Interferon-: an overview of signals, mechanisms and functions

Kate Schroder,*, Paul J. Hertzog,, Timothy Ravasi,*, and David A. Hume*,,1 *Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane, Australia; CRC for Chronic Inammatory Diseases, Parkville, Victoria, Australia; and Molecular Genetics Group, Institute of Reproduction and Development, Monash Medical Centre, Monash University, Clayton, Victoria, Australia

Abstract: Interferon- (IFN-) coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. This article reviews the current understanding of IFN- ligand, receptor, signal transduction, and cellular effects with a focus on macrophage responses and to a lesser extent, responses from other cell types that inuence macrophage function during infection. The current model for IFN- signal transduction is discussed, as well as signal regulation and factors conferring signal specicity. Cellular effects of IFN- are described, including up-regulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation and effects on apoptosis, activation of microbicidal effector functions, immunomodulation, and leukocyte trafcking. In addition, integration of signaling and response with other cytokines and pathogen-associated molecular patterns, such as tumor necrosis factor-, interleukin-4, type I IFNs, and lipopolysaccharide are discussed. J. Leukoc. Biol. 75: 163189; 2004.
Key Words: macrophage cytokine lipopolysaccharide Toll-like receptor inammation

Macrophages respond to a range of different cell products during the innate and acquired immune response. Of these, IFN- (originally called macrophage-activating factor) is among the most important. Macrophage stimulation with IFN- induces direct antimicrobial and antitumor mechanisms as well as up-regulating antigen processing and presentation pathways. IFN- orchestrates leukocyte attraction and directs growth, maturation, and differentiation of many cell types [2 4], in addition to enhancing natural killer (NK) cell activity [5] and regulating B cell functions such as immunoglobulin (Ig) production and class switching [4, 6]. This review provides a brief overview of IFN- biology with respect to the wellcharacterized responses that alter macrophage function during infectious challenge.

THE IFNs
The IFNs were originally discovered as agents that interfere with viral replication [7]. Initially, they were classied by the secreting cell type but are now classied into type I and type II according to receptor specicity and sequence homology. The type I IFNs are comprised of multiple IFN- subtypes (14 20, depending on species), IFN-, IFN-, and IFN-, all of which are structurally related and bind to a common heterodimeric receptor (IFNAR, comprised of IFNAR1 and IFNAR2 chains). Although type I IFNs are secreted at low levels by almost all cell types, hematopoietic cells are the major producers of IFN- and IFN-, whereas broblasts are a major cellular source of IFN- [8]. IFN- is also produced by macrophages under appropriate stimulus (discussed later). Viral infection is the classic stimulus for IFN- and IFN- expression [8, 9]. Secretion of IFN- has only been reported in ruminants [10]. IFN- is the sole type II IFN. It is structurally unrelated to type I IFNs, binds to a different receptor, and is encoded by a separate chromosomal locus. Initially, it was believed that CD4 T helper cell type 1 (Th1) lymphocytes, CD8 cytotoxic lymphocytes, and NK cells exclusively produced IFN- [8, 11]. However, there is now evidence that other cells, such

INTRODUCTION
This review focuses on the interplay between interferon- (IFN-) and macrophages in inammation and acquired immunity during infection. Macrophages are extremely versatile cells involved in a number of complex functions in disease and health. A pathogen encounters macrophages soon after host entry, and the result of these encounters is fundamental to the hosts ability to mount an effective immune response. Macrophage activation, broadly dened as acquisition of competence to execute a complex function [1], is among the rst actions to occur in innate immunity to potential pathogens. In most situations, macrophages are activated to acquire microbicidal effector functions and secrete proinammatory cytokines, resulting in inammation and recruitment of immune cells and subsequent elimination of the microbe by phagocytosis or release of toxic metabolites.

1 Correspondence: Institute for Molecular Bioscience, University of Queensland, St. Lucia, Brisbane 4072, Australia. E-mail: D.Hume@imb.uq.edu.au Received June 2, 2003; revised July 25, 2003; accepted July 27, 2003; doi: 10.1189/jlb.0603252.

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as B cells, NKT cells, and professional antigen-presenting cells (APCs) secrete IFN- (reviewed in refs. [5, 1216]). IFN- production by professional APCs [monocyte/macrophage, dendritic cells (DCs)] acting locally may be important in cell self-activation and activation of nearby cells [12, 13]. IFN- secretion by NK cells and possibly professional APCs is likely to be important in early host defense against infection, whereas T lymphocytes become the major source of IFN- in the adaptive immune response [12, 17]. IFN- production is controlled by cytokines secreted by APCs, most notably interleukin (IL)-12 and IL-18. These cytokines serve as a bridge to link infection with IFN- production in the innate immune response [18 24]. Macrophage recognition of many pathogens induces secretion of IL-12 and chemokines [e.g., macrophage-inammatory protein-1 (MIP1); ref. 25]. These chemokines attract NK cells to the site of inammation, and IL-12 promotes IFN- synthesis in these cells [25, 26]. In macrophages, NK and T cells, the combination of IL-12 and IL-18 stimulation further increases IFN- production [20, 23, 24, 27, 28]. Negative regulators of IFN- production include IL-4, IL10, transforming growth factor-, and glucocorticoids [17, 21, 2729]. Given the complexity of IFN- regulation, it is not surprising that inbred mouse strains vary in their ability to secrete this cytokine; for example, T lymphocytes of C57BL/6 and C3H mice secrete signicantly higher amounts of IFN- compared with the T lymphocytes of BALB/c and B10.D2 mice. Increased IFN- production in these strains is associated with greater resistance to bacteria and viruses [30 32]. Many excellent reviews on the regulation of IFN- production have been published recently and the reader is referred to these for further information [1113].

THE IFN- RECEPTOR

Functional IFN- receptor (IFNGR) is comprised of two ligand-binding IFNGR1 chains associated with two signal-transducing IFNGR2 chains and associated signaling machinery. IFNGR1 and IFNGR2 chains belong to the class II cytokine receptor family, a class of receptors that bind ligand in the small angle of a V formed by the two Ig-like folds that constitute the extracellular domain. It is likely that the IFNGR chains and other family members (type I IFN receptor and tissue factor) evolved from primitive adhesive molecules [33, 34]. The IFNGR2 chain is generally the limiting factor in IFN- responsiveness, as the IFNGR1 chain is usually in surplus [8, 35]. The IFNGR2 chain is constitutively expressed, but its expression level may be tightly regulated according to the state of cellular differentiation or activation [8]. For example, some CD4 Th1 populations have very low levels of cell-surface expression of the IFNGR2 chain and thus low expression of active IFNGR, leading to a functional blockade of some aspects of IFN- signaling [36, 37]. As the growth-inhibitory effects of IFN- are blocked, T cells expressing low levels of the IFNGR2 chain continue to proliferate during IFN- treatment. Conversely, IFN- exposure to CD4 Th2 populations displaying high levels of IFNGR2 inhibits proliferation and
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may induce the apoptotic program [35, 38, 39]. This mechanism may aid in the Th2-to-Th1 phenotype switch apparent when CD4 cells are treated with IFN-: the Th2 population decreases as a result of growth-inhibitory and proapoptotic effects of IFN-, and the Th1 population continues to proliferate as a result of blockade of IFN- function. As a consequence, IFN- signaling causes IFNGR2 down-regulation and switching to a Th1 phenotype as a T cell population but not at a single-cell level [37]. Both IFNGR chains lack intrinsic kinase/phosphatase activity and so must associate with signaling machinery for signal transduction. The IFNGR1 intracellular domain contains binding motifs for the Janus tyrosine kinase (Jak)1 and the latent cytosolic factor, signal transducer and activator of transcription (Stat)1. In humans, the Jak1-binding motif LPKS is a membrane-proximal sequence located at residues 266 269 [40 42]. The human Stat1-binding site YDKPH is positioned at residues 440 444 [43]. This motif contains an essential Y440 phosphorylation site that is phosphorylated during signal transduction to allow Stat1 recruitment to the receptor [42 44]. Residues 441DKPH444 are responsible for the binding specicity of IFNGR1 and Stat1 [41, 43, 44]. The Jak1- and Stat1binding motifs are required for receptor phosphorylation, signal transduction, and induction of biological response. Receptor-ligand internalization is mediated by an isoleucine-leucine sequence at residues 270 and 271 of the mature IFNGR1 chain in humans. Mutant receptors with deletion or substitution of this sequence cannot internalize ligand, although signal transduction is not affected [42, 45]. The intracellular region of IFNGR2 contains a noncontiguous binding motif for recruitment of Jak2 kinase for participation in signal transduction. In humans, these sites are 263 PPSIP267 and 270IEEYL274 [46, 47]. The IFNGR2 chain is not tyrosine phosphorylated during signal transduction [46]. Cross-linking experiments with labeled human IFN- demonstrated that IFN- only associates with IFNGR2 when the IFNGR1 chain is present, indicating that the primary interaction between the IFNGR2 chain and the IFN-:IFNGR1 complex is found between IFNGR1 and IFNGR2, although it is likely that IFNGR2 also interacts weakly with the ligand [46, 48]. IFN-:IFNGR1 and IFNGR1:IFNGR2 interactions are species-specic. A number of studies with chimeric receptors in which the intracellular, transmembrane, or extracellular domains were swapped between the human and murine IFNGR chains showed that species specicity in interactions of the ligand:receptor complex is restricted to the receptor extracellular domains [49 53].

