Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas ligand- and perforin-induced tumor apoptosis, respectively.

Systemic administration of rIL-18 protein to mice significantly suppresses the growth of murine tumor cell lines. The antitumor effect of IL-18 appears to be primarily mediated by asialo GM1+ cells. Since IL-18 enhances Fas ligand (FasL) expression on NK cell lines, the IL-18 antitumor effects could be mediated by FasL-induced cross-linking of Fas and subsequent tumor apoptosis. To address this question, rIL-18 or rIL-12 was administered to animals bearing the CL8-1 melanoma inoculated intradermally into wild type (wt), lymphoproliferation gene (lpr) (Fas deficient), or generalized lymphoproliferative disease gene (gld) (FasL deficient) mice. Although rIL-12 treatment retained significant antitumor effects in gld and lpr mice, those of rIL-18 administration were completely abrogated in gld but not lpr or wt mice. In vitro cytotoxicity was significantly enhanced against NK-sensitive YAC-1 cells and CL8-1 cells by rIL-18 administration to wt mice, but not to gld mice. Furthermore, rIL-18 administration augmented the cytotoxicity of liver lymphocytes harvested from perforin-deficient mice, whereas rIL-12 administration did not. Consistent with the role of this pathway, rIL-18 administration also up-regulates the expression of FasL mRNA in splenocytes. Lysis of CL8-1 cells induced by anti-Fas agonistic Ab was enhanced about 1.4-fold by IFN-gamma, a cytokine that is induced by IL-18 in vitro and in vivo. We conclude that the antitumor effect of IL-18 is exerted predominantly through a Fas-dependent pathway. The perforin pathway, however, appears to be the predominant cytolytic pathway mediating IL-12 antitumor effects.     

Synergistic anti-tumor effect of glycosylphosphatidylinositol-anchored IL-2 and IL-12

BACKGROUND: Preclinical and clinical studies have demonstrated that interleukin 2 (IL-2), interleukin 12 (IL-12), and some other cytokines, play important roles in activating host immune responses against tumor growth. However, severe side effects caused by systemic high-dose administration of these cytokines limit their clinical application. In our previous study, local high doses of IL-2 were achieved by a GPI-anchoring technology; therefore, it will be interesting to know if this technology works for other cytokines. METHODS: A fusion gene containing murine IL-12 and the glycosylphosphatidylinositol (GPI) anchor signal sequence was generated and transfected into the murine melanoma tumor cell line B16F0 either alone or together with a vector encoding GPI-anchored IL-2. The GPI-anchored cytokine expression of the selected stable clones was assayed in vitro by ELISA and their anti-tumor effects were analyzed in vivo by tumor lymphocyte infiltration and tumor growth studies. RESULTS: GPI-anchored IL-12 was successfully expressed on the cell surface as indicated by FACS analysis and IL-12 ELISA assay. The GPI-anchored IL-12 enhanced lymphocyte infiltration and significantly inhibited tumor growth. More importantly, when GPI-anchored IL-12 and GPI-anchored IL-2 were co-delivered, a synergistic anti-tumor effect was observed in both subcutaneous and intravenous tumor models. CONCLUSIONS: GPI anchorage of cytokines represents a new approach to locally deliver high doses of cytokines without the severe adverse effects normally accompanied with systematic high-dose administration of these cytokines.     

A recombinant single-chain antibody interleukin-2 fusion protein

Recombinant interleukin-2 (rIL-2) therapy has been shown to be of value in the treatment of some cases of melanoma and renal cell carcinoma. However, its use can be limited by severe systemic toxicity. Targeting rIL-2 to the tumor should improve the antitumor immune response and decrease the systemic toxicity. With this aim, we have employed recombinant DNA techniques to construct a single-chain antibody interleukin-2 fusion protein (SCA-IL-2). The protein used in this model system consists of the variable domains of the antilysozyme antibody D1.3 fused to human IL-2 and is expressed inE. coli. It retains antigen-binding specificity and has the full biological activity of rIL-2. This approach can be taken to generate SCA-IL-2 proteins that bind to appropriate cellular antigens. In vivo administration of tumor-binding SCA-IL-2 should result in a localized high concentration of rIL-2 in the tumor tissues, maximizing the antitumor response while keeping systemic side effects to a minimum.     

