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The Journal of Immunology, 1999, 163: 4246-4252.
Copyright © 1999 by The American Association of Immunologists

Role of IL-12 in the Induction and Potentiation of IFN-{gamma} in Response to Bacillus Calmette-Guérin1

Michael A. O’Donnell2,*, Yi Luo*, Xiaohong Chen*, Akos Szilvasi*, Sharon E. Hunter{dagger} and Steven K. Clinton{ddagger}

* Division of Urology, Beth Israel Deaconess Medical Center, Boston, MA 02215; {dagger} Genetics Institute, Inc., Cambridge, MA 02140; and {ddagger} Division Hematology and Oncology, Arthur G. James Cancer Hospital, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been accepted as the most effective agent in clinical use against superficial bladder cancer, its mechanism of action remains incompletely understood. A kinetic analysis in assessing the potential role of cytokines from BCG-stimulated murine splenocytes showed that IL-12 expression preceded that of other cytokines. Experiments subtracting endogenous BCG-driven IL-12 using neutralizing Ab or augmenting its activity with supplemental rIL-12 revealed not only that IL-12 plays a dominant role in IFN-{gamma} induction but also that it is normally dose limiting. A striking increase in IFN-{gamma} production could be generated in both mouse and human immunocompetent cell culture by the addition of even a small amount of rIL-12. Moreover, this same synergistic effect could be replicated during in vivo administration of BCG plus rIL-12 into the mouse bladder and was observed in a patient receiving intravesical combination therapy. In costimulation cultures, this synergy appeared to partially rely on IL-18 and IL-2 and could be down-regulated by IL-10. This suggests that a dynamic interplay between Th1 and Th2 cytokines is responsible for net IFN-{gamma} production. The ability of supplemental exogenous IL-12 to strongly shift this balance toward Th1 provides an immunological basis for using it in conjunction with intravesical BCG for bladder cancer immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacillus Calmette-Guérin (BCG)3 is the most effective intravesical agent for the treatment of superficial bladder cancer and prophylaxis against recurrent disease (1, 2, 3). Despite clinical success, the mechanism of BCG action remains poorly understood (4). Among the many immunological events associated with BCG stimulation, induction of an array of cytokines appears to be a major effect. Cytokines IL-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12, interferon-inducible protein 10, TNF-{alpha}, GM-CSF, and IFN-{gamma} have been documented to be readily detectable in intravesical BCG-treated patients’ urine as well as in BCG-stimulated human and murine immunocompetent cell cultures (5, 6, 7, 8, 9, 10, 11, 12). Although the specific role each of these cytokines plays in orchestrating the antitumor action of BCG is not clear, IFN-{gamma}, a major Th1 cytokine, has consistently been shown to up-regulate important cell surface proteins such as MHC Ags, B7 immune costimulators, ICAMs, and apoptosis orchestrators (FAS and TNF-{alpha} receptors) (13, 14, 15, 16, 17, 18, 19). Furthermore, in clinical practice, a massive burst of urinary IFN-{gamma} along with other Th1 cytokines (IL-12 and IL-2) after BCG instillation has been observed to be a common feature in BCG responders, whereas higher levels of Th2 cytokines IL-10 and/or IL-6 appear to be associated with BCG failure. This suggests that antitumor immunity induced by intravesical BCG in bladder cancer therapy largely depends on proper activation of the Th1 immune pathway.

Despite the efficacy of current BCG therapy in human superficial bladder cancer, 30–50% of patients either fail to respond initially or relapse within the first 5 yr of treatment (20). Without a clearer understanding of the mechanism of action of BCG, efforts to improve its clinical efficacy have been largely empirical. Because IL-12 is known as a primary initiator of Th1 immune response and acts as an upstream positive regulator for IFN-{gamma} production from NK and Th1 cells (21, 22, 23, 24, 25, 26, 27), this study was undertaken to investigate the role of IL-12 in the BCG-mediated immune response and to determine whether such an immune response could be enhanced by supplementation of exogenous rIL-12. Because IFN-{gamma} is the most predominant Th1 cytokine observed in clinical responders undergoing BCG therapy (10), the expression of this cytokine was evaluated in this study to define the acquisition of a Th1 immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human subjects