IFN- SYSTEM DYSFUNCTION

IFN-/ and IFNGR1/ mice showed no overt developmental defects, and their immune system appeared to develop normally [54]. However, these mice show deciencies in natural resistance to bacterial, parasitic, and viral infections such as vaccinia virus, Theilers murine encephalomyelitis virus, Leishmania major, Toxoplasma gondii, Listeria monocytogenes, and several poorly virulent mycobacteria species [54 59].
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Some viruses (e.g., vaccinia virus, Theilers murine encephalomyelitis virus, and lymphocytic choriomeningitis virus) appear to require both type I and II IFN pathways, whereas viruses such as Semliki forest virus and vesicular stomatitis virus require a predominantly type I IFN response for efcient clearing [60]. These observations have prompted the suggestion that the two IFN systems may have evolved to complement each other in overlapping but nonredundant activities to defend against a broad spectrum of pathogens [60, 61]. Historically, the type I IFNs were considered to be primarily antiviral agents with limited immunomodulatory activity, whereas IFN- was considered to be primarily an immunomodulator with limited antiviral activity [8]. It is now apparent that both types of IFN possess antiviral and immunomodulatory activities to some degree [62]. The antiviral activity of both types of IFNs is often important in the early stages of viral infection, but their immunomodulatory activities become important in later stages of infection when the adaptive immune response becomes critical [60]. Patients with inactivating mutations of the human IFNGR1 or IFNGR2 chains show clinical presentation similar to the mouse models. Human loss-of-function mutations in the IFNGR1 or IFNGR2 chain are closely associated with severe susceptibility to poorly virulent mycobacteria, often presenting as early onset bacillus Calmette-Guerin infection and fatality in childhood [6371]. Partial deciency of either chain gives a milder phenotype and a much better prognosis [63 65]. Loss of functional IFNGR1 appears to be associated only with increased susceptibility to infection with some viruses and intracellular bacteria; increased susceptibility to other common bacterial and fungi pathogens have not been reported [72]. In addition to recurrent infection, infants with decient production of IFN- exhibited decreased neutrophil mobility and NK cell activity, highlighting the importance of IFN- in the inammatory response and immunoregulation [73]. It is interesting that natural IFN- polymorphisms have been correlated with increased longevity [74]. It has been proposed that a slightly dampened inammatory status caused by an IFN- polymorphism, while not enough to signicantly impact on the individuals ability to clear infection, may prevent or defer inammation-related diseases such as cardiovascular disease, neurodegeneration, osteoarthritis, osteoporosis, and diabetes [74]. Aside from functions in host defense, IFN- may also contribute to autoimmune pathology. Although IFN- production was shown to be disease-limiting in autoimmune models such as murine experimental allergic encephalomyelitis (EAE) [75, 76], it may contribute to autoimmune nephritis [77, 78]. In humans, IFN- is implicated in pathology of diseases such as systemic lupus erythematosus [79, 80], multiple sclerosis [81], and insulin-dependent diabetes mellitus [82 84]. IFN- function is signicant in tumor surveillance. IFNGR1 knockout (KO) mice, cells with dominant-negative IFNGR1 mutations, and cells treated with IFN--neutralizing antibodies display compromised tumor rejection [85 87]. IFN- protects against tumor development and directs the immunogenic phenotype of tumors that arise in an immunocompetent host (the cancer immunoediting concept; reviewed in refs. [88, 89]).

SIGNAL TRANSDUCTION
IFN- primarily signals through the Jak-Stat pathway, a pathway used by over 50 cytokines, growth factors, and hormones to affect gene regulation [90]. Jak-Stat signaling involves sequential receptor recruitment and activation of members of the Janus family of kinases (Jaks: Jaks 13 and Tyk2) and the Stats (Stats 1 6, including Stat5a and Stat5b) to control transcription of target genes via specic response elements. As this signaling mechanism is a recurring theme amongst members of the cytokine receptor superfamily, IFN--induced Jak-Stat signaling has emerged as the global paradigm for class II cytokine receptor signal transduction. The IFNGR1 and IFNGR2 subunits of the IFN- receptor were thought not to strongly associate with each other in the absence of ligand [13, 40, 47, 91, 92], but new techniques allowing the study of receptor chain interactions in intact cells have shown the receptor complex is assembled before ligand binding [93]. Upon ligand binding, the intracellular domains of the receptor chains open out to allow association of downstream signaling components. Biologically active IFN- is a noncovalent homodimer formed by the self-association of two mature polypeptides in an antiparallel orientation [94, 95] and thus binds IFNGR1 in 2:2 binding stoichiometry [47, 9597]. Ligand binding induces Jak2 autophosphorylation and activation, which allows Jak1 transphosphorylation by Jak2 (Fig. 1) [98]. The activated Jak1 phosphorylates functionally critical tyrosines on residue 440 of each IFNGR1 chain to form two adjacent docking sites for the SH2 domains of latent Stat1 [41, 99 101]. The receptor-recruited Stat1 pair is phosphorylated near the C terminus at Y701, probably by Jak2 [98]. Phosphorylation induces dissociation of a Stat1 homodimer (also known as -IFN activation factor) from the receptor [100]. The four critical tyrosines (contained by Jak1, Jak2, IFNGR1, and Stat1) are phosphorylated within 1 min of IFN- treatment [41, 99]. The dissociated Stat1 homodimer enters the nucleus and binds to promoter elements to initiate or suppress transcription of IFN--regulated genes (reviewed in refs. [102104]). Stat1 homodimers bind DNA at GAS elements of consensus sequence TTCN(2-4)GAA [105]. The key function of Stat1 in mediating IFN- signal transduction is indicated by the phenotype of Stat1/ mice, which largely phenocopy IFNGR1/ mice during IFN- stimulation [106]. The rst wave of IFN--induced transcription occurs within 1530 min of IFN- treatment [107]. Many of the induced genes are in fact transcription factors (for example, IRF-1), which are activated by IFN- and are able to further drive regulation of the next wave of transcription. Transcription of many IFN--responsive genes is controlled by a GAS element or an ISRE [108].

Stat1
The crystal structure for tyrosine phosphorylated Stat1 bound to DNA has been determined [109]. The structure veried that the SH2 domain necessary for dimerization is also involved in

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Fig. 1. The current paradigm for IFN- signal transduction. Ligand binding causes a conformational change in the IFN-R (IFNGR1, yellow; IFNGR2, green), such that the inactive Jak2 kinase undergoes autophosphorylation and activation, which in turn allows Jak1 transphosphorylation by Jak2. The activated Jak1 phosphorylates functionally critical tyrosines on residue 440 of each IFNGR1 chain to form two adjacent docking sites for the Src homology (SH)2 domains of latent Stat1. The receptor-recruited Stat1 pair is phosphorylated near the C terminus at Y701. Phosphorylation induces dissociation of a Stat1 homodimer from the receptor. To a lesser extent, IFN- signaling also produces Stat1:Stat1:IFN regulatory factor (IRF)-9 and Stat1:Stat2:IRF-9 [IFN-stimulated gene factor 3 (ISGF3)] complexes. Stat1 homodimers travel to the nucleus and bind to promoter IFN--activation site (GAS) elements to initiate/suppress transcription of IFN--regulated genes. Many of IFN--regulated genes are in fact transcription factors (e.g., IRF-1), which are activated by IFN- and are able to drive regulation of the next wave of transcription (e.g., induction of IFN-). Stat1:Stat1:IRF-9 heterodimers, ISGF3, and IRF-1 are able to bind to IFN-stimulated response element (ISRE) promoter regions in target genes to regulate transcription. IRF-1 is also able to promote transcription of Stat1 through an unusual ISRE site (IRF-E/GAS/IRF-E). Signaling molecules activated by IFN- are depicted in red text. ICAM-1, Intercellular adhesion molecule-1; MIG, monokine induced by IFN-; iNOS, inducible nitric oxide synthase.

DNA binding. The C-terminal transactivation domain contains the Y701 phosphorylation site and also a S727 phosphorylation site, both of which are functionally important for efcient signaling [110 113]. All Stat proteins possess a SH2 domain that when tyrosine phosphorylated, mediates homodimerization or heterodimerization with other Stat molecules [114, 115]. Although the simplistic model described above suggests that IFN- signaling produces active Stat1 homodimers alone, other active complexes such as Stat1 heterodimers (e.g., Stat1:Stat2) and heterotrimers (e.g., Stat1:Stat1:IRF-9, Stat1:Stat2:IRF-9) form during signaling [102, 116 118]. Stat2 is the only Stat that does not contain a DNA-binding domain [119]. The Stat1:
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Stat2:IRF-9 complex (known as ISGF3) was thought to be the typical type I IFN transcription factor. It is now evident that type I IFN signals primarily through ISGF3 but also uses Stat1 homodimers and conversely, type II IFN signals primarily through Stat1 homodimers but also uses ISGF3 to activate transcription [116]. This may explain many of the overlapping effects of the two types of IFNs. Phosphorylation of Stat1 at S727 is essential for maximal ability to activate transcription of target genes [111113, 120 122]. A number of different stimuli induce Stat1 serine phosphorylation, including types I and II IFN, lipopolysaccharide (LPS), IL-2, IL-12, tumor necrosis factor (TNF-), and
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platelet-derived growth factor [110 112, 123, 124]. This may be a mechanism whereby S727 serves as an avenue for modulation of IFN- signaling by independent extracellular cues. For example, LPS signaling increases Stat1 S727 phosphorylation independently of Y701 phosphorylation in macrophages, thereby augmenting cellular responses to IFN- [110]. The ability of Stat1 to activate or repress gene transcription depends on the presence of other transcription factors binding to the promoter element and Stat1 interaction with other factors. Stat1 activation is necessary but not sufcient for transcription of a number of genes [125, 126]. Likewise, factors such as oncostatin M, which participate in Jak-Stat signaling to produce activated Stat1, do not induce transcription of IFN-inducible genes [127]. Stat1 interaction with transcription factors such as IRF-9, upstream stimulatory factor-1, specicity protein-1, and heat shock factor-1 have been reported (reviewed in ref. [119]) and are likely to inuence specicity of DNA binding and transactivator ability. Phosphorylation of S727 is necessary for interaction with some proteins, such as BRCA1 and MCM-5, and this interaction potentiates Stat1mediated transcription [128, 129]. In some promoters, transcription is maximal when more than one GAS-binding transcriptional activator binds to tandem GAS sites in the target gene promoter [130, 131]. In the absence of IFN-induced tyrosine phosphorylation, Stat1 drives constitutive transcription of genes such as caspases [132, 133]. The Stat1 KO mouse showed increased lymphocyte survival and proliferation, which was attributed to insufcient caspase levels to carry out the apoptotic program [133]. Stat1 also constitutively regulates expression of the low molecular protein 2 (LMP2) gene [134]. A complex of unphosphorylated Stat1 and IRF-1 may mediate this. The mechanism of Stat1 entry into the nucleus is still controversial, but the involvement of importin--1 (NPI-1) is implied [135]. Two major models have been suggested, one being the classic nuclear importin mechanism and the other involving a requirement for intracellular IFN-. Nuclear entry of Stat1 is apparent at 15 min and almost complete after 30 min exposure to IFN- [136]. Stat1 nuclear translocation does not appear to be a result of liberation from a cytoplasmic anchor or transport by the cellular cytoskeleton or microtubules [136]. Experiments with green uorescent protein-tagged Stat1 implied that it travels through the cytoplasm to the nucleus by random walk movement [136]. A model was proposed whereby Stat1 dimerization allows interaction with importin NPI-1, which then mediates translocation through the nuclear pore [135]. The nuclear membrane forms an efcient barrier to inactivated Stat1, and entry requires a gain of nuclear localization sequence (NLS) function [136 138]. To date, no classic NLS (single stretch of basic residues or bipartite motif; ref. [139]) has been identied for Stat1, indicating an as-yet unidentied, novel NLS is unmasked during Stat1 homodimerization, or the NLS of a Stat1 homodimer-binding partner mediates nuclear translocation. The second model for Stat1 nuclear entry involves a requirement for intracellular IFN-. IFN enters the cell using a mechanism that is unclear at present. Nuclear accumulation of IFN- is apparent shortly after cellular exposure to IFN- [140, 141]. Nuclear translocation is mediated by a C-terminal