IL-23 and IL-12 have overlapping,but distinct,effects on murine dendritic cells

IL-23 is a recently discovered heterodimeric cytokine that shares biological properties with proinflammatory cytokines. The biologically active heterodimer consists of p19 and the p40 subunit of IL-12. IL-23 has been shown to possess biological activities on T cells that are similar as well distinct from those of IL-12. We have constructed single-chain IL-23 and IL-12 fusion proteins (IL-23-Ig and IL-12-Ig) and have compared the two recombinant proteins for effects on murine dendritic cells (DC). Here we show that the IL-23-Ig can bind a significant proportion of splenic DC of both the CD8alpha(-) and CD8alpha(+) subtypes. Furthermore, IL-23and IL-12-Ig exert biological activities on DC that are only in part overlapping. While both proteins induce IL-12 production from DC, only IL-23-Ig can act directly on CD8alpha(+) DC to promote immunogenic presentation of an otherwise tolerogenic tumor peptide. In addition, the in vitro effects of IL-23-Ig did not appear to require IL-12Rbeta2 or to be mediated by the production of IL-12. These data may establish IL-23 as a novel cytokine with major effects on APC.     

Antiangiogenic and antitumor activities of IL-27

IL-27 is a novel IL-6/IL-12 family cytokine playing an important role in the early regulation of Th1 responses. We have recently demonstrated that IL-27 has potent antitumor activity, which is mainly mediated through CD8(+) T cells, against highly immunogenic murine colon carcinoma. In this study, we further evaluated the antitumor and antiangiogenic activities of IL-27, using poorly immunogenic murine melanoma B16F10 tumors, which were engineered to overexpress single-chain IL-27 (B16F10 + IL-27). B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis. Similar antitumor and antimetastatic activities of IL-27 were also observed in IFN-gamma knockout mice. In NOD-SCID mice, these activities were decreased, but were still fairly well-retained, suggesting that different mechanisms other than the immune response are also involved in the exertion of these activities. Immunohistochemical analyses with Abs against vascular endothelial growth factor and CD31 revealed that B16F10 + IL-27 cells markedly suppressed tumor-induced neovascularization in lung metastases. Moreover, B16F10 + IL-27 cells clearly inhibited angiogenesis by dorsal air sac method, and IL-27 exhibited dose-dependent inhibition of angiogenesis on chick embryo chorioallantoic membrane. IL-27 was revealed to directly act on HUVECs and induce production of the antiangiogenic chemokines, IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma. Finally, augmented mRNA expression of IP-10 and monokine induced by IFN-gamma was detected at the s.c. B16F10 + IL-27 tumor site, and antitumor activity of IL-27 was partially inhibited by the administration of anti-IP-10. These results suggest that IL-27 possesses potent antiangiogenic activity, which plays an important role in its antitumor and antimetastatic activities.     

An anti-IL-12p40 antibody down-regulates type 1 cytokines, chemokines, and IL-12/IL-23 in psoriasis

Psoriasis is characterized by activation of T cells with a type 1 cytokine profile. IL-12 and IL-23 produced by APCs are essential for inducing Th1 effector cells. Promising clinical results of administration of an Ab specific for the p40 subunit of IL-12 and IL-23 (anti-IL-12p40) have been reported recently. This study evaluated histological changes and mRNA expression of relevant cytokines and chemokines in psoriatic skin lesions following a single administration of anti-IL-12p40, using immunohistochemistry and real-time RT-PCR. Expression levels of type 1 cytokine (IFN-gamma) and chemokines (IL-8, IFN-gamma-inducible protein-10, and MCP-1) were significantly reduced at 2 wk posttreatment. The rapid decrease of these expression levels preceded clinical response and histologic changes. Interestingly, the level of an anti-inflammatory cytokine, IL-10, was also significantly reduced. Significant reductions in TNF-alpha levels and infiltrating T cells were observed in high responders (improvement in clinical score, > or =75% at 16 wk), but not in low responders. Of importance, the levels of APC cytokines, IL-12p40 and IL-23p19, were significantly decreased in both responder populations, with larger decreases in high responders. In addition, baseline levels of TNF-alpha significantly correlated with the clinical improvement at 16 wk, suggesting that these levels may predict therapeutic responsiveness to anti-IL-12p40. Thus, in a human Th1-mediated disease, blockade of APC cytokines by anti-IL-12p40 down-regulates expression of type 1 cytokines and chemokines that are downstream of IL-12/IL-23, and also IL-12/IL-23 themselves, with a pattern indicative of coordinated deactivation of APCs and Th1 cells.     

