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. Author manuscript; available in PMC: 2009 Dec 7.
Published in final edited form as: J Immunol. 2009 Jun 17;183(1):740–748. doi: 10.4049/jimmunol.0804166

Coexpression of IL-18 Strongly Attenuates IL-12-Induced Systemic Toxicity through a Rapid Induction of IL-10 without Affecting its Antitumor Capacity1

Maria Cecilia Rodriguez-Galan *,2, Della Reynolds *, Silvia G Correa ‡, Pablo Iribarren †, Morihiro Watanabe *, Howard A Young *
PMCID: PMC2789653  NIHMSID: NIHMS161036  PMID: 19535628

Abstract

IL-12 is an excellent candidate for the treatment of cancer due to its ability to drive strong antitumor responses. Recombinant IL-12 protein is currently used in cancer patients; however, systemic expression of rIL-12 presents disadvantages including cost and dose limitation due to its toxicity. In this study, we used hydrodynamic shear of cDNA as a tool to achieve systemic expression of IL-12. We found that sustained but toxic levels of serum IL-12 could be generated in 6- to 7-wk-old B6 mice after a single injection of the cDNA. Unexpectedly, we observed that when IL-12 cDNA is coinjected with IL-18 cDNA, IL-12 antitumor activity was maintained, but there was a significant attenuation of IL-12 toxicity, as evidenced by a greater survival index and a diminution of liver enzymes (ALT and AST). Interestingly, after IL-12 plus IL-18 cDNA administration, more rapid and higher IL-10 levels were observed than after IL-12 cDNA treatment alone. To understand the mechanism of protection, we coinjected IL-12 plus IL-10 cDNAs and observed an increase in survival that correlated with diminished serum levels of the inflammatory cytokines TNF-α and IFN-γ. Confirming the protective role of early IL-10 expression, we observed a significant decrease in survival in IL-10 knockout mice or IL-10R-blocked B6 mice after IL-12 plus IL-18 treatment. Thus, our data demonstrate that the high and early IL-10 expression induced after IL-12 plus IL-18 cDNA treatment is critical to rapidly attenuate IL-12 toxicity without affecting its antitumor capacity. These data could highly contribute to the design of more efficient/less toxic protocols for the treatment of cancer.

Interleukin 12 is a proinflammatory cytokine that presents multiple antitumoral properties. Tumor growth depends mainly on the inability of the organism to elicit a potent immune response and on the formation of new blood vessels that enable tumor nutrition. IL-12 therapy can target both processes mainly to its ability to enhance type 1 immunity, to induce IFN-γ expression (1, 2), and to inhibit tumor angiogenesis mainly through IFN-γ-dependent production of the chemokine IP-10 (3). Moreover, IL-12 can strongly synergize with other cytokines including IL-2 and IL-18 to enhance both cytolytic activity and IFN-γ production by T, NKT, and NK cells (1, 2). The therapeutic effects of IL-12 have been extensively demonstrated in a variety of animal tumor models when it is administrated either systemically or locally at the tumor site (4–7). Moreover, the pleiotropic antitumoral characteristics of IL-12 have encouraged the initiation of several clinical trials in patients with different types of cancer like T cell lymphoma (6), non-Hodgkin lymphoma (8, 9), melanoma (10), ovarian cancer (11), Kaposi's sarcoma (12), renal carcinoma (13), and so on.

Although some disappointing results occurred in most trials because the clinical outcome has not been as successful as the therapeutic benefits observed in the laboratory, IL-12 administration is still under extensive investigation in animal models of cancer, either administrated alone or as an adjuvant (4, 14, 15).

An additional difficulty in the clinical use of IL-12 is the cytokine-associated toxicity when administrated systemically that limits the doses tolerated by patients (16 –19). Thus, in an attempt to attenuate systemic side effects, several laboratories have focused on expressing IL-12 at the site of the tumor (14, 20 –22). Even when local delivery represents an effective and less toxic alternative, in the case of metastasis or tumors that are difficult to reach, systemic expression of IL-12 still remains a more practical alternative.

In the present work, we compared survival and antitumoral responses in two different tumor models (B16 and 3LL) when IL-12 is systemically expressed, either alone or in combination with IL-18, an IL that synergizes IL-12 antitumor effects (23). To perform the experiments, we utilized hydrodynamic shear as a tool to induce cytokine cDNA expression. As previously reported, larger amounts of protein can be produced in the sera of B6 mice with this procedure compared with recombinant protein administration (24). Our data demonstrate that when high concentrations of IL-12 alone or IL-12 plus IL-18 are expressed in young (6- to 7-wk-old) C57BL/6 mice, a similar and highly efficient antitumor activity occurs. However, a significant increase in survival is observed in IL-12 plus IL-18 mice along with an early production of IL-10, lower hepatic function enzymes (ALT and AST), and a diminished inflammatory infiltrate into vital organs. As the rapid production of IL-10 could be attenuating the overall inflammatory response mediated by IL-12 (8, 11), we evaluated this protective effect in young IL-10 knockout (KO)3 mice or wild-type mice treated with a neutralizing IL-10R Ab. Our data demonstrate that the early IL-10 expression induces the premature control of the inflammatory cytokines IFN-γ and TNF-α and support the advantage of the combined IL-12 plus IL-18 administration as a tool in cancer therapy.