sequence very similar to the Simian virus-40 T antigen NLS, RKRKRSR132 in mice and 128KTGKRKR134 in humans [139, 142145]. This sequence is a functional NLS and is essential for the full biological activity of IFN- [144 148]. A deletion mutant of the putative NLS in human IFN- is still able to bind with high afnity to the IFNGR1 chain, but induction of biological response is absent [149]. The addition of a heterologous NLS is able to rescue biological responsiveness in this system. Another study tracked the cellular localization of components of the IFNGR complex following ligand exposure [150]. IFN-, Stat1, and IFNGR1 were found to be quickly internalized, colocalized, and accumulated in the nucleus, while the majority of the IFNGR2 subunit remained at the cell surface throughout signaling. Stat1 nuclear translocation is mediated by NPI-1 [135], and immunoprecipitation of Stat1 following IFN- treatment showed that IFN-, IFNGR1, Stat1, and NPI-1 form a complex at the nuclear pore during translocation [142, 143]. Subramaniam et al. [90] proposed a model suggesting a function for intracellular ligand in Stat1 nuclear localization. In this model, extracellular ligand binds to the IFNGR1 chain, causing endocytosis of the complex and recycling of the IFNGR2 chain to the cell surface. The intracellular domain of IFNGR1 associates with activated Stat1 and intracellular IFN-, and the IFN:IFNGR1:Stat1 complex is actively transported through the nuclear pore via IFN-NLS interaction with the NPI-1 importin. Activated Stat1 may then bind to promoter elements to activate transcription. Functional Stat1 is crucial to host response to infection. KO of Stat1 renders mice extremely sensitive to infection by viral and microbial pathogens (e.g., vesicular stomatitis viruses, mouse herpes virus, L. monocytogenes) [106, 151]. This susceptibility has been attributed to defective immune system function as a result of the combined loss of types I and II IFN responses [151]. NO production in Stat1/ macrophages primed with types I or II IFN and treated with LPS is greatly impaired compared with the wild type (WT), resulting in reduced macrophage microbicidal function [106]. Impaired macrophage microbicidal function may contribute to the increased susceptibility to infection apparent in Stat1/ mice. Stat1 KO mice also demonstrated the existence of Stat1independent mechanisms in IFN- signaling. IFN- may exert pro- and antiproliferative signals through Stat1-independent and Stat1-dependent pathways, respectively [152154]. Although IFN- treatment of lymphocytes and broblasts inhibited cell growth, the same treatment in Stat1/ cells promoted proliferation and decreased apoptotic signals [133, 153, 154]. Microarray analysis revealed that many genes, including the growth-promoting c-myc and c-jun genes, were induced in response to IFN- in Stat1/ cells [152]. This signaling was not dependent on the Stat1-docking site of IFNGR1. Although much more susceptible than their WT counterparts, Stat1/ mice are signicantly more resistant to viral infection than IFNGR1/IFNAR1 double-KO mice, indicating the existence of IFN-dependent, Stat1-independent, antiviral mechanisms [155]. A loss-of-function, heterozygous mutation in the Stat1 gene was recently described in humans, resulting in a dominantnegative effect over the WT allele for Stat1 activation [156]. Although these patients demonstrated increased susceptibility
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to bacterial infection, viral infections took the normal clinical course. This suggests that IFN--induced, physiologically signicant, Stat1-independent control of antiviral responses also occurs in humans. Mechanisms of Stat1-independent IFN- signaling are currently unclear. One possibility is the existence of an unknown IFN- receptor or IFN- receptor complex linked to an alternative signaling pathway. This hypothesis was suggested when it was found that anti-IFN- antibodies affected the clinical course of EAE in IFNGR1 KO mice [75].

IRF-1
The IRF gene family is intimately involved in mediating the type I and II IFN signal cascades. Members of the IRF family share similar structure, including an amino-terminal DNAbinding domain containing multiple tryptophan residues and a carboxy-terminal transcriptional activation or repression domain (reviewed in ref. [157]). IRF-1, IRF-2, and IRF-9 (p48, ISGF3-) all participate in IFN- signaling. Basal IRF-1 expression has functions in constitutive gene expression [134], but Stat1 and nuclear factor (NF)-B interaction with promoter elements dramatically increases IRF-1 transcription [158 160]. IRF-1 expression is up-regulated in response to types I and II IFN, virus, or cytokines. IRF-1 transcriptional regulatory activity is regulated independently in response to IFN-, virus, and dsRNA [161]. The physiological relevance of IRF-1 function in transcriptional control of IFN--regulated genes is highlighted by IRF-1 KO mice, in which many genes are submaximally induced by IFN- when compared with their WT littermates [162, 163]. IRF-1 functions are not limited to IFN- signaling; IFN--independent activities in viral/bacterial infection, lymphocyte development, regulation of cell cycle, growth inhibition, apoptosis and tumor suppression have been reported [107, 123, 135, 159, 164 168]. IRF-1 drives inducible expression of many target genes through interaction with the IRF-E site, consensus G(A)AAA G T /C /CGAAAG/CT/C [102, 164]. This specicity overlaps with the ISRE consensus site A/GNGAAANNGAAACT, recognized by ISGF3, which is induced by type I IFN and to a lesser extent, type II IFN [102]. In this way, IRF-1 is able to induce a subset of the full spectrum of IFN-inducible genes. IRF-2 also binds to the IRF-E site and largely functions to repress transcription of IRF-1-inducible genes through its transcriptional repressor domain [102, 159].

Negative regulation of IFN- signaling

Stat1 activation is inhibited within 1 h of IFN- treatment, despite the continued presence of extracellular IFN-, and so mechanisms must exist to control the extent of ligand stimulation of IFN- signaling (Fig. 2) [117, 169]. These mechanisms involve every level of the pathway. Following signal transduction, the IFN-:IFNGR1 complex internalizes and enters the endosomal pathway, where the complex dissociates [170]. In many cell types, the IFNGR1 chain eventually recycles to the cell surface in its uncoupled, dephosphorylated form, and the ligand is degraded [45, 171,
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172]. In some cell types, IFN- signaling may induce degradation of the internalized receptor, thereby down-regulating IFNGR1 surface expression [45]. This may be a mechanism by which these cells can prevent overstimulation of the pathway through cellular desensitization. Such a mechanism may also allow differential regulation of IFN--induced responses in different cell types [37]. One of the most inducible targets of IFN- is a specic feedback inhibitor, SOCS-1, which associates with Jak1/2, interfering with tyrosine kinase activity and inhibiting downstream IFN- signaling [173177]. SOCS-1 may also promote degradation of signaling machinery by binding to and targeting signaling molecules for the ubiquitin-proteasome pathway [178]. Overexpression of SOCS-1 in cells in vitro or in transgenic mice causes loss of responsiveness to IFN- [176, 179]. Conversely, SOCS-1/ mice are hyper-responsive to microbial infection, exhibit enhanced macrophage cytocidal activity, and die of IFN--dependent, multisystem inammatory tissue destruction in the absence of infection [180, 181]. This indicates that IFN- is normally present and performs important functions in vivo, even in the absence of infection. Another SOCS protein, SOCS-3, is also induced by IFN- and negatively regulates IFN- signaling, although perhaps less effectively than SOCS-1 [182]. Aside from SOCS protein function, IFN- signal downregulation can occur by receptor and Jak dephosphorylation by the PTPs Shp1 and Shp2 [183187]. Shp2 null cells exhibit elevated tyrosine phosphorylation and DNA-binding ability of Stat1 and show enhanced suppression of cell growth with IFN- treatment [184]. Shp1 KO mice (motheaten mice, me/ me) die from a condition caused by dysregulation of a large number of cytokines, possibly including IFN- [188]. Stat1 phosphorylation may also be controlled by nuclear dephosphorylation. Stat1 homodimers enter the nucleus and are actively retained by a mechanism dependent on the kinase function of Jak1 [189]. McBride et al. [137] proposed a model in which nuclear export is mediated by a leucine-rich nuclear export signal (NES) at residues 197205, which is hidden when Stat1 is bound to DNA. Stat1 dephosphorylation by a nuclear protein tyrosine phosphate (PTP) allows the release of DNA and subsequent recognition of the NES by chromosome region maintenance/exportin 1 (CRM1), an export receptor for NES-containing proteins [190, 191]. CRM1 then mediates Stat1 translocation through the nuclear pore in a Ran-GTPasedependent manner. This model is supported by work from other groups, demonstrating Stat1 tyrosine dephosphorylation in the nucleus and subsequent recycling of inactive Stat1 to the cytoplasm, and is consistent with the crystal structure of Stat1 bound to DNA [137, 189, 192, 193].