In accordance with approved institutional review board guidelines at the Beth Israel Deaconess Medical Center, blood and urine samples were collected from patients with superficial bladder cancer and normal volunteers. Urine for cytokine analysis was stabilized before freezing by the addition of a 10-fold concentrated buffer containing 2 M Tris-HCl (pH 7.6), 5% BSA, 0.1% sodium azide, and the following protease inhibitors (Sigma, St. Louis, MO): aprotinin, pepstatin, leupeptin at 0.01 mg/ml, and AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) at 0.1 mg/ml. Samples were routinely stored at -70°C before batch analysis for cytokines by ELISA.

Mice

Female C57BL/6J mice were obtained at 6–8 wk of age from The Jackson Laboratory (Bar Harbor, ME) and were housed at 20°C with a 12-h light cycle in the Animal Research Facility at Beth Israel Deaconess Medical Center. Mice were acclimated for at least 1 wk before use. Animal care was provided in accordance with procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 86-23, 1985).

BCG

A Pasteur strain of live BCG that had been previously transfected with the kanamycin resistance plasmid 261 was used in all experiments (28, 29). BCG was kept in log rate growth at 37°C in 7H9 Middlebrook broth (Difco, Detroit, MI) containing 0.5% BSA and 0.05% Tween 80 (Sigma) under conditions of continuous shaking. Log phase cultures of viable BCG were quantified using the absorbance at 600 nm (1 A600 unit = 2.5 x 107 CFU). Previous experiments had shown that responses to this BCG were very similar to those obtained using commercial lyophilized preparations.

Cytokines, Abs, and ELISA

Highly purified, endotoxin-free, recombinant murine and human IL-12 were provided by Genetics Institute (Cambridge, MA). Goat polyclonal neutralizing anti-murine IL-12 p70, rabbit anti-human IL-12 p40, and monoclonal anti-human IL-12 p40 (clone C11.5.14) Abs were also obtained from Genetics Institute. Recombinant murine IL-10 and neutralizing anti-murine IL-2 were purchased from PharMingen (San Diego, CA). Paired monoclonal ELISA capture and detecting Abs for murine (IL-2, IL-4, and IL-10) and human (IL-4 and IL-10) were also obtained from PharMingen. Paired human and murine IFN-{gamma} Abs and a human IL-2 ELISA kit were obtained from Endogen (Boston, MA). Hayashibara (Okayama, Japan) supplied polyclonal neutralizing Ab to murine IL-18, while polyclonal neutralizing Ab to human IL-18 was purchased from R&D Systems (Minneapolis, MN). ELISAs were performed in a sandwich format according to the manufacturer’s instructions and typically provided sensitivities in the 10–20 pg/ml range with interwell variances of <15%.

In vitro murine splenocyte and human PBMC cultures

Murine spleens were removed under sterile conditions, minced, filtered through a fine nylon mesh, and placed in ACK lysing buffer (0.15 M NH4Cl, 1.0 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.4) to remove RBC. Pooled cells from usually three to five animals were then resuspended in complete RPMI 1640 medium containing kanamycin (30 µg/ml) and transferred at a final concentration of 1–4 x 106 cells/ml, depending on the experiment, to 24- or 96-well tissue culture plates containing the appropriate stimulus to be tested. Viability by trypan blue exclusion always exceeded 90%. Human PBMCs were prepared from buffy coat leukocytes purified on Ficoll-Paque (Pharmacia, Uppsala, Sweden) and cultured under similar conditions as mouse splenocyte cultures. Supernatants were harvested after 72 h of stimulation unless otherwise stated and were frozen at -70°C before batch testing in cytokine ELISAs.

Intravesical drug administration and urine recovery in mice

Mice were anesthetized by the i.p. administration of ketamine/xylazine/acepromazine stock solution at a dose of 0.2 ml/10 g of body weight. The stock solution was prepared by combining 1.5 ml of ketamine with 0.75 ml of xylazine and 0.5 ml of acepromazine (all products from J. A. Webster), and the resulting 2.75-ml volume was then mixed with 35.75 ml of sterile water and kept at room temperature before use. Under these conditions mice remain asleep for ~1–2 h.