Autocrine regulation of IL-12 receptor expression is independent of secondary IFN-gamma secretion and not restricted to T and NK cells.

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rbeta1 and IL-12Rbeta2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rbeta2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rbeta2 expression can be prevented by treatment with IFN-gamma, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rbeta1 and beta2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-gamma secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-gamma secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-gamma production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-gamma secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.     

Nasal IL-12p70 DNA prevents and treats intestinal allergic diarrhea

OVA-induced allergic diarrhea occurs as a consequence of over-expression of Th1 inhibitory IL-12p40 monomers and homodimers in the large intestine, establishing a dominant Th2-type environment. In this study, we demonstrate that intranasally administered murine IL-12p70 naked DNA expression plasmids resulted in the synthesis of corresponding cytokine in the large intestinal CD11c(+) dendritic cells, leading to the inhibition of Ag-specific Th2-type response for the prevention of allergic diarrhea and the suppression of clinical symptoms including OVA-specific IgE Ab synthesis. The nasal IL-12p70 DNA treatment proved effective even after the establishment of allergic diarrhea. These results suggest that the mucosal administration of naked IL-12p70 DNA plasmid should be considered as a possible preventive and therapeutic treatment for Th2 cell-mediated food allergic diseases in the intestinal tract.     

Adjuvanticity of an IL-12 fusion protein expressed by recombinant deltaG-vesicular stomatitis virus

The remarkable immunomodulatory and adjuvant properties of rIL-12 have been well described. Many early studies documenting the adjuvanticity of IL-12 were performed using the murine model of Listeria monocytogenes infection. In this report, we describe the construction of an attenuated recombinant vesicular stomatitis virus (VSV-deltaG) that encodes a single-chain IL-12 fusion protein (IL-12F), and the use of this virus as an expression vector to produce large quantities of IL-12F. VSV-expressed IL-12F (vIL-12F) was then co-administered to mice along with a poorly immunogenic listerial antigen preparation as a vaccine regimen and the resulting immune responses were monitored. The vIL-12F was found to have adjuvant properties similar to those observed for rIL-12. Co-administration of vIL-12F and listerial antigen elicited powerful cell-mediated immune responses that conferred long-lived protective listerial immunity. These studies demonstrated that VSVdeltaG-IL12F-infected cells secrete bioactive single-chain IL-12, and laid the foundation for studies using VSVdeltaG-IL12F as a vector for delivery of IL-12F in vivo.     

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.     

Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis

Cyclooxygenase-2 (COX-2), the enzyme at the rate-limiting step of prostanoid production, has been found to be overexpressed in human lung cancer. To evaluate lung tumor COX-2 modulation of antitumor immunity, we studied the antitumor effect of specific genetic or pharmacological inhibition of COX-2 in a murine Lewis lung carcinoma (3LL) model. Inhibition of COX-2 led to marked lymphocytic infiltration of the tumor and reduced tumor growth. Treatment of mice with anti-PGE2 mAb replicated the growth reduction seen in tumor-bearing mice treated with COX-2 inhibitors. COX-2 inhibition was accompanied by a significant decrement in IL-10 and a concomitant restoration of IL-12 production by APCs. Because the COX-2 metabolite PGE2 is a potent inducer of IL-10, it was hypothesized that COX-2 inhibition led to antitumor responses by down-regulating production of this potent immunosuppressive cytokine. In support of this concept, transfer of IL-10 transgenic T lymphocytes that overexpress IL-10 under control of the IL-2 promoter reversed the COX-2 inhibitor-induced antitumor response. We conclude that abrogation of COX-2 expression promotes antitumor reactivity by restoring the balance of IL-10 and IL-12 in vivo.     

