Elsevier

Cytokine

Volume 21, Issue 1, 7 January 2003, Pages 17-26
Cytokine

Role of Th1 and Th2 cytokines in BCG-induced IFN-γ production: cytokine promotion and simulation of BCG effect

https://doi.org/10.1016/S1043-4666(02)00490-8Get rights and content

Abstract

Induction of a T-helper-type 1 (Th1) immune response is indispensable for successful treatment of superficial bladder cancer with BCG. In this study possible involvement of various cytokines in BCG action as well as their potential roles in enhancing and mimicking BCG effect were explored. In immunocompetent cell cultures, IFN-γ, a major Th1 cytokine, appears to be a late responsive cytokine to BCG stimulation. Its induction requires involvement of various endogenously produced Th1 and Th2 cytokines. Functional abolishment of any one of these cytokines (IL-2, IL-6, IL-12, IL-18, GMCSF, TNF-α, or IFN-α, except IL-10) by neutralizing antibodies leads to reduced IFN-γ production (19–82% inhibition in mouse and 44–77% inhibition in human systems, respectively). In mice cytokines IL-2, IL-12, IL-18, and GMCSF are observed to synergize with BCG for IFN-γ production, whereas in human cytokines IL-2, IL-12, TNF-α, and IFN-α exhibit similar synergistic effects. Rational combinations of these Th1-stimulating cytokines (IL-12 plus IL-18 in mice and IL-2 plus IL-12 in humans, respectively) dramatically up-regulate IFN-γ production that is incomparably superior to BCG for induction of this cytokine. These results suggest that combined Th1-stimulating cytokines and combinations of BCG plus selected Th1-stimulating cytokines are rational candidates for further study in the treatment of bladder cancer patients.

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 (1A600

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.

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