Bladders were catheterized with a 24-gauge Teflon i.v. cannula lubricated in glycerol. After aspiration of all remaining urine, 0.1 ml of drug was administered. Drug was retained by maintaining the cannula within the bladder with a 1-ml Tb syringe attached. After 1 h the cannula was removed, and mice were allowed to void normally. Mice were placed in metabolic cages overnight (15 h) with ample water but no solid food. Urine was collected in a recovery tube on ice containing 0.1 ml/mouse of a 10x urine stabilizer solution (2 M Tris-HCl (pH 7.6), 5% BSA, 0.1% sodium azide, plus 1/2 COMPLETE protease inhibitor tablet (Boehringer Mannheim, Indianapolis, IN)). Mice produced an average of 0.5–1.0 ml of urine/mouse during this time. After collection the urine was spun to remove any solid debris and was stored at -70°C until batch ELISA cytokine measurements were performed.

Data presentation and statistical evaluations

All figures are derived from representative experiments repeated a minimum of two or three times with similar results. Error bars represent SDs from the mean of a minimum of two independent measurements.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-12 dependence in BCG-induced IFN-{gamma} production

IL-12 is thought to be a prime initiator of the Th1 response (26). In BCG-stimulated murine splenocyte culture, the majority of IL-12 was detectable within the first 24 h, preceding the rise of other Th1 and Th2 cytokines (Fig. 1GoA). Because IFN-{gamma} is an immediate downstream product of IL-12, this early expression of IL-12 might be important for polarizing the immune response to BCG toward the Th1 pathway. This conjecture was substantiated by addition of exogenous neutralizing Ab to IL-12 into the splenocyte culture derived from BCG-primed mice (Fig. 1GoB). Approximately 80% of BCG-induced IFN-{gamma} production was diminished by the Ab. Although neutralizing Ab to IL-2 also showed an inhibitory effect (50%), BCG-driven IL-12 appeared to be more associated with the effect of BCG on IFN-{gamma} induction than did IL-2. Similar results were also obtained using splenocytes from naive mice, with the exception that the IFN-{gamma} response to BCG was significantly lower and much less IL-2 dependent (data not shown). Exogenous rIL-10, an antagonist of the Th1 response, abolished BCG-induced IFN-{gamma} production by 86% largely by suppressing the induction of IL-12, but not IL-2 (data not shown).



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  		FIGURE 1. A, Kinetics of cytokine production from BCG-stimulated murine splenocytes. Splenocytes (2 x 106/ml) from naive mice were incubated with 2.5 x 106 CFU/ml of BCG for up to 3 days. Minimal cytokine was detected in unstimulated cultures (data not shown). Data were represented as a percentage of the maximum value. B, Th1 cytokine dependence in BCG-induced IFN-{gamma} production. Splenocytes (4 x 106/ml) were prepared from BCG-primed mice (three doses of BCG over 3 wk at 2.5 x 106 CFU/dose) 2 wk after the last priming and incubated with 5 x 104 CFU/ml of BCG in the presence or the absence of neutralizing Ab (3 µg/ml) or murine rIL-10 (2.5 ng/ml) for 72 h. BCG-primed splenocytes were selected because they made substantially more cytokines in response to BCG in culture than nonprimed splenocytes. Control rabbit and goat Abs had no effect on IFN-{gamma} production (data not shown). Values represent the mean ± SD from two independent determinations.

 
Enhancement of BCG-induced IFN-{gamma} production from murine splenocytes and human PBMCs by supplemental exogenous rIL-12

Because the antitumor activity of BCG in humans is enhanced by repetitive BCG exposure, perhaps the requisite time to acquire a mature immune response can be hastened by the rational addition of specific cytokines, resulting in improved antitumor activity. To determine whether IL-12 would have such an ability, cultures of splenocytes from naive mice were incubated with increasing amounts of BCG and/or rIL-12 (Fig. 2Go). While either agent alone produced only modest gains in IFN-{gamma} synthesis, combination treatment was clearly synergistic. At an optimal combination of BCG with rIL-12, IFN-{gamma} production was increased ~5-fold over what could be expected from the simple summation of each agent alone.