Targeting IFN-alpha to B cell lymphoma by a tumor-specific antibody elicits potent antitumor activities

IFN-alpha, a cytokine crucial for the innate immune response, also demonstrates antitumor activity. However, use of IFN-alpha as an anticancer drug is hampered by its short half-life and toxicity. One approach to improving IFN-alpha's therapeutic index is to increase its half-life and tumor localization by fusing it to a tumor-specific Ab. In the present study, we constructed a fusion protein consisting of anti-HER2/neu-IgG3 and IFN-alpha (anti-HER2/neu-IgG3-IFN-alpha) and investigated its effect on a murine B cell lymphoma, 38C13, expressing human HER2/neu. Anti-HER2/neu-IgG3-IFN-alpha exhibited potent inhibition of 38C13/HER2 tumor growth in vivo. Administration of three daily 1-microg doses of anti-HER2/neu-IgG3-IFN-alpha beginning 1 day after tumor challenge resulted in 88% of the mice remaining tumor free. Remarkably, anti-HER2/neu-IgG3-IFN-alpha demonstrated potent activity against established 38C13/HER2 tumors, with complete tumor remission observed in 38% of the mice treated with three daily doses of 5 microg of the fusion protein (p = 0.0001). Ab-mediated targeting of IFN-alpha induced growth arrest and apoptosis of lymphoma cells contributing to the antitumor effect. The fusion protein also had a longer in vivo half-life than rIFN-alpha. These results suggest that IFN-alpha Ab fusion proteins may be effective in the treatment of B cell lymphoma.     

Critical role of Kupffer cell-derived IL-10 for host defense in septic peritonitis

Intra-abdominal infection in patients following major visceral surgery is associated with high mortality. Using a macrophage depletion technique, we demonstrate that in murine septic peritonitis, Kupffer cells are a major source of systemic IL-10 levels. Kupffer cell-depleted mice were highly susceptible to the lethal effects of septic peritonitis and exhibited an increased bacterial load. Kupffer cell-depleted mice were protected by the administration of an IL-10-Fc fusion protein. Loss of Kupffer cell-derived IL-10 was associated with a weak increase in serum IL-12 levels, whereas TNF, IL-1alpha, and IL-18 levels were not significantly elevated, suggesting that the loss of Kupffer cell-derived IL-10 did not result in a toxic cytokine release syndrome. Instead, loss of Kupffer cell-derived IL-10 was associated with a reduced splenocyte production of IFN-gamma that is required for immune protection in murine septic peritonitis. Therefore, the results suggest that the protective function of IL-10 in septic peritonitis may not be restricted to the anti-inflammatory activities of IL-10.     

Antitumor and antimetastatic activity of IL-23

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 +/- 333 mm(3), then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8(+) T cells, but not CD4(+) T cells or NK cells, were crucial for the antitumor activity of IL-23.     

Regulation of Staphylococcus epidermidis-induced IFN-gamma in whole human blood: the role of endogenous IL-18, IL-12, IL-1, and TNF

Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-gamma) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-gamma synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-gamma and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-gamma and IL-1beta levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-gamma (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-gamma production. Blocking endogenous IL-12 and TNF reduced IFN-gamma production by 69 and 36%. S. epidermidis-induced TNF-alpha was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-gamma which is IL-18, IL-12 and TNF-dependent, but IL-1 independent.     

IL-12 and IL-10 expression synergize to induce the immune-mediated eradication of established colon and mammary tumors and lung metastasis

Preclinical studies demonstrated that certain cytokines are potentially useful for the induction of antitumor immune responses. However, their administration in clinical settings was only marginally useful and evoked serious toxicity. In this study, we demonstrate that the combination of autologous inactivated tumor cells expressing IL-12 and IL-10 induced tumor remission in 50-70% of mice harboring large established colon or mammary tumors and spontaneous lung metastases, with the consequent establishment of an antitumor immune memory. Mice treatment with tumor cells expressing IL-12 was only marginally effective, while expression of IL-10 was not effective at all. Administration of the combined immunotherapy stimulated the recruitment of a strong inflammatory infiltrate that correlated with local, increased expression levels of the chemokines MIP-2, MCP-1, IFN-gamma-inducible protein-10, and TCA-3 and the overexpression of IFN-gamma, but not IL-4. The combined immunotherapy was also therapeutically effective on established lung metastases from both colon and mammary tumors. The antitumor effect of the combined immunotherapy was mainly dependent on CD8+ cells although CD4+ T cells also played a role. The production of IFN-gamma and IL-4 by spleen cells and the development of tumor-specific IgG1 and IgG2a Abs indicate that each cytokine stimulated its own Th pathway and that both arms were actively engaged in the antitumor effect. This study provides the first evidence of a synergistic antitumor effect of IL-12 and IL-10 suggesting that a Th1 and a Th2 cytokine can be effectively combined as a novel rational approach for cancer immunotherapy.     

