: new prospects for an old warhorse
Surg Oncol Clin North Am
(1995) - et al.
Elevations of cytokines interleukin-1, interleukin-2 and tumor necrosis factor in the urine of patients after intravesical bacillus Calmette-Guérin immunotherapy
J Urol
(1990) - et al.
Urinary cytokines during intravesical bacillus Calmette-Guérin therapy for superficial bladder cancer: processing, stability and prognostic value
J Urol
(1996) - et al.
Interleukin-6 production by bladder tumors is upregulated by BCG immunotherapy
J Urol
(1995) - et al.
Radio-immunoassay detection of interferon-gamma in urine after intravesical Evans BCG therapy
J Urol
(1990) - et al.
Urinary Interleukin-8 and 18 predict the response of superficial bladder cancer to intravesical therapy with bacillus Calmette-Guérin
J Urol
(2000) - et al.
BCG-induced urinary cytokines inhibit microvascular endothelial cell proliferation
J Urol
(2000) - et al.
T-cell subsets required for intravesical BCG immunotherapy for bladder cancer
J Urol
(1993) Long-term results of intravesical therapy for superficial bladder cancer
Urol Clin North Am
(1992)