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  		FIGURE 2. Dose-dependent synergy between IL-12 and BCG for IFN-{gamma} production. Splenocytes (2 x 106/ml) from naive mice were incubated with various concentrations of murine rIL-12 and/or BCG for 72 h. Values represent the mean ± SD from two independent determinations.

 
Such synergy between BCG and rIL-12 for IFN-{gamma} production was also evidenced in human PBMC cultures (Fig. 3Go). The fold increase in IFN-{gamma} production with combination treatment ranged from 4–128, with BCG-naive individuals (HW and MO) displaying the greatest degree of IFN-{gamma} amplification. This indicates that exogenous rIL-12 could accelerate the Th1 immune response to BCG stimulation. The absolute amount of IFN-{gamma} produced, however, was dependent on prior BCG or tuberculosis exposure and greatest in patients AS, DF, MK, and SB.



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  		FIGURE 3. Enhancement of BCG-induced IFN-{gamma} production by IL-12. Human blood samples were collected from six patients (HW and MO were BCG-naive individuals, and AS, DF, MK, and SB had prior BCG or tuberculosis exposure history). PBMCs (3 x 106/ml) were stimulated with human rIL-12 (200 pg/ml) and/or BCG (2.5 x 104 CFU/ml) for 72 h. The amount of IFN-{gamma} in the nonstimulated PBMCs as well as in BCG or rIL-12-stimulated PBMCs together with the BCG/Tb history information are indicated below the graph. The fold increase in IFN-{gamma} was calculated by dividing the combination value by the sum of individual values. Interwell variation for duplicates was <20%. No significant amount of IFN-{gamma} was observed from unstimulated PBMCs.

 
Synergy of intravesical IL-12 with BCG in inducing urinary and serum IFN-{gamma}

The ability of rIL-12 to synergize with BCG to produce urinary IFN-{gamma} in the murine bladder was investigated. Mice were treated intravesically every other day with BCG, rIL-12, or BCG mixed with rIL-12 for a total of six treatments. Urinary IFN-{gamma} mass per mouse for the 15-h collections was recorded for treatments 3, 4, and 5 (Fig. 4GoA). While at treatment 3 no synergy of IL-12 with BCG was observed, by treatments 4 and 5 the amount of urinary IFN-{gamma} in the combined therapy group significantly exceeded that in the BCG or IL-12 monotherapy group. When the BCG dose was fixed, a dose-dependent synergy of rIL-12 on BCG was apparent (Fig. 4GoB). Mouse serum was collected 6 h after completing the last dose of treatment for IFN-{gamma} measurement (Fig. 5Go). The IFN-{gamma} level in the sera of the mice receiving BCG plus IL-12 combination therapy was 3-fold higher than that in mice receiving BCG monotherapy and 6-fold higher than that in mice receiving IL-12 monotherapy.



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  		FIGURE 4. Synergy of intravesical IL-12 with BCG in inducing urinary IFN-{gamma}. Mice were treated intravesically every other day with either BCG (2.5 x 106 CFU/dose), murine rIL-12 (5 µg/dose), or BCG (2.5 x 106 CFU/dose) mixed with indicated doses of rIL-12 for a total of six treatments. Urinary IFN-{gamma} mass per mouse for the 15-h collections was recorded. Synergistic effect (A) and dose dependence (B) of IL-12 on BCG for IFN-{gamma} induction are represented. Values represent the mean ± SD from two independent determinations.

 


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  		FIGURE 5. Induction of elevated serum IFN-{gamma} by intravesical administration of IL-12 plus BCG. Mice were treated intravesically every other day with either BCG (2.5 x 106 CFU/dose), murine rIL-12 (4 µg/dose), or BCG (2.5 x 106 CFU/dose) mixed with murine rIL-12 (4 µg/dose) for a total of six treatments. Serum was obtained for IFN-{gamma} measurement 6 h after completing the last dose of treatment. Values represent the mean ± SD from two independent determinations.