Signaling specicity
The many cytokines that use Jak-Stat signaling direct transcription through specic but not necessarily unique combinations of Jaks and Stats. This raises the question of how signal specicity is maintained when many cytokines use the same signaling machinery. Evidence for a devoted role of Jaks in signal transduction of specic cytokines is controversial. Mutant cell lines lacking Jak1 or Jak2 have demonstrated deciency in types I and II
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IRF-2

Fig. 2. Negative regulation of IFN- signaling. A number of factors limit the extent and duration of IFN- signal transduction. Receptor IFNGR2 (green) expression is tightly regulated in some cell types, whereas IFNGR1 (yellow) surface concentration may be regulated by the extent of recycling following receptor:ligand internalization. Protein tyrosine phosphatases (PTPs), such as SH2-containing tyrosine phosphatase-2 (Shp2), dephosphorylate Jak1, Jak2, and IFNGR1, and suppressors of cytokine signaling-1 (SOCS-1) and SOCS-3 interfere with Jak activity to turn off signaling after ligand binding. Stat1 activation is down-regulated by Stat1 dephosphorylation in the nucleus. IRF-2 antagonizes transcriptional activation of many (IRF-1-inducible) genes containing ISRE or IRF-E promoter elements by competing for binding sites without promoting gene expression. Signaling molecules activated by IFN- are depicted in red text.

IFN signaling (Jak1) or IFN- signaling (Jak2) [194, 195]. These deciencies are reected in the Jak1 and Jak2 KO mice, suggesting that these Jaks are functionally specic to their cytokine systems, and other Jaks cannot substitute for them in vivo. Jak1 gene KO mice exhibit a number of abnormalities, all attributable to signaling decits in three cytokine receptor families: type II cytokine receptors (i.e., types I and II IFN and IL-10 receptors); all cytokines that use the c subunit for signaling, thereby promoting lymphopoiesis (e.g., IL-2, IL-4, IL-7 receptors); and gp130 receptor family members (e.g., IL-6, IL-11, leukemia inhibitory factor receptors) [196]. These results showed that in vivo, the role of Jak1 is essential and nonredundant in signal transduction and induction of biological response for a specic range of cytokine receptors. Jak2 gene KO in mice is embryonic lethal as a result of deciencies

in erythropoiesis [197, 198]. Other than suppression of IFN- signaling, the cytokine response decit of Jak2 KO mice is largely nonoverlapping compared with Jak1 KO mice [197, 198]. This indicates that other Jaks cannot functionally substitute for Jak2 in vivo, although Jak3 can substitute for Jak2 in vitro to produce Stat1 homodimers and activate transcription of class I major histomcompatibility complex (MHC) [199]. Although specic Stats appear to be dedicated to specic subsets of cytokine signaling, the same Stat or combination of Stats can be activated in different cytokine systems to give completely different biological effects (e.g., IFN- and oncostatin M as described earlier). Possible mechanisms giving rise to specicity of response with respect to Stats include: different thresholds/timeframes of Stat activation may be required for specic biological responses, and specic Stat com-

Schroder et al. Signals, mechanisms, and functions of IFN- 169

STA T1

IRF-1

STA T1
STAT1

Nucleus

STA T1

IFN-regulated gene

Transcriptional repression by IRF-2 competition for ISRE/IRF-E sites

plexes induced by cytokines may give different biological effects. It is widely agreed that signal specicity is largely conferred by the particular Stats activated in cytokine signaling [106, 151, 200 205]. This is supported by the Stat1 KO mouse, which displays loss of only IFN signaling, despite multiple reports of Stat1 activation in response to a range of other cytokines in vitro [106, 151]. Similarly, KO mice of the other Stat proteins showed a loss of biological responsiveness to a single cytokine, indicating that Stat activation may be specic for a single cytokine in vivo if not in vitro [169]. In addition to the Stats themselves, other factors (e.g., intracellular IFN- as described above, or cell-specic transcription factors), which associate with Stats may contribute to the specicity of the signal.

Cross-talk between IFN-/ and IFN- pathways

The IFN- and IFN-/ signal pathways cross-talk at multiple levels. The signal pathways and target genes used by types I and II IFN are partially overlapping, which gives the opportunity for cross-talk to synergize or antagonize particular functions within the cell. This cross-talk is biologically relevant, as cells in vivo are not stimulated with one cytokine in isolation, rather a cytokine cocktail instructs gene expression through the integration of multiple signaling pathways. Although IFN- primarily signals through Stat1 homodimers in the initial signal cascade, additional signaling molecules are activated. As noted above, the archetypal type I IFN signaling molecule ISGF3 is activated by IFN-, thus providing a mechanism for cross-talk between the types I and II IFN pathways [116]. ISGF3 is able to induce expression of type I IFN, thereby further amplifying the response of IFN-/-induced genes [206, 207]. Conversely, type I IFN can elicit classic type II IFN signaling molecules such as active Stat1 homodimers, which are able to bind to GAS sites to activate transcription of target genes [17, 208]. Takaoka et al. [209] published data indicating that IFN- signaling in mouse embryonic broblasts requires a constitutive subthreshold IFN-/ signal. They have suggested that IFN-/ may promote the interaction of the IFNGR2 and IFNAR1 chains in the caveolar membrane domains of the cell membrane. In this model, low level IFN-/ signal is necessary for maintaining its receptor in the phosphorylated form, thus providing a functional aid for efcient assembly of IFN-activated Stat1 homodimers.

IFN- stimulation induces a replacement of the constitutive proteasome subunits with immunoproteasome subunits. In unstimulated cells, the proteasome enzymatic subunits are 1, 2, and 5, encoded outside of the MHC locus. IFN- induces expression of new subunits, LMP2, MECL-1, and LMP7, which competitively replace 1, 2, and 5 subunits of the proteasome, respectively [210 216]. In this way, new species of proteasome are formed, consisting of LMP2:MECL-1:LMP7 (the immunoproteasome), LMP2:MECL-1:5, or 1:2:LMP7, as well as low levels of the 1:2:5 constitutive proteasome [216]. Inducible proteasome replacement is thought to be a mechanism by which IFN- can increase the quantity, quality, and repertoire of peptides for class I MHC loading. The quantity is increased, as overall expression levels of proteasome are increased. The cleavage specicity of the immunoproteasome may allow production of peptides better able to bind class I MHC and thereby increase efciency in the system. Peptide diversity is thought to increase as a result of differences in cleavage specicities between different species of proteasome. As a whole, this serves to increase levels and diversity of epitopes presented for CD8 T cell recognition in the context of class I MHC and thus increase immune surveillance (reviewed in ref. [216]). This mechanism may have evolved to ensure LMP2/LMP7/MECL-1-dependent epitopes are only produced in sites of inammation and thus avoid autoimmunity without compromising appropriate T cell stimulation [216]. The efciency of peptide generation is further increased with the IFN--induced PA28, which is composed of PA28 and PA29 subunits and associates with the proteasome and alters proteasome proteolytic cleavage preference. It is thought to gear antigen processing toward more efcient generation of TAP- and class I MHC-compatible peptides to increase overall efciency of class I MHC peptide delivery [217, 218]. The IFN--inducible TAP transporter is vital in peptide transport from the cytosol to the ER lumen [307]. TAP transiently associates with class I MHC to aid in efcient peptide loading [307, 308]. Genes encoding the TAP subunits are located within the class II MHC locus and are coordinately expressed with MHC class I [220, 221]. The class I MHC complex is composed of a heavy chain (consisting of 1, 2, and 3 domains) and a light chain (2 microglobulin) and is up-regulated by IFN- stimulation [4, 222, 224 232]. Chaperones such as tapasin and GP96 implicated in aiding in the efcient assembly of peptide:MHC I complexes are also up-regulated by IFN- [4, 309, 310].

Class II antigen presentation pathway CELLULAR EFFECTS OF IFN- Class I antigen presentation pathway
Types I and II IFN up-regulate multiple functions within the class I antigen presentation pathway to increase the quantity and diversity of peptides presented on the cell surface in the context of class I MHC. Up-regulation of cell-surface class I MHC by IFN- (Table 1) is important for host response to intracellular pathogens, as it increases the potential for cytotoxic T cell recognition of foreign peptides and thus promotes the induction of cell-mediated immunity.
170 Journal of Leukocyte Biology Volume 75, February 2004

Of the IFNs, IFN- alone can efciently up-regulate the class II antigen presenting pathway and thus promote peptide-specic activation of CD4 T cells [4, 234]. IFN- treatment further up-regulates class II MHC molecules in cells constitutively expressing class II MHC, such as B cells, DCs, and cells of the monocyte-macrophage lineage (professional APCs) [234]. IFN- is also able to induce class II MHC expression in cells that do not constitutively express these genes (nonprofessional APCs) [76]. IFN- up-regulates the quantity of peptide: MHC II complexes on the cell surface by promoting expression of several key molecules (Table 1): the Ii chain and the components of the class II MHC complex [234 239]; cathephttp://www.jleukbio.org

TABLE 1. Antigen Processing and Presentation Class I antigen presentation pathway Gene/protein up-regulated by IFN- LMP-2, LMP-7, MECL-1

IFN--Regulated Genes and Their Function in Producing IFN- Effects

Function IFN--inducible, enzymatic proteasome subunits, which replace the constitutive proteasome 1, 5, and 2 subunits. May function to optimize peptide diversity and loading to class I MHC and thus alter epitopes presented to the immune system. LMP-2 and LMP-7 map to class II MHC, and multicatalytic endopeptidase complex-like-1 (MECL-1) is encoded outside the MHC Proteasome activator (PA)28:PA28 dimer is a nonenzymatic proteasome subunit, which alters the specicity of peptides generated to increase efciency of class I MHC peptide delivery. The transporter associated with antigen processing (TAP) is a heterodimer consisting of TAP-1 and TAP-2 subunits and belongs to the adenosine 5triphosphate-binding cassette transporter family. TAP functions as a transmembrane pump to transfer peptides from the proteasome into the endoplasmic reticulum (ER) lumen. It also aids in peptide delivery to class I MHC. TAP-1 and TAP-2 map to class II MHC. The heavy chain associates with 2-microglobulin to form the MHC class I complex (MHC I). MHC I displays foreign and self-peptides on the cell surface for immune surveillance by cytotoxic T cells. The class I heavy chain is encoded by the class I MHC locus. Light chain that associates with the class I MHC heavy chain to form the MHC I. MHC I displays foreign and self-peptides on the cell surface for immune surveillance by cytotoxic T cells. The 2-microglobulin light chain is not MHC-encoded. Tapasin is a chaperone that aids in the retention of empty MHC I in the ER and peptide loading into MHC I peptide-binding cleft.

