It has recently been demonstrated that in vivo administration of murine interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocyte-activated killer cell activity, enhancement of cytolytic T cell generation, and induction of interferon gamma secretion. In this study, the in vivo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growth of the B16F10 melanoma were markedly reduced in mice treated intraperitoneally with IL-12, resulting in an increase in survival time. The therapeutic effectiveness of IL-12 was dose dependent and treatment of subcutaneous tumors could be initiated up to 14 d after injection of tumor cells. Likewise, established experimental hepatic metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local peritumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell-deficient beige mice or in mice depleted of NK cell activity by treatment with antiasialo GM1, suggesting that NK cells are not the primary cell type mediating the antitumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8+ T cells in mediating the antitumor effects against subcutaneous tumors.

IL-12 is a cytokine that can exert regulatory effects on T and NK cells and promote Th1 responses. To delineate further the physiologic role of IL-12 in immunity, mice deficient for this cytokine were generated. IL-12-deficient mice were impaired but not completely lacking in the ability to produce IFN gamma following endotoxin administration and to mount a Th1 response in vivo, as measured by antigen-induced IFN gamma secretion by immune lymph node cells in vitro. In contrast, secretion of IL-4 was enhanced, while proliferation and secretion of IL-2 and IL-10 were normal following antigen stimulation. DTH responses were significantly reduced in IL-12-deficient mice, but no defect in allogeneic CTL responses was observed. These results indicate that IL-12 plays an essential role in regulating IFN gamma production and in facilitating normal DTH responses. However, other phenomena associated with Th1 responses and cell-mediated immunity, i.e., IL-2 secretion and CTL generation, were not compromised in the absence of IL-12.

T cell cytokines are known to play a major role in determining protection and pathology in infectious disease. It has recently become clear that IL-12 is a key inducer of the type 1 T cell cytokine pattern characterized by production of IFN-␥ . Conversely, IL-10 down-regulates IL-12 production and type 1 cytokine responses. We have investigated whether IL-12 and IL-10 might be involved in a chronic inflammatory reaction, atherosclerosis. In atherosclerotic plaques, we found strong expression of IFN-␥ but not IL-4 mRNAs as compared to normal arteries. IL-12 p40 mRNA and IL-12 p70 protein were also found to be abundant in atherosclerotic plaques. IL-12 was induced in monocytes in vitro in response to highly oxidized LDL but not minimally modified LDL. The cross-regulatory role of IL-10 was indicated by the expression of IL-10 in some atherosclerotic lesions, and the demonstration that exogenous rIL-10 inhibited LDL-induced IL-12 release. These data suggest that the balance between IL-12 and IL-10 production contributes to the level of immune-mediated tissue injury in atherosclerosis. (

IL-12 is a heterodimeric cytokine that has been shown to enhance natural killer (NK) and cytotoxic T lymphocyte (CTL) responses, and to induce IFN-gamma production in vitro. In this study, we have examined the effects in vivo of administering purified murine rIL-12 to normal mice. Daily injections of rIL-12 i.p. (1 ng to 10 micrograms/day) caused dose-dependent enhancement of NK cell lytic activity in the spleens and livers of treated mice. Histologic examination of the livers of IL-12-treated mice revealed focal mononuclear cell infiltrates, and flow cytometry studies indicated that the livers of IL-12-treated mice contained increased numbers of NK cells, CD8+ T cells, and monocytes. Liver and splenic lymphoid cells from IL-12-treated mice, unlike liver and splenic lymphoid cells from control mice, spontaneously secreted IFN-gamma in vitro, suggesting that they had been induced by IL-12 to produce IFN-gamma in vivo. Consistent with this, IFN-gamma could be detected in the serum of IL-12-treated mice. In mice which had been immunized by footpad injection of allogeneic splenocytes, daily administration of rIL-12 i.p. was shown to enhance the specific CTL response in the draining lymph nodes. Thus, these studies demonstrate that IL-12 can enhance NK and CTL activity and induce IFN-gamma production in vivo, as well as in vitro, and suggest possible mechanisms by which IL-12 may exert therapeutic effects in the treatment of some tumors and infectious diseases.

Collagen-induced arthritis (CIA) is an animal model for rheumatoid arthritis. The disease is elicited by immunization of genetically susceptible DBA/1 mice with type II collagen, resulting in a debilitating arthritis characterized by inflammation and involvement of multiple joints. We investigated the role of endogenous interleukin (IL)-12 in the pathogenesis of this disease by undertaking an analysis of IL-12-deficient mice on the DBA/1 genetic background after immunization with type II collagen. Both the incidence and severity of disease were significantly reduced in mice unable to produce biologically active IL-12. Concomitant decreases were observed in serum levels of pathogenic, collagen-specific IgG2a antibodies and collagen-induced secretion of interferon-gamma by immune splenocytes in vitro, consistent with an impaired T helper-1 response. There were, however, a few animals which developed severe disease in a single paw in spite of this highly diminished Th1 response. Taken together, these results demonstrate an important role for IL-12 in the pathogenesis of CIA, although it is not absolutely required for disease development.

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

Interleukin-12 (IL-12) has been described as a pivotal molecule in the immune response based in part on its ability to influence the differentiation of T helper (Th) cells into a type 1 (Th1) phenotype. This event is crucial in that appropriate differentiation of naive T cells can determine susceptibility or resistance to given pathogens by influencing the balance between cellular and humoral immunity. In order to further delineate the role of IL-12 in the immune response, we generated mice deficient for this cytokine. IL-12 knockout mice were viable, fully fertile, and displayed no obvious developmental abnormalities. Upon immunological analysis, these mice demonstrated an impaired ability to effect a Th1 response as well as an impaired ability to produce interferon-gamma in response to endotoxin in vivo. These data establish an essential role for IL-12 in the generation of optimal Th1 responses in vivo, but weak responses can occur independently of IL-12.

The interleukin-12 receptor (IL-12R)beta1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rbeta1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rbeta1 and IL-12 binding occurred on days 3-4. Anti-CD3-induced expression of IL-12Rbeta1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rbeta1 expression and IL-12 binding on resting PBMC, whereas IL-1alpha and tumor necrosis factor-alpha had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-beta2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rbeta1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-beta2, IL-10 and IL-4; however, TGF-beta2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-beta2 or IL-10 failed to produce IFN-gamma or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rbeta2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-beta2 and IL-10 than is expression of IL-12Rbeta1.