 
Such up-regulation of urinary IFN-{gamma} production by combined intravesical BCG plus IL-12 was also manifested in a clinical bladder cancer patient (MN). During the initial unsuccessful course of intravesical BCG plus IFN-{alpha} 2B therapy (once a week for a total of eight treatments), MN showed a gradually increasing pattern of urinary IFN-{gamma} production following each of the first five treatments but a much reduced amount of IFN-{gamma} by the end of the latter three treatments (Fig. 6GoB). To investigate whether IFN-{gamma} production could be enhanced by supplementation with Th1-stimulating cytokines such as IL-2, IL-12, GM-CSF, and IFN-{alpha}, MN's PBMCs were incubated in vitro with BCG plus each of these cytokines for IFN-{gamma} induction (Fig. 6GoA). Among the combinations, only the costimulation of BCG plus rIL-12 evoked an ample amplification of IFN-{gamma}. When MN was retreated intravesically with the combination of BCG plus rIL-12, he showed an immediate massive induction of urinary IFN-{gamma} in the first treatment and in the following two treatments (Fig. 6GoB). His urinary IFN-{gamma} response was amplified up to 15- to 30-fold higher than that of the highest level seen in the fifth treatment during the prior BCG plus IFN-{alpha} therapy. Unfortunately, the discovery of metastatic disease forced discontinuation of this treatment. Nevertheless, the results obtained from both murine and human studies clearly indicate that rIL-12 has the ability to efficiently enhance in vivo Th1 immune responses to BCG via the intravesical route of administration.



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  		FIGURE 6. Enhancement of BCG-induced IFN-{gamma} by supplementation of rIL-12 in a clinical patient (MN). A, Synergy between IL-12 and BCG in induction of IFN-{gamma} from MN's PBMCs. PBMCs (4 x 106/ml) were incubated for 72 h in the presence or the absence of single agent BCG (3 x 105 CFU/ml), human rIFN-{alpha} (104 IU/ml), human rGM-CSF (1000 pg/ml), human rIL-2 (1000 pg/ml), human rIL-12 (500 pg/ml), or the same doses of two combined agents BCG plus rIFN-{alpha}, BCG plus rGMCSF, BCG plus rIL-2, or BCG plus rIL-12. B, Kinetics of MN's urinary IFN-{gamma} production. MN was initially treated with intravesical BCG (full dose) plus human rIFN-{alpha} (50 mu/dose) for a total of eight treatments (I1 to I8). He was later treated with reduced doses of BCG (one-third of the full dose) plus human rIL-12 (2.5 or 5 µg/dose) for a total of three treatments (I'1 to I'3). Urine was collected over the first 12 h following each treatment. Urinary IFN-{gamma} mass is represented. All values represent the mean ± SD from two independent determinations.

 
Roles of IL-18 and IL-2 in IFN-{gamma} induction by costimulation of BCG plus IL-12

The possible involvement of IL-18 and IL-2 in the synergy pathway of costimulation was investigated. Murine splenocytes from naive (Fig. 7GoA) or BCG-primed mice (Fig. 7GoB) were incubated with BCG plus rIL-12 in the presence or the absence of neutralizing Ab to IL-12, IL-2, or IL-18. As expected, anti-IL-12 Ab completely abolished the synergistic effect of exogenous rIL-12 on BCG for IFN-{gamma} induction. Neutralizing endogenous IL-18 or IL-2 in the costimulation cultures reduced IFN-{gamma} production by 72% for anti-IL-18 Ab and by 20% for anti-IL-2 Ab in BCG-naive mice, but by only 17% for both anti-IL-18 and anti-IL-2 Abs in BCG-primed mice. A similar suppression pattern for IFN-{gamma} production by neutralizing these two cytokines in the same costimulation cultures was observed in human PBMCs (Fig. 8Go). Neutralizing endogenous IL-18 or IL-2 reduced IFN-{gamma} production by 33 and 17%, respectively. Addition of exogenous rIL-10 into the costimulation cultures inhibited IFN-{gamma} synthesis by 70% in murine splenocytes (see Fig. 7GoB) and by 33% in human PBMCs (see Fig. 8Go). Boiled IL-10 had no appreciable effect (data not shown).