Materials and Methods

Mice

C57BL/6 (B6), BALB/c, RAG 2−/− (B6 background, Taconic Farms), IFN-γ−/− (B6 background, contains a disrupted exon 2 of the IFN-γ genomic DNA), IL-10−/−, and iNOS−/− mice (B6 background, The Jackson Laboratory) were used in this study. All mice were 6- to 7-wk-old (young) or 12-wk-old (old) of age and were maintained under specific pathogen-free conditions. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health-Publication No. 86-23, 1985).

Cell lines

B16-F10 melanoma cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and nonessential amino acids at 37°C, 5% CO2.

3LL cells from a murine lung carcinoma cell line were maintained in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine at 37°C in 5% CO2.

Antibodies

For multicolor staining, PE-, Cy-Chrome-, or allophycocyanin-conjugated anti-CD3, anti-IFN-γ, and anti-NK1.1 (BD Pharmingen) were used in various combinations.

For in vivo NK and NKT cell depletion, 200 μg/mouse of neutralizing Ab (clone PK136) obtained from ascites was administrated i.p. 1 day before and 6 days post cDNA injections as previously described (25). For in vivo CD4 and CD8 cell depletion, mice were injected i.p. with 200 μg/mouse of Abs clone GK1.5 or clone 19/178, respectively, on day 0 and every 3 days up to day 15 post cDNA treatment (26). The IL-10R Ab was obtained from ascites and injected i.p. on days −1 and 1 of the protocol at a concentration of 250 μg/mouse. Regarding the cell depletion protocols, NK/NKT cells cannot be detected up to 7 days post each PK136 Abs injection and the same depletion kinetics are observed for CD4 and CD8 T cells when GK1.5 or clone 19/178, respectively, are injected every 3 days.

Hydrodynamic cDNA injections

The hydrodynamic gene transfer procedure was described previously (27). Animals were injected in the tail vein with the cDNAs in 1.6 ml of sterile 0.9% sodium chloride solution in <8 s and separated in four groups: 1) control (15 μg of ORF empty vector control cDNA), 12 plus 18 (5 μg of IL-12 cDNA) (pscIL-12, p40-p35 fusion gene) plus 10 μg of IL-18 cDNA (pDNA elongation factor pro-IL-18), 12 (5 μg of IL-12 cDNA) (pscIL-12, p40-p35 fusion gene), and 18 (10 μg of IL-18 cDNA) (pDNA elongation factor pro-IL-18). All the expression plasmids utilize the human elongation 1-α promoter to drive transcription of the respective cytokines.

This method does not elicit liver damage at the time when control mice were examined. Liver enzyme levels (ALT and AST) in cDNA control-injected mice are low and similar to the levels in untreated mice.

Serum and spleen samples

Sera were obtained and used for cytokine measurement by ELISA and AST and ALT detection by a dry slide colorimetric methodology using a Vitros 250 (Ortho-Clinical Diagnostics).

Spleens were smashed, depleted of red cells by incubation in ACK lysing buffer (BioWhittaker), washed, and resuspended in supplemented media (RPMI 1640, 10% FBS, 2 mM l-glutamine, 5 × 10−5 M 2-ME, 1 mM sodium pyruvate, and nonessential amino acids).

Pulmonary and hepatic metastasis assay

C57BL/6 mice were i.v. injected with either B16-F10 melanoma cells or 3LL cells (0.5 × 106 cells/0.2 ml 0.9% sodium chloride solution) into the tail vein for pulmonary metastasis analysis or by intrasplenic injection (0.5 × 106 cells/0.5 ml 0.9% sodium chloride solution) for hepatic metastasis analysis. Three days later, cDNAs were delivered by hydrodynamic injection. Eleven days later, lungs and livers were collected and the number of metastases was determined under a dissecting microscope.

Subcutaneous tumor growth

C57BL/6 mice were shaved and injected s.c. in the left flank with 1 × 106 B16-F10 melanoma cells in 0.1 ml of sterile 0.9% sodium chloride solution. After ∼10–14 days, when solid tumors were visible (4–5 mm diameter), mice were hydrodynamically injected with the designated cDNAs. Tumor growth was monitored with a caliper.

Cytokine assays

Sera were assayed for cytokine production by ELISA according to the manufacturers' instructions. The kits utilized were: mIL-12 (p70), mIL-10, mIFNγ, mTNFα (R&D Systems), and mIL-18 (MBL International).

Flow cytometry analysis

Multiple-color analysis was performed using FACSort (BD Pharmingen) as previously reported (28). Anti-mouse CD16/CD32 mAb (BD Pharmingen) was used to block non-specific binding. Cells were stained for surface markers for 30 min at 4°C, and washed twice before analysis. For IFN-γ intracellular staining, spleen leukocytes were left in culture for at least 3 h in the presence of brefeldin A (10 μg/ml), then surface stained, fixed, intracellular stained, and analyzed by flow cytometry.