Refs. [210216]

PA28, PA28 TAP-1, TAP-2

[4, 217, 218, 219] [4, 215, 220223]

Class I MHC heavy chain

[4, 224231]

2-microglobulin

[222, 230, 232]

Tapasin

[4, 233]

Class II antigen presentation pathway Gene/protein up-regulated by IFN- 1, 2, 1, 2 MHC II chains Ii chain Function Constituents of the heterodimeric MHC II. MHC II displays foreign and selfpeptides on the cell surface for immune surveillance by CD4 T cells. The MHC II and chains are encoded by the class II MHC locus. The invariant (Ii) chain is a transmembrane chaperone, which trafcs MHC II from the ER to the MHC II endosomal compartment (MIIC). An Ii-derived peptide, CLIP (class II-associated invariant chain peptide), binds to the MHC II peptide-binding groove to prevent inappropriate peptide binding. The DMA:DMB dimer forms DM, a class II-like heterodimeric protein resident in the MIIC. DM functions to remove CLIP from the peptide-binding cleft of MHC II so that it is accessible for peptide loading. DM is encoded by the class II MHC locus. Lysosomal proteases implicated in peptide production for class II MHC loading. Class II transactivator (CIITA) is a transactivator with no DNA-binding motif. It is the limiting component of a complex that induces transcription of key genes involved in class II MHC induction, including constituents of the class II MHC complex, the Ii chain, and DM. Refs. [234237] [237239]

DMA, DMB

[235, 237]

Cathepsins B, H, L CIITA

[240, 241] [234]

Antiviral Effect Gene/protein up-regulated by IFN- PKR Function Protein kinase dsRNA-regulated (PKR) is a serine/threonine kinase activated by dsRNA, which inhibits viral protein synthesis by phosphorylating the subunit of eukaryotic translation initiation factor (eIF-2). PKR is also implicated in NF-B activation, TNF- mRNA splice regulation, induction of apoptosis, and regulation of Stat1 and Stat3 activity. The dsRNA-specic adenosine deaminase (ADAR) catalyzes the deamination of adenosine to form inosine on dsRNA substrates and thus may be responsible for the generation of edited viral mRNA. The cellular translational machinery treats inosine as guanosine, and thus, A 3 I editing of viral mRNA may cause mistranslation into nonfunctional viral proteins to inhibit viral replication. The guanylate-binding proteins (GBP) are GTPases with antiviral properties that function by an unknown mechanism. Refs. [154, 242247]

ADAR

[248]

GBP1, GBP2

[61, 249, 250]

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171

TABLE 1. Antiproliferative Effect Gene/protein up-regulated by IFN- PKR p21, p27 Rb Function

(Continued)

Refs. [154, 242247] [251] [252] [253256] [257]

p202 Mad1

PKR is an antiviral enzyme, which functions as a serine/threonine kinase when activated by dsRNA. PKR inhibits cellular proliferation by phosphorylating the subunit of eIF-2, thereby halting protein synthesis. May also suppress c-myc function. p21 and p27 are cyclin-dependent kinase (CDK) inhibitors of the Cip/Kip family. p21 and p27 inhibit the activity of CDK2 and CDK4, respectively, causing cell cycle arrest at the G1/S checkpoint. IFN- inhibits the activity of the G1 cyclin:CDK complexes [via up-regulation of CDK inhibitors (CKIs), up-regulating CDK-activating kinase activity, and down-regulating CDC25A activity]. Decreased G1 cyclin:CDK activity results in suppression of retinoblastoma (Rb) phosphorylation, thereby increasing levels of the active (E2F-repressing) form of Rb and preventing transcription of E2F-dependent genes required for S phase. p202 is a strong cell cycle repressor that can bind to E2F and inactivate its DNA-binding activity, thereby preventing transcription of E2F-dependent genes required for S phase. Mad1 antagonizes c-myc function, thereby inhibiting proliferation. Mad1 competes with myc for max binding, forming the mad1:max heterodimer, which has similar DNA sequence specicity to myc:max but suppresses (instead of activating) transcription of genes required for S-phase progression. High levels of mad1 are often associated with differentiation. Function c-myc controls G1/S transition by activating cyclin:CDK complexes and inducing transcription of genes required for S phase. Expression of c-myc is induced by a number of growth factors and cytokines and is down-regulated by antiproliferative agents such as IFN-. IFN- regulates c-myc expression by Rb-dependent down-regulation of E2F activity, as well as Rb-independent pathways that may be a result of PKR action. c-myc is active when associated with max. c-myc activity is antagonized by mad1.

[155, 258] [259262]

Gene/protein downregulated by IFN- c-myc

Refs. [263] [264] [154] [251] [265] [259]

Apoptotic Effect Gene/protein up-regulated by IFN- IRF-1 Caspase 1 (IL-1-converting enzyme) PKR DAPs Cathepsin D Fas/Fas ligand TNF- receptor Function IRF-1 is a tumor-suppressor gene required for the induction of apoptosis by signals such as DNA damage. Many of the proapoptotic effects of IRF-1 are mediated by the IRF-1-induced caspase 1. Caspase-1 is a cysteine protease involved in the generation of bioactive IL-1 and IL-18 and implicated in mediating macrophage apoptosis. PKR is an antiviral enzyme that appears to mediate TNF--induced apoptosis. Mechanisms by which PKR affects apoptosis are still unclear but may involve induction of Fas. The death-associated proteins (DAP15) are mediators of IFN--induced apoptosis by poorly dened mechanisms. Mediates IFN-, Fas/apolipoprotein, and IFN--induced cell death by poorly dened mechanisms. IFN- may increase cellular sensitivity to apoptosis by up-regulating expression of Fas and Fas ligand. IFN- may promote cellular sensitivity to the proapoptotic effects of TNF- by promoting surface expression of a TNF- receptor on tumor cells. Refs. [165, 266, 267] [267269] [267269] [270272] [273276] [277] [278, 279] [280]

Activation of Microbial Effector Functions Production of NO intermediates Gene/protein up-regulated by IFN- iNOS/NOS2 Function The NOS enzymes (NOS1, iNOS, NOS3) catalyze the reduced nictonimide adenine dinucleotide phosphate (NADPH)-dependent conversion of L-arginine to L-citrulline, forming NO as a by-product. Of these, iNOS is the only isoform inducible by cytokine and/or microbial stimulus. Argininosuccinate synthetase produces the L-arginine substrate required for the NO-liberating reaction catalyzed by iNOS. Guanosine 5-triphosphate (GTP)-cyclohydroxylase I supplies the tetrahydrobiopterin cofactor required for NO production by iNOS. Refs. [281]

Argininosuccinate synthetase GTP-cyclohydroxylase I

[282284] [285, 286]

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TABLE 1. Production of reactive oxygen species (ROS) Gene/protein up-regulated by IFN- gp91
phox

(Continued)

Function gp91 is a subunit of the NADPH oxidase, which associates with gp22 , gp47phox, and gp67phox to form the active complex capable of the generation of ROS during the respiratory burst. gp67phox is a subunit of the NADPH oxidase found in the cytosol in resting cells. Upon cell activation, it translocates to the phagosome membrane and associates with gp22phox, gp47phox, and gp91phox to form the active complex capable of the generation of ROS during the respiratory burst.
phox phox

Refs. [287, 288] [289]

gp67phox

Other antimicrobial mechanisms Gene/protein up-regulated by IFN- NRAMP1 FcRI C2, C4, Factor B Complement receptor CR3 (Mac-1) Function The natural resistance-associated macrophage protein (NRAMP1) confers resistance to macrophage intracellular pathogens by largely unknown mechanisms. Expression of the high-afnity Fc receptor (FcRI) is increased in myeloid cells by IFN- stimulation. FcRI binds extracellular pathogens via IgG in the adaptive phase of the immune response. Complement proteins are secreted by macrophages and broblasts in response to IFN-. Complement functions to opsonize extracellular pathogen for receptor-mediated phagocytosis by mononuclear phagocytes. Complement receptors of mononuclear phagocytes are up-regulated by IFN- to promote receptor-mediated phagocytosis of opsonized extracellular pathogens. Refs. [4, 290] [291] [292294] [295]

Immunomodulation, th Development, and Leukocyte Trafcking Gene/protein up-regulated by IFN- IL-12 IFN-inducible protein 10 (IP-10; CXCL10) Monocyte chemoattractant protein-1 (MCP)-1/JE (CCL2) MIG (CXCL9) MIP-1, MIP-1 (CCL3, CCL4) Regulated on activation, normal T expressed and secreted (RANTES; CCL5) ICAM-1 Vascular cell adhesion molecule-1 (VCAM-1) B7.2 Function NK cell activator and differentiation factor driving CD4 cell development to a Th1 phenotype. Chemoattractant for monocytes and T cells. Chemoattractant for monocyte/macrophages. Chemoattractant for T cells. Chemoattractant for CD4, CD8, and memory T cells. Chemoattractant for memory CD4 T cells and monocyte/macrophages. Adhesion molecule-binding to lymphocyte function-associated antigen-1 and Mac1. Adhesion molecule-binding to very late antigen-4. Surface molecule on APCs that provide costimulus for antigen-specic T cell activation. Refs. [296, 297] [155, 298] [299] [155] [155, 300] [301] [155] [155, 302] [303] [304] [305, 306]

sins B, H, and L, lysosomal proteases implicated in production of antigenic peptides for class II MHC loading [240, 241]; and DM, a regulator of peptide accessibility to the peptide-binding cleft of class II MHC [235, 237]. The CIITA mediates coordinate transcriptional control over these genes. Targeted disruption of CIITA in mice causes complete loss of constitutive or inducible display of class II MHC molecules [311]. Likewise, loss of constitutive/inducible class II MHC display is found in humans with CIITA mutation (Bare Lymphocyte Syndrome) [312314]. As CIITA is the limiting factor in a complex that directs transcriptional regulation, it acts as a switch for rapid up-regulation of class II MHC-related genes by IFN- [315].