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  		FIGURE 7. Effects of IL-2, IL-18, and IL-10 on IFN-{gamma} production in response to costimulation with BCG plus IL-12 in two independent experiments using either BCG naive (A) or BCG-primed (B) mouse splenocytes. Splenocytes (4 x 106/ml) were incubated with BCG (2.5 x 105 CFU/ml) plus murine rIL-12 (500 pg/ml) in the presence or the absence of neutralizing Ab (3 µg/ml) or murine rIL-10 (2000 pg/ml). As controls for the costimulation, splenocytes were incubated with medium, BCG (same dose), or rIL-12 (same dose). Control rabbit and goat Abs as well as boiled (heat-inactivated) rIL-10 had no effect on IFN-{gamma} production (data not shown). Values represent the mean ± SD from two independent determinations.

 


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  		FIGURE 8. Effects of IL-2, IL-18, and IL-10 on IFN-{gamma} production from human PBMCs in response to costimulation with BCG plus IL-12. PBMCs (4 x 106/ml) were incubated with BCG (2.5 x 105 CFU/ml) plus human rIL-12 (200 pg/ml) in the presence or the absence of neutralizing Ab (3 µg/ml) or human rIL-10 (1000 pg/ml). As controls for the costimulation, PBMCs were incubated with medium, BCG (same dose), or rIL-12 (same dose). Control rabbit and goat Abs as well as boiled (heat-inactivated) rIL-10 had no effect on IFN-{gamma} production (data not shown). Values represent the mean ± SD from two independent determinations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
BCG has been used as an antitumor agent for bladder cancer immunotherapy for over 2 decades (30). Although BCG antitumor activity has been clinically demonstrated, its successful use has been tempered by its systemic toxicity, unreliable efficacy, and prognostic unpredictability (4, 31). However, since BCG remains the most efficacious topical therapy for superficial bladder cancer, additional efforts to define an improved BCG regimen in both efficacy and safety should be pursued. Although the precise mechanism of BCG action remains to be defined, observations from both clinical and experimental studies suggest that BCG therapy requires an intact immune system, works via a localized process of stimulation, mediates an interacting network of immune cells and cytokines, and induces a nonspecific immune response (4, 31, 32, 33). As a potent immune stimulator, BCG elicits both humoral and cellular responses. However, antitumor immunity induced by intravesical BCG in bladder cancer therapy appears to primarily rely on cellular immune pathways (32, 33). A positive correlation between bladder tumor response to BCG therapy and urinary Th1 cytokine expression has been documented (6, 34).

IL-12, a cytokine primarily derived from APC, plays an important role in natural immune surveillance and has been shown to be a strong polarizer of the Th1 immune response (21, 25, 27). It functions as a growth factor to activate T lymphocytes and NK cells and induces the secretion of IFN-{gamma} from these cells (35, 36). It has been documented that IL-12 is readily inducible in the urine of patients receiving intravesical BCG as well as in human PBMC and murine splenocyte cultures (37, 38, 39, 40). At least in the murine system such accessibility for mounting an IL-12 response to BCG is common regardless of genetic background (our unpublished observations).

A dominant role for IL-12 in inducing IFN-{gamma} after BCG stimulation is suggested from this paper. From experiments using mixed cultures of cells, the source of IFN-{gamma} cannot be precisely determined. However, our preliminary cell fractionation and reconstitution experiments show that CD4+ or CD8+ {alpha}ß T cells are unable to produce IFN-{gamma} after BCG stimulation unless supplied with adherent cells (primarily macrophages), which are known to produce IL-12. Moreover, it has recently been reported that the bladder is ready endowed with both macrophages and CD1a-positive dendritic cells, an even more robust source of IL-12 (41). During the response to BCG, induction of IFN-{gamma} appears to be mediated by the early release of IL-12. Adding neutralizing anti-IL-12 Ab into the mixed cultures of murine splenocytes with BCG at the beginning of the experimental setting inhibited IFN-{gamma} production by ~80%. Analysis of the time course for cytokine production from BCG-stimulated murine splenocytes also showed that induction of IL-12 is an early event during BCG stimulation. This observation agrees with others reporting IL-12 mRNA detection as early as 3 h after BCG stimulation of human monocyte-derived macrophages (42). As expected, exogenous IL-10, a potent inhibitor of IL-12 production (37), strongly suppressed BCG-induced IFN-{gamma} production.