Statistical analysis

Statistical significance was analyzed by means of Student unpaired t test with p < 0.05 considered as significant. Mouse survival curves were plotted as Kaplan-Meier plots (Prism Version IV, GraphPad Software) and a p < 0.05 was considered as significant.

Results

Expression of IL-12 plus IL-18 cDNAs strongly attenuates IL-12 toxicity

In the initial experiments in this study, we performed a kinetic analysis of cytokine levels in the serum of young C57BL/6 mice following hydrodynamic delivery of the cDNAs. Fig. 1A (left) shows that a single injection of IL-12 cDNA is sufficient to produce large amounts of the cytokine with higher levels at 24 h postinjection and a rapid decrease over the following 10 days post-treatment. We also treated a separated group of mice with IL-12 plus IL-18 cDNAs since it is well known that both cytokines synergistically increase IFN-γ production and cytolitic activity (23). In this group, IL-12 kinetics is similar to the mice injected with IL-12 cDNA alone, although higher levels of the cytokine are observed (Fig. 1A, right). Serum IL-18 showed higher levels at 48 h postinjection and then decreased until day 10 (Fig. 1A, right).

FIGURE 1.

FIGURE 1

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Cytokine serum levels and toxicity after hydrodynamic injection of IL-12 and IL-18 cDNAs. C57BL/6 mice (6- to 7-wk-old) were hydrodynamically injected with control, IL-12, IL-18, or IL-12 plus IL-18 cDNAs. Up to 50 days postinjection, animals were bled and the sera were assayed for cytokine levels by ELISA (A) and for alanine aminotransferase (ALT) (C) or aspartate aminotransferase (AST) (D) liver enzyme levels. Mice injected with the cDNAs were also monitored for survival (B). Data are expressed as the mean of three different experiments. 12 and 12 plus 18 vs control: *, p < 0.05; 12 vs 12 plus 18: #, p < 0.05.

Considering the elevated amounts of IL-12 induced in the sera, we decided to monitor the toxicity of the treatment in B6 mice of different ages. Surprisingly, we found that mice older than 12 wk of age completely tolerated the treatment (100% survival, data not shown), however, when young mice (between 6 and 7 wk of age) were treated, almost all mice died (Fig. 1B, left, 10% survival, n > 40). More interestingly, when IL-12 cDNA was coinjected with IL-18 cDNA, mortality was significantly lower (Fig. 1B, right, 60% survival, n > 40), even when IL-12 serum levels were higher than observed when IL-12 cDNA was injected alone (p = 0.003). These findings were unexpected as it has been reported that simultaneous administration of rIL-12 and rIL-18 proteins led to 100% mortality of mice due to the synergistic action of the individual cytokines (29, 30).

One of the clinical manifestations of systemic IL-12 treatment is hepatotoxicity (16 –18, 24, 29, 30). Based on these reports, we measured serum levels of the enzymes ALT and AST as indicators of liver damage at early (day 10) and late (day 50) time points after injection of the cDNAs in young B6 mice since they are more susceptible to the cytokine induced toxicity (Fig. 1, C and D). Mice treated with IL-12 or IL-12 plus IL-18 cDNAs demonstrated a significant increase of ALT and AST on day 10 post treatment that diminished but still remained higher than normal levels by day 50 (p < 0.05). In agreement with the survival data, AST and ALT levels were significantly lower in IL-12 plus IL-18 cDNA treated mice compared with IL-12 cDNA injected group on days 10 and 50, respectively (Fig. 1, C and D, p < 0.05).

Taken together, these data demonstrate that when IL-18 cDNA is coinjected with IL-12 cDNA, a significant increase in survival is observed that correlates with lower serum levels of ALT and AST.

IL-12 and/or IL-18 cDNA expression induces a potent antitumor effect

We next evaluated if the high and persistent serum levels of IL-12 ± IL-18 were able to elicit a protective antitumor response. We tested both a moderate-mild aggressive tumor model (3LL) or an aggressive tumor model (B16). As hydrodynamic delivery results in hepatic expression of the injected cDNAs, we evaluated possible antitumor effects in a local (liver) or in a distant (lungs) site. As can be clearly seen in Fig. 2A and, after the counting of metastases (data not shown), the expression of IL-12 or IL-12 plus IL-18 significantly reduced the number of B16 liver and lung metastases compared with cDNA control-injected young B6 mice (control vs 12 or 12 plus 18; p < 0.05). However, similar numbers were observed in liver and lungs of IL-12 and IL-12 plus IL-18 treated mice, thus indicating that the combination of cytokines did not result in a synergistic antitumor response.

FIGURE 2.

FIGURE 2

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IL-12 alone or coexpressed with IL-18 mediates strong antitumoral activity against B16 lung and liver metastasis formation and s.c. tumor growth. C57BL/6 mice (6- to 7-wk-old) were injected on day 0 with 0.5 × 106 B16 melanoma cells either intrasplenic for liver metastases or i.v. for lung metastases (A). For s.c. tumor growth, B16 cells (1 × 106) were injected s.c. and between days 10–14, when tumors reached a size of approximately 5 mm2, mice were hydrodynamically injected with the designated cDNAs (B). In the days post cDNA injection, tumor growth was measured with a caliper. *, p < 0.005 vs control.