IFN- and development of Th1 response

IFN- is a major product of Th1 cells and further skews the immune response toward a Th1 phenotype. IFN- achieves this by promoting characteristic Th1 effector mechanisms: innate cell-mediated immunity (via activation of NK cell effector functions), specic cytotoxic immunity (via T cell:APC interactions), and macrophage activation (discussed below) [4]. IFN--induced, specic cytotoxic immunity is promoted by direct and indirect mechanisms. IFN- promotes specic cytotoxic immunity by indirect mechanisms, such as growth inhibition of Th2 populations and up-regulation of antigen processing, presentation, and APC costimulatory molecules,
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thereby increasing CD4 differentiation. IFN- also inuences na ve CD4 cell differentiation toward a Th1 phenotype more directly. The phenotype adopted by a naive T cell during T cell activation is heavily inuenced by the cytokine milieu present at the time of T cell receptor engagement. IFN- and IL-12 are the prototypic cytokines directing Th1 differentiation during the primary response to antigen, and IL-4 directs differentiation of Th2 populations. IFN- induces IL-12 production in phagocytes [296] and inhibits IL-4 secretion by Th2 populations [39], which may further drive Th1 differentiation in vivo. IL-12 and IFN- coordinate the link between pathogen recognition by innate immune cells and the induction of specic immunity, by mediating a positive feedback loop to amplify the Th1 response [4]. LPS and other pathogen-associated molecular patterns directly trigger IL-12 production upon recognition by macrophages, DCs, and neutrophils [316 319], which in turn induces IFN- secretion in antigen-stimulated, naive CD4 T cells and NK cells [320, 321]. IL-12-induced IFN- participates in positive feedback by further promoting IL-12 production in macrophages [296, 297]. This amplication may be important in initiation or stabilization of the Th1 response (reviewed in refs. [4, 322]). IL-18 is highly synergistic with IL-12 for IFN- production and has important functions in the in vivo Th response [23, 24]. Secretion of large amounts of IFN- or IL-4 is the dening feature of Th1 or Th2 cells, respectively [323]. These cytokines function to directly promote cell-mediated immunity (IFN-) or humoral immunity (IL-4) and to reciprocally antagonize each others actions, further polarizing the response (reviewed in ref. [108]). For example, IL-4 inhibits IFN--dependent activation of macrophage-effector functions [324, 325]. Conversely, IFN- antagonizes IL-4-dependent induction of the IgE receptor, FcRII [326].

chinery. Phosphorylation by PKR prevents recycling of eIF-2 GTP from its guanosine 5-diphosphate-bound form, thereby inhibiting viral and cellular protein synthesis [242]. PKR is implicated in numerous other functions, including activation of NF-B [243, 334], TNF- transcript splice regulation [244], induction of fas-mediated apoptosis [245, 246], and regulation of Stat1 activity [154, 247]. It is likely that IFNs induce many as-yet undiscovered antiviral proteins, as the enzymes above and described in Table 1 do not account for all IFN-induced antiviral effects [335, 336]. Also, the host of factors mediating the profound growth-inhibitory and proapoptotic effects of IFN- would have no small part in limiting viral replication within the cell and spread to other cells.

Cell cycle, growth, and apoptosis

Macrophages are produced in large amounts by the bone marrow, and many die shortly after production by a process of programmed cell death (apoptosis) [337]. After release from the bone marrow, macrophages proliferate, differentiate, acquire specialized functions (e.g., microbicidal activity), or die by apoptosis depending on the presence or absence of extracellular cues. Many reports indicate that IFN- arrests the macrophage cell cycle and provides a survival signal, and others suggest that it serves as a proapoptotic signal. Although recent advances in cell cycle control and apoptosis have greatly increased our understanding in these functions in isolation, the inter-relationship between these intimately related processes is still somewhat confusing. For this reason, the effects of IFN- on cellular proliferation and apoptosis are described here separately. One of the most easily observed effects of IFN- is cell growth inhibition. Types I and II IFN are able to protect against pathogen-induced apoptosis and suppress colony stimulating factor type 1 (CSF-1)-dependent growth of bone marrow-derived macrophages (BMM) [252]. Granulocyte macrophageCSF, which is usually released by T cells at the same time as IFN-, is able to protect against the growth-inhibitory effects of IFN- on macrophages, and this may be physiologically important in vivo [338]. The IFNs most commonly arrest the cell cycle at the G1/S checkpoint, although blockages of other cell cycle stages have been reported [339 341]. It has been suggested that IFN-induced G1 arrest may actually reect exit from the cell cycle into G0 [342]. IFNs inhibit proliferation primarily by increasing protein levels of Ink4 and Cip/Kip CKIs [252255, 343, 344]. IFN- transcriptionally induces p21 and p27 CKIs (Table 1) [256, 345, 346]. p21 and p27 inhibit the activity of cyclin E:CDK2 and cyclin D:CDK4 complexes, respectively, thereby arresting the cell cycle at the G1/S boundary. Decreased cyclin:CDK activity during G1 results in hypophosphorylation of the tumor suppressor Rb. In its hypophosphorylated state, Rb sequesters the E2F family of transcription factors from activation of genes (e.g., c-myc) required for cell cycle progression from G1 to S phase [292]. c-myc controls G1/S transition by activating cyclin:CDK complexes and inducing transcription of genes required for S phase (reviewed in ref. [263]). Expression of c-myc is induced rapidly by a number of growth factors and cytokines and is down-regulated by IFN- [154, 264]. IFN- inuences
http://www.jleukbio.org

The IFN-induced antiviral state

The IFN system regulates innate and adaptive immunity to viral infection. Viral invasion directly triggers induction of type I IFNs, through a mechanism involving IRF-3 and IRF-7 (reviewed in ref. [327]). Although both types of IFN are crucial in the immediate cellular response to viral infection, the immunomodulatory activities of IFN- (discussed elsewhere) become important later in the response in coordinating the immune response and establishing an antiviral state for longer term control [54, 60, 328, 329]. IFN--induced antiviral mechanisms include induction key antiviral enzymes (Table 1), most notably PKR, which is a serine/threonine kinase greatly induced by types I and II IFN stimulation [242, 330]. PKR is inactive in its constitutive form and requires an activating signal for autophosphorylation. dsRNA, a necessary intermediate in replication of RNA viruses, is the best characterized activator of PKR, although other agents such as heparin are able to activate PKR [331]. Association of PKR with dsRNA is likely to cause a conformational change that unmasks the catalytic domain responsible for PKR autophosphorylation [242, 332]. PKR is then activated for dsRNA-independent phosphorylation of specic cellular substrates [333]. One of these substrates is the eIF-2 subunit, a rate-limiting factor in the normal cellular translational ma174 Journal of Leukocyte Biology Volume 75, February 2004

c-myc expression through multiple pathways, including suppression of Rb phosphorylation, resulting in decreased E2F activity as well as Rb-independent pathways, which may be a result of PKR action [251]. In its active form, c-myc is associated with max, and the myc:max heterodimer activates transcription of genes required for cell cycle progression [265]. The level of active myc is negatively regulated by mad1, which sequesters the max coactivator away from myc and suppresses transcription of myc-inducible genes [259]. In this way, the mad1-to-c-myc ratio determines whether a cell will proliferate (as a result of high levels of myc:max) or arrest in G1 (as a result of high levels of mad1:max) [259, 260]. In addition to decreasing the abundance of myc, IFN- increases mad1 levels, thereby further antagonizing myc activity and inhibiting CSF-1-dependent proliferation in BMM [347]. Apoptosis is a process in which the cell directs its own demise by activating intrinsic suicide machinery (reviewed in refs. [337, 348]). In macrophages, apoptosis may be desirable to prevent colonization by intracellular pathogens. Conversely, many pathogens actually induce host-cell death through secretion of macrophage-specic toxins. Induction of apoptosis by signals such as DNA damage requires the IRF-1 tumor-suppressor gene [165, 266, 267]. Levels of IRF-1 may be a deciding factor in whether IFN- induces or protects from apoptosis on treated cells [35, 349, 350]. It is proposed that IFN- treatment of cells with high levels of functional IFNGR very rapidly activates Stat1, thereby producing high levels of IRF-1 that are able to induce apoptosis. In contrast, IFN- treatment of cells with low levels of functional IFNGR may activate Stat1 more slowly, thereby producing lower levels of IRF-1 that are not sufcient to induce apoptosis [35]. Experiments in which overexpression of functional IFNGR on normally low-level IFNGR-expressing cells changed the IFN- response of these cells from an antiapoptotic/proliferative phenotype to a proapoptotic phenotype are consistent with this hypothesis [35]. This may also explain why myeloid cells are more sensitive to the proapoptotic actions of IFN- than other cells such as T cells, as myeloid cells express relatively high numbers of functional IFNGR on the cell surface [35]. Many of the proapoptotic effects of IRF-1 are mediated by the IRF-1-induced caspase 1 (IL-1-converting enzyme) [267 269]. Caspase 1 is a cysteine protease implicated in mediating macrophage apoptosis by various stimuli including LPS [351 353] and is involved in generation of bioactive IL-1 and IL-18 [354]. Caspase 1 expression and resulting apoptosis can be IFN--inducible [269] or can occur through IFN--independent IRF-1 activity [267]. IFN- also induces a number of other proapoptotic molecules. These include PKR, the DAPs, cathepsin D, and surface expression of Fas and the TNF- receptor (Table 1).