IL-2 has also been reported to contribute to BCG-stimulated IFN-{gamma} production, possibly by stabling its mRNA (29, 43). However, IL-2 appears to be less critical than IL-12 and more dependent on prior BCG exposure. Neutralizing Ab to endogenous IL-2 from BCG-primed murine splenocytes reduced IFN-{gamma} production by 50%. The effect on unprimed mice was negligible, consistent with our other observations that little IL-2 can be detected in such cultures (data not shown). This suggests that sufficient IL-2 is not available to functionally contribute to IFN-{gamma} output until a population of BCG memory T cells is expanded, as would occur with repetitive BCG treatment. Indeed, a strong parallel relationship between urinary IL-2 and IFN-{gamma} has been reported clinically during successive weekly BCG administration (6, 10).

However, while IL-12 appears to be functionally dominant as an IFN-{gamma} inducer, endogenous IL-12 expression after BCG exposure is nonsaturating. In the present study supplemental exogenous rIL-12 showed a dose-dependent capacity to synergistically promote BCG-induced IFN-{gamma} production both in vivo in mouse voided urine after intravesical BCG administration and in vitro in the immune-competent cell cultures. Such synergy was specific, because neutralizing anti-IL-12 Ab nearly completely abolished the effect of rIL-12 in both murine splenocyte and human PBMC cultures. In addition, the clinical compassionate use of intravesical low dose BCG with IL-12 in one patient (MN) did result in marked 15- to 30-fold increases in urinary IFN-{gamma} over those seen with BCG plus IFN-{alpha} 2B, a weaker Th1-polarizing cytokine in more widespread clinical use (44). Furthermore, a saturating dose of exogenous rIL-12 added to BCG also appears to diminish the relative importance of endogenous IL-2. This synergistic effect of IL-12 on BCG suggests that rIL-12 may be able to work in patients who fail other BCG-cytokine combinational treatments or who are at the highest risk for tumor recurrence because of low endogenous IL-2 release (6).

BCG plus IL-12 synergy also appeared to be partially dependent on IL-18. IL-18 is a recently reported novel cytokine with a potent capacity to induce IFN-{gamma} production (45, 46). Whether BCG alone could induce IL-18 expression from the immune cells is not clear from this study. Induction of IL-18 by IL-12 alone and synergy between the two for IFN-{gamma} production have been previously documented (47, 48). Furthermore, in an IL-18-deficient mouse model marked reduction of IFN-{gamma} expression induced by LPS was observed despite normal IL-12 induction (49). Thus, the synergistic effect of IL-12 on BCG in both murine and human systems may depend somewhat on coincident IL-18 synthesis. For unclear reasons naive splenocytes appear more IL-18 dependent than BCG-primed splenocytes. Exogenous IL-10 antagonizes the effect of BCG plus IL-12 costimulation, but whether it works directly to dampen IFN-{gamma} production or indirectly on IL-18 or other endogenous cofactors cannot be determined from our current experiments. However, because IL-10 is produced endogenously after BCG stimulation (50), it also probably plays an important dynamic role in regulating the net Th1 immune response to BCG.

The present study demonstrates that intravesical coadministration of BCG plus rIL-12 augments urinary IFN-{gamma} production much more strongly than either single agent alone. This in vivo observation together with the others from this study provide an immunological basis for immunotherapy of superficial bladder cancer using combination intravesical IL-12 plus BCG.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R29CA64230 and a grant from Genetics Institute. Back

2 Address correspondence and reprint requests to Dr. Michael A. O’Donnell, Division of Urology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail address: Back

3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; Tb, tubercle bacillus. Back

Received for publication December 14, 1998. Accepted for publication August 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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