The expression of IL-18 cDNA alone was unable to significantly decrease liver or lung metastasis (Fig. 2A). We next studied if cytokine expression could control not only metastasis but also solid tumor formation. We found that in young B6 mice treated with IL-12 or IL-12 plus IL-18 cDNAs, the tumor growth was totally abolished compared with control cDNA injected mice (p < 0.005) resulting in a complete regression of the tumors. Moreover, in both groups, tumors showed a necrotic morphology 2 wk after the treatments (data not shown). Similar results were observed with 3LL cells (data not shown). Thus, we conclude that the systemic expression of IL-12 or IL-12 plus IL-18 cDNAs has a potent and similar protective effect on liver and lung metastases and is also effective in controlling s.c. tumor cell growth.

Controversial role of IFN-γ in IL-12 mediated toxicity

Data presented by Nakamura et al. indicate that the high levels of IFN-γ generated after treating mice with rIL-12 and rIL-18 proteins were ultimately responsible for the severe adverse effects observed in those animals (30). Moreover, in a phase II IL-12 clinical trial, unacceptable toxicity resulting from IFN-γ overproduction was observed (4). Based on these reports, we hypothesized that differences in the susceptibility observed between IL-12 and IL-12 plus IL-18 treatments could be explained by differential expression of IFN-γ. We observed that administration of IL-12 and IL-12 plus IL-18 cDNAs induced different levels and kinetics of IFN-γ expression (Fig. 3A). Whereas IFN-γ levels in young B6 mice were detected 24 h post IL-12 plus IL-18 cDNA treatment reaching higher levels on day 3, when IL-12 cDNA was injected alone, IFN-γ was detected 48 h post injection with higher levels on day 7, reaching levels 2-fold higher than observed in 12 plus 18 cDNA treated mice.

FIGURE 3.

FIGURE 3

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Role of IFN-γ during IL-12-induced toxicity. C57BL/6 mice (6- to 7-wk-old) were injected on day 0 with the cDNAs and up to 50 days postinjection IFN-γ serum levels were measured by ELISA (A). IFN-γ serum levels in 6-wk-old B6 or BALB/c mice 72 h post IL-12 or IL-12 plus IL-18 cDNA injection (B). Survival monitored in 12-wk-old B6 or IFN-γ KO (GKO) mice following IL-12 or IL-12 plus IL-18 cDNA injections (C). 12 and 12 plus 18 vs control: *, p < 0.05; BALB/c vs B6: #, p < 0.05; and 12 and 12 plus 18 B6 vs 12 and 12 plus 18 GKO: **, p < 0.05.

To better understand if the IFN-γ levels correlated with survival, we injected the cDNAs into mice of different genetic backgrounds as different mouse strains are known to preferentially express a Th1 vs a Th2 cytokine background (B6 = Th1 vs BALB/c = Th2). As shown in Fig. 3B, young BALB/c mice produce significantly less IFN-γ than young B6 mice either after IL-12 plus IL-18 or IL-12 treatments (12 plus 18 B6 vs 12 plus 18 BALB/c or 12 B6 vs 12 BALB/c; p < 0.05). Consistent with this result, 100% of young BALB/c mice survive IL-12 or IL-12 plus IL-18 treatments compared with 10 and 60% survival observed in young B6 mice, respectively (data not shown). If the differential expression of IFN-γ between mouse strains accounts for the different survival outcome, then IFN-γ null mice should be resistant to IL-12 or IL-12 plus IL-18-induced toxicity. Surprisingly, we observed that susceptibility to IL-12 cDNA or IL-12 plus IL-18 cDNAs in IFN-γ deficient mice was similar to B6 mice when animals were young (data not shown). However, when old mice were tested, a considerably decrease in survival was observed both in IL-12 or IL-12 plus IL-18 treated IFN-γ KO mice compared with complete survival of old B6 mice (Fig. 3C, p < 0.05).

Toxicity after IL-12 treatment is partially mediated by NK1.1+, CD4+, and CD8+ T cells

It has been reported that IL-12-induced toxicity can be abolished by simultaneous depletion of T and NK cells as possible sources of IFN-γ (31). Thus, we next determined if those cell types were the main producers of IFN-γ in our model. We confirmed that both, NK/NKT and T cells can produce IFN-γ after IL-12 and IL-12 plus IL-18 cDNA expression (Fig. 4A). As IL-12 and IL-18 can rapidly act upon NK/NKT cells due to constitutively expressed receptors (32), we depleted NK/NKT cells with a specific neutralizing Ab and evaluated survival after cDNAs injections. Fig. 4B shows a significant increase in survival in NK1.1-depleted young mice at early time points between days 11–20 (day 20, percent survival NK1.1-depleted vs B6 = 86 vs 28%, respectively). Next, to address the role of T cells in the response to treatments, we depleted T cells subsets with either anti-CD4 or anti-CD8 neutralizing Abs. After IL-12 cDNA treatment, we found no increase in survival when CD4+ or CD8+ cells were depleted individually (data not shown). However, when both subsets were codepleted, we observed a significant increase in survival that mainly covers a later time point between days 13–27 post-treatment (day 27, percent survival CD4/CD8 depleted vs B6 = 63 vs 7%, respectively), demonstrating that both CD4 and CD8 T cells are involved in IL-12-mediated toxicity (Fig. 4C). Interestingly, after simultaneous depletion of NK/NKT and CD4/CD8 T cells, we observed 100% survival up to day 27, consistent with a protective additive effect based on the observation in NK1.1 and CD4 plus CD8-depleted mice (Fig. 4D).

