Within the vast array of bioactive cytokines, the interleukin-12 (IL-12) family is unique. It is the only heterodimeric cytokine family and this endows them with several unique and distinctive features. It also presents a number of molecular and functional realities that provide unprecedented opportunities for positive and negative feedback control, some of which may remain unrealized. Chain pairing promiscuity is a common feature of this heterodimeric cytokine family which currently includes IL-12, IL-23, IL-27 and IL-35. Despite many structural similarities in the cytokines, their receptors and downstream signaling components, they possess vastly contrasting biological activities which belie their common features. Each of the IL-12 family cytokines consists of two subunits: an alpha chain (p19, p28, or p35) with a four alpha-helix bundle structure and a beta chain (p40 or EBI3) that is homologous to the soluble class I cytokine receptor chains.
Members of IL-12 family
Table 1. IL-12 family related products
|IL-12 Family Ligands||IL12||IL12A / IL35||IL12B|
|IL-12 Family Receptors||IL12RB1||IL12RB2||IL23R|
Interleukin 12 (IL-12) is an interleukin that is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) in response to antigenic stimulation. IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (referred to as 'p70'), and a homodimer of p40 are formed following protein synthesis. A positive feedback loop is established where IL-12 induces IFNγ production by T cells which primes additional APCs for IL-12 production and facilitates Th1 differentiation. IL-12 can also induce production of IFNγ by NK cells.
Figure 1. IL-12 protein
Interleukin-23 (IL-23) is a heterodimeric cytokine composed of an IL12B (IL-12p40) subunit (that is shared with IL12) and the IL23A (IL-23p19) subunit. A functional receptor for IL-23 (the IL-23 receptor) has been identified and is composed of IL-12R β1 and IL-23R. IL-23 was first described by Robert Kastelein and colleagues at the DNAX research institute using a combination of computational, biochemical and cellular immunology approach. Seminal studies in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis, showed that IL-23 was responsible for the inflammation observed, not IL-12 as previously thought.
Interleukin 27 (IL-27) is a member of the IL-12 cytokine family. It is a heterodimeric cytokine that is composed of two distinct genes, Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28. IL-27 is expressed by antigen presenting cells and interacts with a specific cell-surface receptor complex known as IL-27 receptor (IL-27R). This receptor consists of two proteins, IL-27ɑ and gp130. IL-27 has been reported to have both pro-stimulatory and inhibitory functions, although accumulating data supports a role for IL-27 in inhibiting immune responses. Activation of APCs with TLR agonists (TLR3, 4 and 7/8) induces IL-27 and IFNβ synergies with TLR mediated IL-27 expression. It is often generated during the resolution phase of an autoimmune response by local APCs. Furthermore, diverse stimuli including Tregs, IFNγ and statins can induce IL-27 production by APCs and thereby limit induction of inflammation. Although IL-27 alone has no apparent stimulatory properties, it can induce IFNγ production by T cells and NK cells in combination with IL-12 or IL-2.
Interleukin 35 (IL-35) is an IL-12 family cytokine produced by regulatory, but not effector, T-cells and plays a role in immune suppression. It is a dimeric protein composed of IL-12α and IL-27β chains, which are encoded by two separate genes called IL12A and EBI3, respectively. Secreted by regulatory T-cells (Tregs), IL-35 suppresses inflammatory responses of immune cells. IL-35 is not constitutively expressed in tissues, but the gene encoding IL-35 is transcribed by vascular endothelial cells, smooth muscle cells and monocytes after activation with proinflammatory stimuli. Genetic deletion or antibody-mediated blockade IL-35 blocks nTreg-mediated suppression via soluble factors. IL-35 suppresses T cell proliferation by inducing cell cycle arrest in G1 without inducing apoptosis. Like TGFβ and IL-10, IL-35 can induce the development of an induced regulatory T cell (iTreg) population, iTr35, that suppress via IL-35 but does express Foxp3, IL-10 or TGFβ.
IL-12 is involved in the differentiation of naive T cells into Th1 cells. It is known as a T cell-stimulating factor, which can stimulate the growth and function of T cells. It stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells, and reduces IL-4 mediated suppression of IFN-γ. T cells that produce IL-12 have a coreceptor, CD30, which is associated with IL-12 activity. It plays an important role in the activities of natural killer cells and T lymphocytes. IL-12 mediates enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. There also seems to be a link between IL-2 and the signal transduction of IL-12 in NK cells. IL-2 stimulates the expression of two IL-12 receptors, IL-12R-β1 and IL-12R-β2, maintaining the expression of a critical protein involved in IL-12 signaling in NK cells. Enhanced functional response is demonstrated by IFN-γ production and killing of target cells.
