Interferons (IFNs) were first described in 1957 by Issacs and Lindenmann as substances that restrict viral replication (Isaacs and Lindernmann 1957). In mid-1960, Wheelock reported that human leukocytes stimulated with phytohemagglutinin expressed IFN-like inhibitors (Wheelock 1965). However, these IFN-like substances had less resistance to heat and acid than the interferons described previously. In the 1970s, these substances were further characterized based on the inducing properties and cell-type expression patterns, and first named Immune IFN, then later Type II IFN. This nomenclature was originally a subject of some debate as the Type II IFN was thought to be physicochemically and also biologically different from Type I IFNs (IFN-α, IFN-β, IFN-ω, and IFN-τ) (Billiau 2009). In 1980, a panel of experts acknowledged the differences between Type I and Type II IFNs, and gave the Type II IFN the name IFN-γ.
IFN-γ and IFN-γ Receptor Complex
The biological activity of IFN-γ is initiated upon binding of the dimer to its receptor. The IFN-γ receptor consists of two ligand-binding IFN-γR1 chains and two signal-transducing IFN-γR2 chains. These proteins are encoded by separate genes (IFNGR1 and IFNGR2, respectively) that are located on different chromosomes (Bach et al. 1997). Both chains are constitutively expressed on most cells. While the expression level of IFN-γR1 is usually strong, the expression level of IFN-γR2 is determined by the cell types, differentiation stages, and activation status of the cells. T cells have a lower IFN-γR2 expression level than B cells and monocytes. Compared to CD4+ Th2 cells, Th1 cells have significantly lower expression of IFN-γR2. IFN-γ first binds to IFN-γR1, and the IFN-γ: IFN-γR1 oligomerization prompts its association with IFN-γ R2 that then initiates the downstream signaling events (Schroder et al. 2004). The interaction between IFN-γ: IFN-γR1 and IFN-γR1: IFN-γR2 has been shown to be species specific, e.g., mouse IFN-γ does not interact with the human IFN-γ receptors.
The primary gene products of IFN-γ stimulation are transcription factors, e.g., interferon-regulated factors (IRFs), which will then initiate the secondary transcription of ISGs. Both IRF-1 and IRF-2 interact with the interferon-stimulated response element (ISRE, NGAAANNGAAAG/CN) of ISGs. While IRF-1 initiates the transcription of genes, such as NOS2, IL-12, and CIITA, IRF-2 suppresses gene expression. Interestingly, IRF-8 and IRF-9 cannot bind to an ISRE and they regulate ISG transcription by binding to other transcriptional factors, e.g., IRF-8 forms a dimer with IRF-1 and IRF-9 forms a trimer with STAT1 and STAT2 (Fig. 2) (Savitsky et al. 2010).
IFN-γ also can activate STAT1-independent pathways, although it still requires the activation of JAKs. The profile of proteins recruited to IFN-γR1 shows that IFN-γ stimulation triggers the MAP kinase pathway. JAKs activate IFN-γR1 associated Raf1 and Rap1 serine kinases, which then phosphorylate ERK kinase. p38 MAP kinase is the other target of the MAP kinase pathway that has been shown to be activated after IFN-γ treatment. PI3K pathway is another pathway stimulated by IFN-γ that activates protein kinase C (PKC) α, δ, and ε isotypes. PKCα directly magnifies the transcription of ISGs. In contrast, PKCε amplifies transcription by phosphorylating MAP kinase, while PKCδ does so by phosphorylating ser727 on STAT1. It also has been shown that IFN-γ stimulation activates NF-κB pathway by degrading the inhibitor of κB (IκB) and triggering the phosphorylation of the IKKα and β subunits (Gough et al. 2008).
As stated above, the JAK-STAT pathway is the primary signaling pathway initiated by IFN-γ stimulation. With the activation of JAKs, the signal is further amplified by initiating other signal transduction pathways and maximizing STAT1 activity. To prevent undesirable outcomes, such as uncontrolled expression of specific genes resulting in tissue damage and autoimmunity, IFN-γ signaling needs to be tightly regulated. This is accomplished by the existence of a negative feedback mechanism that results in the IFN-γ induced expression of the suppressor of cytokine signaling genes ( SOCS). SOCS proteins negatively regulate IFN-γ signaling by inhibiting JAK catalytic activity. The IFN-γ signaling pathway is then suppressed within hours of IFN-γ treatment (Croker et al. 2008).