FIGURE 4.

FIGURE 4

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Toxicity is dependent upon NK/NKT cells and T cells. C57BL/6 mice (6- to 7-wk-old) were injected on day 0 with the cDNAs and 3 days postinjection the percentage of IFN-γ+ cells was evaluated in CD3+ (T cells) or NK1.1+ (NK/NKT cells) splenocytes by intracellular staining (A). Survival monitored post-IL-12 cDNA injections in mice depleted of NK/NKT cells (B), CD4 and CD8 T cells (C), and NK/NKT and CD4/CD8 T cells (D). 12 vs 12-cell depleted: *, p < 0.05.

Early IL-10 expression after IL-12 plus IL-18 cDNA treatments is essential to ameliorate IL-12 induced toxicity

Next, we hypothesized that differences in IFN-γ expression in IL-12 cDNA-treated young B6 mice might be part of a subtle balance between Th1 and Th2 cytokines that are regulated in a different manner when IL-18 is present. Although IL-18 has been initially associated to a Th1 profile, we and other investigators have demonstrated that IL-18 is able to induce a Th1 (IFN-γ, TNF-α) or a Th2 (IL-5, IL-13, IL-10) phenotype in T and NK cells (33, 34). We speculated that in the present model, the antiinflammatory cytokines IL-13 and/or IL-10 could be participating as protective mediators produced after IL-12 plus IL-18 treatment and that the expression of either of these cytokines may account, at least in part, for the difference in survival observed between IL-12 and IL-12 plus IL-18 cDNA-treated mice. As IL-13 was undetectable either in the sera or in the supernatants of splenocytes from young IL-12 or IL-12 plus IL-18 treated mice (data not shown), we focused our attention on IL-10 expression. In this context, it has been previously postulated that IL-10 is produced after IL-12 expression to protect the host from an uncontrolled and damaging inflammatory response (35, 36). To test this hypothesis, we first evaluated the kinetics of IL-10 production in IL-12 and IL-12 plus IL-18 cDNA treated young B6 mice on the days post cDNA administration. Interestingly, we observed a different kinetic in IL-10 expression when comparing both treatments. As shown in Fig. 5A, while IL-12 plus IL-18 mice induced a rapid and higher expression of IL-10 early after cDNA injection (day 2), mice receiving IL-12 cDNA alone expressed higher levels of IL-10 on day 8 post-treatment, by the time at which most of the mice appeared ill.

FIGURE 5.

FIGURE 5

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Early expression of IL-10 in IL-12 plus IL-18 mice is important in controlling IL-12 toxic side effects. C57BL/6 mice (6- to 7-wk-old) were injected on day 0 with the cDNAs and IL-10 serum levels were measured by ELISA on days post-treatment (A). Survival was monitored in IL-10 KO and B6 mice injected with IL-12 cDNA (B) or IL-12 plus IL-18 cDNAs (C). Survival was monitored in B6 mice injected with IL-12 plus IL-18 cDNAs where IL-10 biological effects were blocked in vivo by i.p. injection of a monoclonal anti-IL-10R Ab (D) after cDNAs injections. 12 vs 12 plus 18: *, p < 0.05; 12 and 12 plus 18 (B6) vs 12 and 12 plus 18 (IL-10 KO): **, p < 0.05; 12 plus 18 vs 12 plus 18 (IL-10R Ab): #, p < 0.05.

To confirm if early IL-10 expression is able to attenuate IL-12-induced inflammation, we evaluated survival in IL-10 KO mice after cDNA treatments. In support of our hypothesis, while significant changes in survival were seen in young IL-10 KO mice versus young B6 mice after IL-12 cDNA treatment (Fig. 5B), the lack of IL-10 in animals injected with IL-12 plus IL-18 provoked a more dramatic death curve that started as early as day 1 post-treatment compared with the one observed in B6 mice (Fig. 5C). To further verify the protective role of IL-10 in IL-12 plus IL-18 cDNA treated mice, we performed experiments in young B6 mice treated in vivo with a neutralizing IL-10R Ab. As can be seen in Fig. 5D, when the biological effects of IL-10 were blocked by the Ab, 100% of IL-12 plus IL-18 mice rapidly died by day 7 post-treatment.