IL-23, like IL-12, is a pro-inflammatory cytokine that is also produced by activated DCs and macrophages in response to microbial pathogens which is enhanced by CD40-CD40L interactions. It also induces expression of IL23R creating a positive feedback loop that enhances IL-23 expression. However, it differs from IL-12 in playing a key role in Th17 development by stabilizing IL-17 expression and the Th17 phenotype, but is not a differentiation factor for Th17 cells. In addition, IL-23 is critical for inducing a pathogenic phenotype in Th17 cells. TLR2 and Dectin-1 signaling by fungal products like zymosan induces IL-23 induction and promotes Th17 differentiation.
There are many different subsets of T cells, such as Th1, Th2, Th17, Tr1, and Treg cells; IL-27 is greatly involved in differentiation through inducing or suppressing of each T cell subset. Th1 cells, which express IFNγ, are promoted by IL-27 through STAT1 and T-bet activation. Th2 cells, which express IL-4, are inhibited by IL-27 through the transcription factor GATA-3. Th17 cells, which express IL-17, IL-22, and granulocyte macrophage colony-stimulating factor (GM-CSF), are inhibited by IL-27 through STAT1 and expression of transcription factor RORγt. Tr1 cells, which express IL-10, are induced by IL-27 through the transcription factor c-Maf. Treg cells are inhibited by IL-27 through STAT1 and STAT3.
Studies in mice show the absence of either IL-35 chain from regulatory Tregs reduces the cells' ability to suppress inflammation; this has been observed during cell culture experiments and using an experimental model for inflammatory bowel disease. To produce its suppressive effects (eg on collagen-induced arthritis), IL-35 has selective activities on different T-cell subsets; it induces proliferation of Treg cell populations but reduces activity of Th17 cell populations.
Figure 2. IL-12 family cytokines as an immunological nexus
Role in disease
In many respects, the IL-12 cytokine family epitomizes the central tenet of immunoregulation. IL-12 and IL-23 are pro-inflammatory/pro-stimulatory cytokines, while IL-27 and IL-35 are inhibitory cytokines. This raises the possibility that there is an unappreciated spectrum of immune balance that is established by, and within, the IL-12 cytokine family. This is impacted by multiple positive and negative feedback loops and interactions that modulate many aspects of immune function. In addition to modulating the behavior of multiple T cell populations, the IL-12 cytokine family has a substantial impact on the direction of immune responses and a wide variety of diseases by also controlling the development of many contrasting T cell populations.
Both endogenous IL-12 and treatment with exogenous IL-12 have been shown to exert profound antitumor and antimetastatic activity in murine models of transplantable and chemically induced tumors. These antitumor activities include innate and adaptive immune mechanisms. In all tumor studies, the antitumor effect of IL-12 was at least partially dependent on IFN-γ. Taken together, all members of the IL-12 family appear to have profound effects in tumor control. For the future, clinical trials will have to reveal how the members of the IL-12 family can be used for treatment of cancer patients without inducing major toxicities.
Psoriasis vulgaris is a T cell-driven disease with Th1 cells predominating in lesional skin. Keratinocytes with transgenic expression of p40 show production of IL-23, but not IL-12, in the skin leading to an inflammatory cutaneous response with elevated numbers of Langerhans’ cells. Moreover, in lesional skin of patients with psoriasis vulgaris, an increase of p40 and p19 but not of p35 mRNA compared with nonlesional skin was detected. This emphasizes a major role of IL-23 in cutaneous chronic inflammatory responses.
Inflammatory bowel disease (IBD) encompasses two disease entitities, Crohn’s disease and ulcerative colitis. Crohn’s disease most commonly affects the terminal ileum and ascending colon. An immunopathological function of IL-12 in IBD was assumed for Crohn’s disease, since patients with Crohn’s disease were found to express IL-12 in the gut. In addition, in mouse models of IBD, treatment with anti-IL-12p40 antibodies prevented or terminated disease. IL-12 might play a role in colitis independent of its ability to generate IFN-γ-producing T cells. With the discovery of IL- 23, the role of IL-12 in IBD needed to be readdressed, since anti-p40 antibodies used before neutralized both IL-12 and IL-23.
Taken together, these observations clearly show that the IL-12 family cytokines act as the immunological playmaker, shaping immune responses by directly inducing the development of T cell subpopulations and altering the function and fate of many cell populations that dictate disease outcome.