Biological Functions of IFN-γ
An antiviral effect was the first observed biological function of IFN-γ and thus accounts for its original designation as an interferon. IFN-γ exercises its antiviral activity by modulating both innate and adaptive immune responses. During virus infection, IFN-γ triggers the expression of protein kinase dsRNA-regulated and dsRNA-specific adenosine deaminase, proteins that inhibit viral protein synthesis. In addition, IFN-γ augments the antiviral state of the cells by enhancing Type I IFN expression and the formation of ISGF3, the main protein complex involved in stimulating Type I IFN-induced gene expression. IFN-γ also plays a role in conveying antiviral signals from the innate to the adaptive immune response. Increased chemokine/chemokine receptors, induced by IFN-γ, recruit T cells to the infection site. Upon receiving the IFN-γ signal, APCs increase MHC class II: peptide complex and costimulatory molecule expression levels, hence facilitating peptide-specific CD4+ T cell activation and initiation of the adaptive immune response against viral infection. IFN-γ can also limit viral infection by upregulating MHC class I pathway allowing cells to present a higher quantity and more diverse peptide repertoire to CD8+ T cells (Schroder et al. 2004). In modulating adaptive immune responses, IFN-γ facilitates and maintains the commitment of CD4+ T cells to the Th1 lineage that is crucial in controlling viral infection. IFN-γ also induces the expression of IL-12 by APCs. IL-12 not only activates NK cells, a major antiviral component of innate immunity, but also drives Th1 development. Additionally, IFN-γ signaling facilitates Th1 development and its own expression by inducing T-bet expression and suppressing the expression of GATA3, a protein that drives T cell Th2 differentiation. To further solidify the commitment to the Th1 lineage, IFN-γ signaling also inhibits the Th2 essential IL-4-STAT6 signaling pathway (Hu et al. 2008).
Another important biological function of IFN-γ is macrophage activation, a critical component in controlling microbial infection by the host. In fact, the original “Macrophage Activating Factor” was later determined to be IFN-γ. Once stimulated with IFN-γ, macrophages are primed and more responsive to pro-inflammatory mediators such as a tumor necrosis factor (TNF) and toll-like receptor (TLR) ligands. IFN-γ priming amplifies TLR signaling in macrophages by both increasing the expression of TLRs and activating the transcription factor NF-kB. As a result, macrophages increase the expression of inflammatory mediators and immune effectors including multiple cytokines and chemokines (Hu et al. 2008). IFN-γ suppresses the anti-inflammatory signaling triggered by IL-10 and TGF-β, thus IFN-γ primed macrophages are able to kill ingested pathogens through the generation of NADPH oxidase and nitric oxide synthase associated with the production of reactive oxygen species and reactive nitrogen intermediates, respectively (Saha et al. 2010). Furthermore, in response to IFN-γ, macrophages upregulate the expression of complement components which opsonize extracellular pathogens.
In contrast to the pro-inflammatory effects described above, modulation of an anti-inflammatory response is another major aspect of IFN-γ biological function. IL-17 is a pro-inflammatory cytokine produced by Th17 cells. IL-17 generates an inflammatory environment by enhancing the production of pro-inflammatory cytokines and chemokines. Thus, Th17 response is vital for protection against extracellular pathogens. However, if the response is not regulated, tissue damage may occur. IFN-γ inhibits the development of Th17 cells by inhibiting the effects of IL-6, IL-1, TGF-β, IL-21, and IL-23, all of which promote Th17 cell development. STAT1 inhibits STAT3, a critical component used by the IL-6, IL-23, and IL-21 signaling pathways. In addition, IFN-γ signaling downregulates the expression of both IL-23 and IL-1 receptors on Th17 cells (Bettelli et al. 2007). IFN-γ also plays a critical role in maintaining the homeostasis of the immune response. When stimulated with IFN-γ, Foxp3+T cells upregulate the expression of T-bet. These Foxp3 + T-bet + regulatory T cells (Tregs) specialize in confining the Th1 immune response (Koch et al. 2009). IFN-γ also induces STAT1-dependent apoptosis in macrophages by upregulating caspases-1 to reduce the inflammation resulting from macrophage infiltration. For T cells, the apoptosis effect is associated with the strength of IFN-γ signaling. When treated with a high level of IFN-γ, T cells stop the internalization of IFN-γR2. The overexpression of IFN-γR2 then induces the expression of Fas ligand (FasL), promoting Fas-dependent apoptosis (Regis et al. 2006).
In summary, the data generated thus far clearly demonstrates that there are two arms of IFN-γ biological functions, i.e., pro- and anti-inflammatory pathways, and both arms are critical for a balanced immune response. The complex yet delicate signaling network allows IFN-γ to tailor the immune response either for defense against infection or toward maintaining the homeostasis of the host.
IFN-γ: Friend or Foe to Autoimmune Diseases
Due to its pro-inflammatory properties, IFN-γ has been associated with promoting different autoimmune diseases. Systemic lupus erythematous (SLE) is a complex autoimmune disease and its main characteristic is the generation of autoantibodies by active B cells. The antibody-complement complex causes local and systemic inflammation and may result in kidney failure. In SLE patients, elevated pSTAT1 was observed in peripheral blood lymphocytes and similar results have also been seen in an SLE mouse model (Mozes and Sharabi 2010). Hodge and colleague has developed a mouse where the RNA stability element (ARE) in the 3′UTR of the IFN-γ mRNA has been removed. Without ARE-mediated decay of IFN-γ mRNA, the animals constitutively express low levels of IFN-γ. A significant finding resulting from the constitutive expression of low levels of IFN-γ is the rapid appearance of SLE-like symptoms (Hodge et al. unpublished data). While IFN-γ has not been shown to cause SLE (the pathogenesis of SLE remains unclear), the presence of IFN-γ exacerbates the disease. IFN-γ upregulates the expression of IFN-α whose signaling induces the transcription of the B-cell activation factor (BAFF). BAFF gives rise to the expansion of B cells and autoantibody production. Additionally, macrophages and fibroblasts increase the secretion of complement proteins in response to IFN-γ, resulting in complement deposition in the kidney. Furthermore, IFN-γ promotes widespread inflammation by inducing chemokines to recruit monocytes to different target organs. As a whole, various novel mouse models of lupus-like disease demonstrate that low levels of IFN-γ, if persistent, are capable of promoting autoimmunity.