Activation of microbicidal effector functions

One of the most important effects of IFN- on macrophages is the activation of microbicidal effector functions. Macrophages activated by IFN- display increased pinocytosis and receptormediated phagocytosis as well as enhanced microbial killing ability. IFN--activated microbicidal ability includes induction of the NADPH-dependent phagocyte oxidase (NADPH oxidase) system (respiratory burst), priming for NO produc-

tion, tryptophan depletion, and up-regulation of lysosomal enzymes promoting microbe destruction (Table 1) [222]. Similar microbicidal mechanisms are activated by IFN- in neutrophils [4]. Macrophages kill bacteria, viruses, protozoa, helminths, fungi, and tumor cells primarily by production of ROS and reactive nitrogen intermediates (RNI) via induction of the NADPH oxidase system and iNOS, respectively (reviewed in refs. [281, 355, 356]). ROS and RNI use the advantages of low molecular weight, reactivity, and lipophilicity to easily penetrate the microbial cell wall/coat to inict injury. The importance of these molecules in host defense is highlighted by the highly pathogen-susceptible phenotype of mice doubly decient in NADPH oxidase and iNOS enzymes [357]. The timeframes and situations in which macrophage use these cytocidal activities are generally different. ROS production becomes apparent within 1 h after stimulus, whereas RNI generation requires 24 h after encountering a pathogen product. In general, ROS are used to target extracellular pathogens during phagocytosis or that are too large for phagocytosis, whereas RNI target intracellular pathogens and upon appropriate stimulus, extracellular pathogens and tumor cells. NO is produced in the NADPH-dependent conversion of L-arginine to L-citrulline by the NOS enzymes (NOS13). The NOS2/iNOS isoform alone is inducible by cytokine and/or microbial stimulus [281]. The NO generated by this enzyme exists in equilibrium among a number of different redox states (called RNI in this review) that have differing biological activities. It should be noted that much of the work presented below applies to mice rather than humans, and there is at present much controversy of the physiological relevance of NO production in human host defense (reviewed in ref. [281]). A wealth of evidence supports a crucial function of iNOS and RNI in host defense (reviewed in ref. [281]). IFN-dependent RNI production is associated with increased ability of phagocytic cells to kill ingested pathogens, and this ability is inhibited by the addition of a substrate analog inhibitor of iNOS such as N-methyl-L-arginine [358 361]. Furthermore, mice in which the iNOS gene has been mutated show greater susceptibility to parasitic infection and a dampened, nonspecic inammatory response [362]. Production of RNI also inhibits replication of a range of viruses, through ill-dened mechanisms [358, 363, 364]. IFN- induces RNI production by up-regulating expression of substrate, cofactor, and catalyst required for NO generation. IFN- up-regulates argininosuccinate synthetase (which produces the L-arginine substrate), GTP-cyclohydroxylase I (which supplies the tetrahydrobiopterin cofactor required for NO production), and the iNOS enzyme [285, 286, 365367]. Maximal induction of iNOS transcription requires priming and triggering stimuli such as priming with IFN- and subsequent triggering with LPS or TNF- [368, 369]. The type I IFNs cannot fully substitute for IFN- in triggering NO production [368]. Superoxide and its reactive products are also important toxic effector molecules of the nonspecic cytotoxic response. The superoxide anion, O2, is generated by the multicomponent avocytochrome enzyme phagocyte oxidase during a process called respiratory burst. This enzyme is composed of two

Schroder et al. Signals, mechanisms, and functions of IFN- 175

cytochrome b558 subunits, gp91phox and gp22phox, concentrated in the phagosome membrane, and two cytosolic components, p47phox and p67phox. Upon appropriate stimulus (e.g., phagocytosis), the cytosolic components translocate to the membrane to form the active complex, which generates superoxide in the phagosome through transfer of a transported electron to molecular oxygen. The superoxide anion generated by the respiratory burst spontaneously reacts to form hydrogen peroxide (H2O2), hydroxyl radicals (OH), and hypochlorous acid (HOCl) [281]. The toxic oxidants produced by the respiratory burst are also able to react with those produced by iNOS, thereby forming a large number of different toxic species (e.g., peroxynitrite) to mediate cytotoxicity by a wide variety of mechanisms [281, 370, 371]. The primary mechanism of IFN--induced up-regulation of ROS production in phagocytes is transcriptional induction of the gp91phox and p67phox subunits of the NADPH oxidase complex [287289, 372]. The biological importance of this pathway in host defense is emphasized by the human genetic disease, chronic granulomatous disease, caused by defects in the NADPH oxidase system and characterized by recurrent and often fatal infections. IFN- also promotes microbe destruction by augmenting surface expression of the high-afnity FcRI on mononuclear phagocytes, thereby promoting antibody-dependent, cell-mediated cytotoxicity [291]. Complement-mediated phagocytosis is also up-regulated by IFN- through increased complement secretion and complement receptor surface expression on mononuclear phagocytes [292]. Tryptophan depletion from cells in response to IFN- may be an antiparasitic mechanism in humans [373]. IFN- upregulates the expression of indoeamine 2,3 dioxygenase, an enzyme responsible for the generation of N-formylkynurenine from tryptophan [374 377]. It is interesting that IFN- also induces expression of tryptophanyl-tRNA synthetase, a housekeeping gene that catalyzes tRNA Trp aminoacetylation during protein synthesis [378, 379]. It is proposed that this enzyme may also aid in tryptophan depletion by incorporating free tryptophan into translating protein and may ensure synthesis of Trp-rich proteins with immunologically important functions (e.g., the IRF family and many MHC molecules) during cellular tryptophan depletion [380].

chemokine milieu to extravasate into the tissue via interactions between adhesion molecules presented on leukocyte and endothelial surfaces (diapedesis) [4]. IFN- regulates this process by up-regulating expression of chemokines (e.g., IP-10, MCP-1, MIG, MIP-1/, RANTES; see Table 1) and adhesion molecules (e.g., ICAM-1, VCAM-1; see Table 1). TNF- and IL-1 synergistically regulate many of these molecules [4].

IFN- priming of the macrophage LPS response

LPS/endotoxin is a cell wall constituent of gram-negative bacteria that activates macrophage microbicidal effector functions and the production of proinammatory cytokines (e.g., TNF-, IL-1, IL-6). LPS:macrophage interaction can be benecial or deleterious to the host. Macrophages sense and are activated to ght and clear bacterial infection through LPS recognition, a mechanism obviously advantageous to the host; however, high doses of LPS that trigger excessive production of inammatory mediators can result in the potentially lethal systemic disorder, septic shock. Macrophages recognition of lipid A (the active moiety of LPS), requires Toll-like receptor (TLR) family member TLR4. The TLR family is a homolog of the Drosophila antifungal protein Toll. To date, 10 mammalian TLRs have been identied (TLR110), and many have proposed or demonstrated functions in innate immunity. LPS:TLR4 interaction triggers a signaling cascade (Fig. 3), which ultimately results in activation of NF-B and activated protein-1 (AP-1) and transcriptional control over genes directing immune function in macrophages (reviewed in refs. [383386]). MyD88 functions as an adaptor to connect the intracellular portion of TLR4 to downstream signaling components and is required for many, but not all, LPS-induced effects [387]. For this reason, a TLR4 signaling paradigm has emerged in which LPS signals through MyD88-dependent and MyD88-independent pathways. MyD88 gene KO mice demonstrated that the MyD88-dependent pathway is necessary for rapid activation of MAPK, AP-1, and NF-B, the production of inammatory cytokines, and septic shock [387]. The MyD88-independent pathway results in slower activation of MAPK, AP-1, and NF-B and does not contribute to the same extent to the inammatory response. IFN- primes macrophages for more rapid and heightened responses to LPS [58, 388, 389] as well as other TLR agonists, such as unmethylated CpG motifs present in bacterial DNA (CpG DNA) [390]. Pretreatment with IFN- is necessary for the induction of some genes in response to LPS. iNOS is the archetypal example of such a gene, although the need for exogenous priming by IFN- can be overcome by adequate production of endogenous type I IFN in response to high doses of LPS [391]. For other genes, IFN- is able to shift the dose-response curve such that transcription is induced at lower concentrations of LPS, thereby superinducing transcription in IFN--primed cells treated with LPS compared with the same concentration of LPS alone [392]. IFN- priming does not occur for all LPS-induced responses; IFN- appears to prime for a select subset of LPS-induced genes. For example, IFN- pretreatment promotes LPS-induced TNF- but not procoagulant activity enzyme production [393]. IFN- receptor KO mice are highly resistant to LPS-induced toxicity [394]. This highlights the physiological signicance of
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Immunomodulation and leukocyte trafcking

The ability of IFN- to coordinate the transition from innate immunity to adaptive immunity distinguishes it from the other IFNs. Mechanisms by which IFN- coordinates this transition include aiding in the development of a Th1-type response (described earlier), directly promoting B cell isotype switching to IgG2a [54, 381, 382], and regulation of local leukocyteendothelial interactions (Table 1). IFN- orchestrates the trafcking of specic immune cells to sites of inammation through up-regulating expression of adhesion molecules and chemokines. Unstimulated leukocytes cycle continuously between the blood and the lymph. IFN- and NO produced at the site of inammation cause local dilation of the blood vessels, thereby decreasing the local blood ow rate and causing gathering of blood in leaky vessels. Specic leukocyte subsets are instructed by the cytokine/
176 Journal of Leukocyte Biology Volume 75, February 2004

LBP

LPS CD14

MD-2

TLR4


Cytosol

MyD88

MyD88-dependent pathway

IRAK TRAF6 NIK IKK

TRIF?