Expression of IFN-gamma upon triggering of activating Ly49D NK receptors in vitro and in vivo: costimulation with IL-12 or IL-18 overrides inhibitory receptors

NK cells can express both activating and inhibitory Ly49 receptors on their cell surface. When cells expressing both receptors are presented with a ligand, inhibition dominates the functional outcome. In this report we demonstrate that costimulation of the activating Ly49D murine NK cell receptor with IL-12 or IL-18 is capable of over-riding the inhibitory Ly49G2 receptor blockade for cytokine production both in vitro and in vivo. This synergy is mediated by and dependent upon Ly49D-expressing NK cells and results in significant systemic expression of IFN-gamma. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial, antiviral, and antitumor immunity and provide a mechanism for the release of activating Ly49 receptors from inhibitory receptor blockade.     

Systemic administration of IL-23 induces potent antitumor immunity primarily mediated through Th1-type response in association with the endogenously expressed IL-12

IL-23, a cytokine, which is composed of the p40 subunit shared with IL-12 and the IL-23-specific p19 subunit, has been shown to preferentially act on Th1 effector/memory CD4+ T cells and to induce their proliferation and IFN-gamma production. The IL-23 is also reported to act on Th17-CD4+ T cells, which are involved in inducing tissue injury. In this study, we examined the antitumor effects associated with systemic administration of IL-23 and their mechanisms in mouse tumor system. Systemic administration of high-dose IL-23 was achieved using in vivo electroporation of IL-23 plasmid DNA into the pretibial muscles of C57BL/6 mice. The IL-23 treatment was associated with significant suppression of the growth of pre-existing MCA205 fibrosarcoma and prolongation of the survival of treated mice without significant toxicity when compared with those of the mice treated with EGFP. Although the therapeutic outcomes were similar to those with the IL-12 treatment, the IL-23 treatment induced characteristic immune responses distinctive to those of IL-12 treatment. The IL-23 administration even at the therapeutic levels did not induce detectable IFN-gamma concentration in the serum. In vivo depletion of CD4+ T cells, CD8+ T cells, or NK cells significantly inhibited the antitumor effects of IL-23. Furthermore, the CD4+ T cells in the lymph nodes in the IL-23-treated mice showed significant IFN-gamma and IL-17 response upon anti-CD3 mAb stimulation in vitro. These results and the ones in the IFN-gamma or IL-12 gene knockout mice suggest that potent antitumor effects of IL-23 treatment could be achieved when the Th1-type response is fully promoted in the presence of endogenously expressed IL-12.     

Effect of IL-12 encoding plasmid administration on tight-skin mouse

The tight-skin (Tsk/+) mutant mice, a putative murine model of scleroderma, are characterized by the excessive deposition of collagen and the presence of antinuclear antibodies. Type 2 cytokines, such as IL-4 and IL-6, are capable of regulating the synthesis of various matrix molecules, including type I collagen, by fibroblasts. IL-12 is well known to induce type 1 cytokine production and to reduce type 2 activity. Here, we examined the effect of IL-12 encoding plasmid (pCAGGSIL-12) on the disease progression of Tsk/+ mice. pCAGGSIL-12 plasmid or pCAGGS parental vector was injected intramuscularly 7 times at 3 week intervals into Tsk/+ mice. One week after the last injection, pCAGGSIL-12 administered Tsk/+ mice exhibited a marked decrease in the skin thickness compared with the mice treated with pCAGGS vector. The serum levels of antinuclear antibodies were diminished in pCAGGSIL-12 treated mice. IL-4 production by spleen cells from pCAGGSIL-12 plasmid treated mice was significantly lower than that from vector treated mice. These results indicate that pCAGGSIL-12 administration into Tsk/+ mice had beneficial effects in preventing the collagen accumulation in the skin and suppressing the autoimmunity via improvement of Th1/Th2 balance. The present study suggests that the IL-12 encoding plasmid administration might have a therapeutic effect on systemic sclerosis.