In contrast to SLE, IFN-γ has been shown to mitigate the severity of disease in multiple sclerosis (MS). MS is an autoimmune disease caused by infiltration of lymphocytes into the central nervous system. As there is increased IFN-γ in the brain of MS patients, it was proposed that self-reactive Th1 cells maybe the culprit in promoting the destruction of the myelin sheath. However, the results of experiments using neutralizing anti-IFN-γ antibodies in an experimental autoimmune encephalomyelitis animal model showed otherwise. Injection with anti-IFN-γ antibodies did not ameliorate the symptoms, but actually aggravated the disease. Furthermore, recent reports have found multiple polymorphisms in the genes encoding IFN-γ/IFN-γR in MS patients. The defect in IFN-γ signaling may result in heightened Th1 immune responses due to the failure to generate Th1-specific Treg cells and induce apoptosis in active immune cells. Also, without the inhibition of IFN-γ, Th1 cells will commit to the Th17 lineage. Due to the defect in IFN-γ signaling found in MS patient, the involvement of Th17 cells in the pathogenesis of MS has been suggested (Billiau and Matthys 2009). However, IFN-γ treatment in MS patients often resulted in disease relapse. A recent report suggested the effect of IFN-γ on patients may differ based on the pathogenesis of demyelination. IFN-γ can be detrimental for patients with remyelination of oligodendrocytes (ODCs) but beneficial for those with ODC death (Lees and Cross 2007).
With respect to another autoimmune disease, rheumatoid arthritis (RA), the effect of IFN-γ depends on the stage of disease. RA is characterized by the accumulation of effector T cells that target the synovial membrane, cartilage, and bone. In the collagen-induced arthritis animal model, there was an acceleration of disease progression when treated with IFN-γ in the early phase of disease induction. As expected, the symptoms improved when animals were treated with anti-IFN-γ antibodies; however, symptoms became worse when the antibodies were given during the later stage of the disease (Saha et al. 2010). It is possible that IFN-γ promotes the activity of effector T cells during initiation of disease, and dampens the immune responses during disease progression by inducting apoptosis of effector T cells and generation of Tregs.
Autoimmune diseases are complex and the systems biology that affects disease development and progression is yet fully comprehended. Experimental studies thus far demonstrate that the effects of IFN-γ vary depending upon the types and progression status of disease. To utilize IFN-γ or antibodies to neutralize IFN-γ as a treatment for autoimmune disease, more work is needed to better understand disease pathogenesis.
IFN-γ and Cancer
The effect of IFN-γ on containing tumor progression and growth is well documented. IFN-γ augments the immunogenicity of tumor cells by increasing tumor antigen presentation to tumor-specific T cells and NK cells. IFN-γ also enhances the antitumor activity of T and NK cells. For example, it has been shown that IFN-γ can induce FasL on tumor cells which would promote Fas-dependent apoptosis. Furthermore, IFN-γ inhibits the growth and proliferation of tumor cells by arresting the cell cycle (Saha et al. 2010). Also, depletion of IFN-γ promotes the growth of chemically induced tumors. However, a recent report suggested that the inflammatory environment driven by IFN-γ signaling may play a role in promoting cancer cell survival, immunoinvasion of the tumor cells, and immunosuppression of the host response in UVB-induced melanoma (Zaidi et al. 2011). The result implies that the effect of IFN-γ on cancer may be dependent on the local tumor environment.
IFN-γ is an extraordinarily pleiotropic cytokine. It can not only heighten both the innate and adaptive immune response against pathogens and tumors, but also has the ability to maintain immune homeostasis. IFN-γ has such diverse functions because its effect differs based on the cell type and the activation level of the response to receptor triggering. Utilizing IFN-γ for clinical trials in autoimmune disease and cancer therapy has been attempted, albeit with only modest success. The experimental data obtained thus far indicates that one needs to consider the fine balance between pro- and anti-inflammatory effects of IFN-γ when considering an effective clinical use for this important immunoregulatory protein. More than five decades after the discovery of IFN-γ, the complexity of the effects of IFN-γ has yet to be fully appreciated. Further investigation into the interactions among the different signal transduction pathways affected by IFN-γ will help to map out the network from a systems biology perspective. This knowledge will be beneficial in understanding the pathogenesis of multiple disease states, and will result in the identification of the appropriate clinical intervention strategy for either administering or neutralizing the effects of this key immunoregulatory protein.
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