MyD88-independent pathway

!
p38 MAPK JNK/SAPK ERK

IRF-3

Rapid activation of NFB and AP-1

Slow activation of NFB and AP-1

"
NF-B AP-1

#
Transcription of IFN/

Nucleus

Transcription of target genes

eg. IL-1, IL-6, TNF

Transcription of target genes

eg. GARG16, IRG-1, IP-10

Fig. 3. The current paradigm for optimal LPS signal transduction through TLR4. The LPS:LPS-binding protein (LBP) complex binds to CD14. The LPS:LBP:CD14 complex probably activates TLR4, and optimal LPS-induced signaling requires the association of the MD-2 accessory component with the TLR4 extracellular domain. Downstream signaling is dependent on sequential recruitment and activation of signaling molecules. In the MyD88-dependent pathway, the signaling adaptor MyD88 is recruited to the receptor, and the signal is relayed via sequential recruitment and activation of the serine/threonine kinase IL-1R-associated kinase (IRAK), TNF receptor-associated factor (TRAF)6, NF-B-inducing kinase (NIK), and IB kinase for the rapid activation of NF-B and AP-1 (IKK). MyD88 adaptor-like/Toll/IL-1 receptor/resistance (TIR) domain-containing adaptor protein also contributes to the MyD88-dependent pathway, through ill-dened mechanisms. The MyD88-independent pathway is less well characterized but involves activation of IRF-3 and more slowly activates NF-B and AP-1. It has recently been suggested that TIR domain-containing adaptor-inducing IFN- (TRIF) participates in the MyD88-independent pathway upstream of IRF-3. Activation of the MyD88-dependent and -independent pathways also activates the mitogen-activated protein kinase (MAPK) pathways, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), p38 MAPK, and extracellular-regulated kinase (ERK) pathways. The MyD88-dependent pathway ultimately controls transcription of target genes, including many of the proinammatory cytokines implicated in the pathophysiology of septic shock. Genes regulated by the MyD88-independent pathway, however, do not contribute to septic shock to the same extent. Possible mechanisms giving rise to the priming effect apparent when LPS-stimulated macrophages are pretreated with IFN- include (numbered on the gure): (1) IFN- up-regulates expression of the TLR4 and MD-2 components of the LPS recognition complex; (2) IFN- upregulates expression of MyD88 and IRAK; (3) LPS activates Stat1, possibly through the MAPK pathway, thus augmenting the IFN- response; (4) IFN- promotes LPS-induced NF-B activation; (5) LPS induces type I IFN, which may participate in cross-talk with the IFN- signaling pathway to augment the macrophage IFN- response; and (6) LPS/TNF-- and IFN--activated/induced signaling molecules can often bind to promoter regions of target genes to synergistically regulate transcription. GARG, Glucocorticoid attenuated response gene; IRG, immune-responsive gene.

IFN- priming in vivo, as it demonstrates that IFN- is normally produced during the response to LPS and acts to amplify LPS-induced cellular responses. The priming phenomenon relies on IFN- and LPS reciprocal cross-regulation of signaling molecules of the other pathway. IFN- inuences LPS-dependent signaling capabilities by promoting ligand-receptor interactions as well as downstream signaling machinery. Efcient LPS:TLR4 interaction requires the MD-2 and CD14 accessory molecules, and subsequent maximal signaling requires MyD88 signaling adaptor. In a number of experiments, IFN- was shown to promote transcription of TLR4, subsequent TLR4 surface expression, and LPSbinding ability in macrophages [395397]. Furthermore, IFN-

inhibits the LPS-dependent down-regulation of TLR4 surface expression apparent in unprimed cells [395]. IFN- stimulation may also promote LPS-dependent signaling by promoting expression of the MD-2 accessory molecule, the MyD88 adaptor, and the IRAK signaling molecule [395, 398]. LPS mediates many of its effects through the NF-B transcription factor. In the macrophage-like cell line RAW264.7, it was found that IFN- pretreatment promoted NF-B activation upon LPS exposure, as well as more rapid DNA-binding kinetics and faster degradation of the NF-B inhibitor, IB- [399]. In human monocytes also, IFN- pretreatment causes superinduction of active NF-B upon LPS treatment [400]. When these cells were unstimulated, they exhibited high levels

Schroder et al. Signals, mechanisms, and functions of IFN- 177

of the p50 subunit of NF-B but only low levels of p65. IFN- priming caused an increase in p65 mRNA as a result of increased transcript stability. Many IFN--inducible genes are also TNF--inducible, and these genes are often superinduced by the combination of these factors [366, 372, 401, 402]. TNF- is a macrophage-derived cytokine secreted in response to LPS, which can act in an autocrine manner to mediate many LPS-induced effects via NF-B [403]. IFN- priming of TNF- responses is thus responsible for the priming of a subset of LPS-induced genes. In many cases, synergistic induction by the combination of these factors may be a result of the combined presence of Stat1 and NF-B-binding sites in the promoter elements of responsive genes [160, 404]. Synergy may be also be a result of cross-talk between the IFN- and TNF- signaling pathways: TNF- stimulation is able to induce transcription of IRF-1 and promotes IFN--induced Stat1 activation [405, 406]; IFN-induced PKR is able to signal to NF-B [407]; and IFN- increases surface expression of the TNF- receptor [280, 408, 409], although the signicance of this is uncertain, as an increased receptor expression level has not been linked with increased response to TNF- [410, 411]. Although probably not complete, these mechanisms of increasing cellular sensitivity to LPS may start to explain the shift in the LPS dose-response curve seen with IFN- priming. Genes that need IFN- and LPS treatment for induction may require a certain threshold of LPS signaling for transactivation, and so IFN- treatment shifts the LPS dose-response curve by increasing cellular sensitivity. Alternatively, these genes may require specic cross-talk between pathways before transcriptional activation can occur. Examples of such cross-talk may include LPS-induced Stat1 serine phosphorylation or crosstalk between IFN-- and LPS-induced type I IFN pathways. LPS is able to promote the IFN- signaling pathway through Stat1. As noted earlier, phosphorylation of S727 of Stat1 occurs in response to IFN- or LPS exposure and is necessary for maximal Stat1 activation. LPS treatment following IFN- exposure increases the percentage of molecules phosphorylated on Y701 and S727 and subsequent Stat1 DNA-binding activity relative to IFN- treatment alone, thereby augmenting IFN-dependent, Stat1-mediated gene expression [123, 223]. IFN- and LPS may use different pathways for Stat1 serine phosphorylation, as LPS-induced Stat1 serine phosphorylation is sensitive to a p38 MAPK inhibitor, whereas the serine phosphorylation induced by IFN- is not [110]. Another study suggested that although p38 MAPK is necessary, it is not sufcient for Stat1 serine phosphorylation by LPS, and protein kinase C- is also required to mediate phosphorylation [412]. In addition to promoting serine phosphorylation, LPS treatment following IFN- exposure augments Stat1 tyrosine phosphorylation of residue 701 [399]. Macrophage exposure to LPS triggers production of endogenous type I IFN that can act on the macrophage population in an autocrine/paracrine manner. This has been identied as the causal agent of LPS-induced Stat1 tyrosine phosphorylation [413]. It has long been recognized that LPS induces transcription of type I IFN [414], but only in recent years has the signicance of autocrine/paracrine functions of type I IFN in mediating LPS-induced gene expression been acknowledged [391, 415,
178 Journal of Leukocyte Biology Volume 75, February 2004

416]. Stat1 is tyrosine-phosphorylated as a result of autocrine/ paracrine type I IFN and is required for the full LPS response in IFN--unprimed macrophages [416]. LPS-induced Stat1 Y701 phosphorylation was found to be essential for iNOS expression [413], and macrophages from mice unable to respond to type I IFN through targeted disruption of the IFNAR1 receptor subunit exhibited limited capacity to induce iNOS in response to LPS [391]. iNOS-producing capacity in response to LPS was able to be rescued by exposure to IFN-, which presumably restored Stat1 activation [391]. LPS induces transcription of type I IFN through an ill-dened, MyD88-independent pathway involving IRF-3 [417]. The contribution of type I IFN to IFN- priming of the LPS response has not been formally studied; however, as IFN- up-regulates expression of IFNAR in other cell types (P. J. Hertzog, unpublished observations) and promotes IFN-/ expression, it is possible that IFN- sensitizes the cells to LPS-induced IFN-/. When the many levels of cross-talk between the IFN- and IFN-/ pathway are considered, it is likely that in vivo IFN- potentiates LPS-induced IFN-/ effects and conversely, LPS augments IFN--mediated effects (e.g., by Stat1 Y701 and S727 phosphorylation). Synergy between LPS- and IFN--induced transcription factors in expression of target genes also contributes to the priming phenotype. Genes such as IRF-1, IP-10, ICAM-1, and iNOS contain Stat1 and NF-B-binding sites in their promoter, and maximal transcription requires both signals [158, 160, 402, 404, 418 423]. This is a demonstrated mechanism in IFN--dependent gene superinduction in response to LPS for a number of genes and is likely to be a global mechanism for synergistic, coordinate regulation of large synexpression groups. Genes that require IFN- and LPS treatment for induction (e.g., iNOS) may also be explained by this mechanism, as it is likely that the IFN-- and LPS-induced transcription factors are limiting for gene induction at a single-cell level. This theory is consistent with the all-or-nothing transcription of iNOS and many other IFN-/LPS-regulated genes observed at a single-cell level (ref. [424] and references therein).

CONCLUDING REMARKS
IFN- is a remarkable cytokine that orchestrates many distinct cellular programs through transcriptional control over large numbers of genes. Many IFN--induced effects resulting in heightened immune surveillance and immune system function during infection have been discussed in this review. As pathogen products such as LPS and CpG DNA augment local IFN- production, and IFN- augments the immune system response to these agonists, an important function of IFN- during in vivo infection is suggested, whereby IFN- participates in an amplication loop to increase immune system sensitivity and response to pathogens. This model is supported by the in vivo and in vitro priming phenotype apparent when LPS-stimulated macrophages are pretreated with IFN-, and also by the high resistance of IFNGR/ mice to LPS-induced septic shock. The ability of IFN- to synergize or antagonize the effects of cytokines, growth factors, and pathogen-associated molecular pattern (PAMP)-signaling pathways (e.g., TNF-, IL-4, CSF-1,
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IFN-/, LPS, and CpG DNA) is particularly important in macrophage biology, as macrophages constantly receive multiple signals and need to integrate them to give a response appropriate to the extracellular milieu. The reductionist approach used by many scientists has greatly furthered our understanding of mechanisms by which IFN- coordinates its pleiotropic effects. However, a cell is not exposed to one stimulus in isolation in vivo, and so, one of the great challenges to IFN biologists today is to understand the mechanisms by which IFN- signaling and cellular effects integrate with other growth factor, cytokine, and PAMP signaling pathways. This understanding will enable a more realistic view of in vivo macrophage function during naturally occurring human infection.

17. 18.

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ACKNOWLEDGMENT
The authors thank Katharine Irvine for critical reading of the manuscript and helpful suggestions.

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