Protective role of IL-10 in IL-12 plus IL-18 cDNA treated mice is associated to a rapid control of the inflammatory cytokines IFN-γ and TNF-α

In an attempt to disclose the mechanism of IL-10 protection observed in IL-12 plus IL-18 mice, we coinjected IL-12 and IL-10 cDNAs in young B6 mice and evaluated serum IL-10 levels early after the injections. Fig. 6A shows that when IL-12 and IL-10 cDNAs are coadministrated, a quick and higher IL-10 production occurs compared with when IL-12 cDNA is injected alone, similar to what we observed in IL-12 plus IL-18 mice. In Fig. 6B, we demonstrated that the early IL-10 expression results in almost 100% survival up to day 17 compared with 60% survival observed in IL-12 cDNA treated mice on that day (p < 0.05). Moreover, the overall survival by day 25 in 12 plus 10 cDNA-treated mice reach 30% compared with 7% in mice injected with IL-12 cDNA alone. Finally, we found that mice receiving IL-12 plus IL-10 cDNAs show a significant diminution in the serum levels of TNF-α and IFN-γ (Fig. 6, C and D, respectively), two cytokines that are highly involved in the fatal inflammatory response observed in septic shock (37, 38).

FIGURE 6.

FIGURE 6

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Coexpression of IL-10 and IL-12 cDNAs increases survival and controls IFN-γ and TNF-α serum levels. C57BL/6 mice (6- to 7-wk-old) were injected on day 0 with IL-12 or IL-12 plus IL-10 cDNAs and IL-10 (A), TNF-α (C), and IFN-γ (D) serum levels were measured by ELISA. Mice were monitored for survival (B) on the days post-treatment. 12 vs 12 plus 10; *, p < 0.05.

Discussion

The clinical development of IL-12 as a single agent for systemic cancer therapy has been hindered by its significant toxicity and disappointing antitumor effects in human patients (6, 8, 13, 16–19, 39). The lack of efficacy was accompanied by declining biological effects of IL-12 in the course of repeated administration of the recombinant protein at doses approaching the maximum tolerated dose (MTD = 500 ng/kg). However, when IL-12 is used as an adjuvant in combination with other drugs or cytokines, it demonstrates a much powerful immunotherapeutic property in multiple animal cancer models (40). Based on these reports, we decided to evaluate IL-12 antitumoral effects in the context of systemic cDNA expression alone or in association with IL-18, since it has been reported that IL-18 and IL-12 have synergistic antitumor activity (23). Moreover, IL-18 can augment antitumor activity independently of IL-12 (35, 41, 42). Previous pharmacokinetic studies demonstrated that a single rIL-12 protein administration resulted in lower serum levels of IL-12 that rapidly declined over time, compared with a persistent and more than 100-fold higher concentration of the cytokine when systemic cDNA IL-12 was administrated (15). Our results indicate that when IL-12 and IL-18 are systemically expressed through a single injection of their respective cDNAs, high serum levels of the cytokines are observed for several days and that these levels are well tolerated in BALB/c mice and B6 mice that were at least 12 wk of age. Although IL-12 and IL-18 expression levels are different between both vectors, we found that the levels induced by both cDNAs are biological relevant in vivo since mice injected with 12 plus 18 present a significant increase in survival and a different cytokine profile compared with 12-cDNA treated mice. Moreover, in cell culture, levels of IL-18 as low as 10 ng/ml are found to be strongly stimulatory to synergize with IL-12 (34).

This situation presents the advantage of avoiding repeated administration of the rIL-12 protein to reach persistent levels of the cytokine. Moreover, the single administration of the cDNAs resulted in a strong antitumor response either at local or distal sites. Interestingly, when IL-12 and IL-18 cDNAs were coexpressed, an important attenuation in IL-12-mediated toxicity was observed as demonstrated by an increase in survival of susceptible young C56BL/6 mice (6- to 7-wk-old) and diminution of the liver enzyme levels ALT and AST. These results were quite surprising since a synergistic effect in toxicity has been previously described when both cytokines were coadministrated as recombinant proteins (29, 30). We believe that the differences observed between both models are based on the origin (exogenous administration versus endogenous induction) of the cytokines and also to the kinetics and amounts of the cytokines induced (IFN-γ, TNF-α, IL-10, etc.) in each case. In this context, the report by Nakamura et al. demonstrates that after IL-12 plus IL-18 recombinant protein administration, mice produce much higher levels of IFN-γ but similar levels of IL-10 than mice injected with rIL-12 alone (30). This later results reveal a clear difference with the kinetics and amounts of those cytokines observed in our model. Moreover, Carson et al. reports that after IL-12 plus IL-18 recombinant protein administration, IFN-γ kinetics are similar but levels are 7-fold higher than in rIL-12 injected mice. In their model, IL-10 peaks much later (around 96 h postinjection) than in our system and they report higher presence of TNF-α in rIL-12 plus rIL-18 than in rIL-12 injected mice (29). The later data are just the opposite of what we observed in our model. In the light of the data presented above, it becomes clear that the source, kinetics, and levels of cytokines administered and induced, are important factors that could lead to totally different survival outcomes. Our results strongly indicate that the expression of IL-18 could account for the attenuation of IL-12 toxicity. IL-18 is a functionally pleiotropic cytokine that is involved in either the Th1 or the Th2 immune response as previously reported (33, 34, 43). For instance, a single injection of IL-18 induces a typical Th1 profile with elevated levels of IFN-γ while multiple IL-18 doses are able to induce high levels of Th2 cytokines, including IL-10 and IL-13 (44).

Because IFN-γ has been extensively postulated to be responsible for the toxic side effects observed after IL-12 and/or IL-18 administration (29, 30, 45), we first analyzed the kinetics of IFN-γ expression in young IL-12 and IL-12 plus IL-18 cDNA-treated mice. Our data show that when IL-12 cDNA is coinjected with IL-18 cDNA, a more rapid, albeit 2-fold lower production of IFN-γ is detected compared with the levels observed when IL-12 cDNA is injected alone, even when IL-12 serum levels are higher in 12 plus 18-treated mice than in IL-12-treated mice. Interestingly, these 2-fold lower IFN-γ levels did not alter the potent antitumor activity observed in response to IL-12 treatment.

To understand the role of IFN-γ in terms of toxicity in our experimental model, we performed a comparative experiment between two genetically different strains of mice (BALB/c, Th2-associated and B6, Th1 associated). The data suggest that the greater survival observed in young BALB/c mice was associated with a significantly lower production of IFN-γ after IL-12 or IL-12 plus IL-18 cDNA administration compared with young B6 mice. Therefore, this observation suggested that the higher levels of IFN-γ induced after IL-12 cDNA treatment in B6 mice may contribute to the greater degree of toxicity. However, when IFN-γ−/− mice (B6 background) were injected with IL-12 or IL-12 plus IL-18 cDNAs, most of the mice succumbed while all control (10- to 12-wk-old) mice survived. Consistent with these findings, it has been reported that IFN-γ-deficient mice treated with 12 plus 18 experience a severe pulmonary edema that is not seen in WT mice (30). Moreover, Car et al. reported that endogenous IFN-γ induced after IL-12 and IL-18 treatment might play a role in preventing fatal pulmonary disease as is observed in IFN-γR−/− mice (45). Based on these reports, it can be concluded that the higher levels of IFN-γ induced after IL-12 cDNA treatment may account for the greater degree of toxicity observed but, at the same time, the total absence of IFN-γ abrogates a compensatory beneficial effect that might be operating post IL-12 or IL-12 plus IL-18 treatments.

Next, we investigated which cell types were producing IFN-γ and thus possibly contributing to the IL-12 toxicity. Different groups have postulated that NK and T cells are partially responsible for IL-12-induced toxicity (31, 45–47). We observed that both populations produced IFN-γ after IL-12 or IL-12 plus IL-18 cDNA treatment. Interestingly, NK/NKT cells and both T cell types, CD4, and CD8 T cells, appear to participate in systemic toxicity at different time points after IL-12 cDNA administration. Although NK/NKT cell depletion induced protection at early time points, depletion of CD4/CD8 seems to protect from IL-12 toxicity at later time points. This was confirmed when 100% survival was observed up to day 27 post-treatment as an additive effect after simultaneous depletion of NK1.1 and T cells. These results are consistent with the fact that NK1.1 cells, through constitutive expression of IL-12R (32), act as an early source of IFN-γ while T cells needs to be first activated to express IL12R and then produce IFN-γ (48).

Based on the results presented in this study, we hypothesized that when IL-18 cDNA is coexpressed, a differential inflammatory and anti-inflammatory cytokine balance is established that may account for the overall protective role observed. In support of this model, we observed that IL-18 serum levels in 12 plus 18 cDNA injected mice persist for several days post injection and, as reported by Kinoshita et al., this could be triggering high levels of IL-10 and IL-13 and possible deviating the balance to a more Th2 environment (44). We found the presence of IL-10 but not IL-13 both in serum and supernatants of splenocytes from young B6 mice injected with IL-12 or IL-12 plus IL-18. Consistent with reports from other laboratories, the attenuation of IL-12 toxic effects in the presence of IL-18 could be at least partially due to the presence of IL-10 since the later cytokine can function as an important feedback regulator controlling the pathology associated with the exacerbated inflammatory responses mediated by IL-12 (35, 36). Because IL-10 was detected in both IL-12 and IL-12 plus IL-18-treated mice, we tested the hypothesis that IL-10 may have a different protective role in mitigating IL-12 mediated toxicity in both groups. After comparing the time of appearance and serum levels of IL-10 in young B6 IL-12 and IL-12 plus IL-18 cDNA treated mice we found that when elevated IL-12 plus IL-18 levels are systemically induced after cDNA expression, IL-10 was rapidly produced at 2-fold higher levels than in mice treated with IL-12 cDNA alone. Two different explanations for this effect can be build on what is already published for rIL-12 and/or rIL-18 administration (30, 49). First, the levels of IL-10 induced after IL-12 treatment are not linear as doubling the concentration of IL-12 injected, resulted in a 4-fold increase in IL-10 serum levels (49). Consistently, we observed higher serum levels of IL-12 in 12 plus 18-treated mice than when IL-12 cDNA is injected alone. Second, it has been described an additive effect in IL-10 expression when both rIL-12 and rIL-18 are coinjected (30). Overall, the rapid IL-10 expression in 12 plus 18 mice seems to be critical in controlling the inflammatory response triggered by IL-12 since in the IL-12 treated group, higher IL-10 levels are seen by day 8 post treatment when most mice already appear ill. In support of this possibility, we observed a more dramatic mortality in young IL-10 KO mice treated with IL-12 plus IL-18 cDNAs than when treated with IL-12 cDNA alone compared with young B6 mice. Moreover, when IL-10R is blocked at the time of cDNA administration, all IL-12 plus IL-18 cDNA treated mice succumbed to the treatment. Finally, to better understand how this early IL-10 expression in 12 plus 18 mice could be attenuating IL-12 toxic effects, we coadministered IL-12 and IL-10 cDNAs. We confirmed that under these conditions, young B6 animals were more protected from IL-12 toxicity. Furthermore, protection from IL-12 toxicity correlated with lower serum levels of IFN-γ and TNF-α, two cytokines strongly associated with a fatal outcome in septic shock (37, 38).

An interesting observation in this model is that mortality cannot be avoided after day 30 post IL-12 expression even when NK/NKT and T cells are depleted. In relation with this finding, we speculate that other cell types could also contribute to IL-12 toxicity. It has been reported that systemic toxicity induced after IL-12 plus IL-18 could be abolished when the macrophage (Mϕ) population is depleted (29). Moreover, we observed that after IL-12 and IL-12 plus IL-18 treatments, the Mϕ population significantly increases and expresses activation markers (data not shown). Since NO is a cytotoxic Mϕ product and has been implicated in the cellular toxicity during sepsis (50), we first evaluated the presence of this mediator in our model. Our experiments demonstrated that even though Mϕs expressed iNOS and produced NO after IL-12 and IL-12 plus IL-18 treatments, no differences in survival were observed between control B6 and iNOS KO mice (data not shown) after cDNA injection. We are currently performing experiments to evaluate if other Mϕ products could be involved in the toxicity observed in this model.

In light of the data presented in this study, it is clear that IL-10 plays a relevant role in preventing the fatal outcome after IL-12 treatment. Based on the published data, there are many cellular sources of IL-10 (36). In this study, we focused our attention in the situation when IL-10 is produced in the presence of IL-12. In this context, it has been previously reported that IL-12 is able to induce IL-10 as a negative feedback mechanism to protect the host from an exacerbated and uncontrolled inflammatory immune response (35). Moreover, in several inflammatory processes with a strong Th1 component (e.g., Toxoplasma gondii or Trypanosoma cruzi infections), high levels of IL-10 are produced to limit the inflammatory pathology observed in those mice (51). Interestingly, Gerosa et al. have reported that IL-12 is able to prime CD4+ and CD8+ T cell clones for high and simultaneous production of both IFN-γ and IL-10 (52). In the same work, the authors demonstrated that neutralizing anti-IFN-γ Abs in these cultures did not prevent IL-12-induced priming for IFN-γ but significantly decreased IL-10 production (52). Based on these reports, we hypothesize that in our model, Th0/Th1 cells may be stimulated by the high levels of IL-12 that in turn are able to trigger both IFN-γ and IL-10. Moreover, we think that IFN-γ and IL-10 are produced from the same cell. We speculate that IL-10 production in our system may be conditioned by IL-12-induced IFN-γ expression (as reported by Gerosa et al.), and that when IL-18 is coexpressed with IL-12, an earlier IFN-γ response is observed than when IL-12 is expressed alone. This early IFN-γ response in 12 plus 18 cDNA-treated mice may thus induce an earlier IL-10 production that will control the inflammation and result in better treatment survival. This model is currently being tested.

Systemic IL-12 expression is currently used in several clinical trials and represents a potential treatment for metastasis growth control or when tumors are difficult to be surgically removed. Then, the use of systemic IL-12 alone, combined with other cytokines or as an adjuvant, still represents a powerful tool in the treatment of cancer. In this context, the importance of this work lies in the fact that, through the use of IL-18, we have demonstrated a role for IL-10 in modulating the toxicity of IL-12 treatment without attenuating the antitumor activity of IL-12. Incorporating these data may support the design of new technologies for utilizing IL-12 as therapeutic tool in the treatment of a variety of diseases.

Acknowledgments

We thank Mike Sanford for performing ELISA and RNase protection assay analysis and John Wine and Tim Back for their support in animal care and experimentation. The anti-NK1.1 neutralizing Ab was provided by Dr. John Ortaldo and the anti-CD4 and anti-CD8 Abs were a generous gift from Jeff Subleski. CRG, SGC, and PI belong to the research staff of CONICET.

Footnotes

1

This project has been funded with federal funds from the National Cancer Institute, National Institute of Health under Contract No. N01-CO-12400.

3

Abbreviations used in this paper: KO, knockout; Mϕ, macrophage.

Disclosures

The authors have no financial conflict of interest.

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