Interferon Gamma

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
Inflammatory 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 influence macrophage function during infection. The current model for IFN-
signal transduction is discussed, as well as signal
regulation and factors conferring signal specificity.
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 trafficking. 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 inflammation
INTRODUCTION
This review focuses on the interplay between interferon-
(IFN-) and macrophages in inflammation 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 defined as acquisition of competence to execute a complex function [1], is among the first
actions to occur in innate immunity to potential pathogens. In
most situations, macrophages are activated to acquire microbicidal effector functions and secrete proinflammatory cytokines, resulting in inflammation and recruitment of immune
cells and subsequent elimination of the microbe by phagocytosis or release of toxic metabolites.
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 classified by the
secreting cell type but are now classified into type I and type
II according to receptor specificity 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 fibroblasts 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
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.
Journal of Leukocyte Biology Volume 75, February 2004 163
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-inflammatory protein-1 (MIP1); ref. 25]. These chemokines attract NK cells to the site of
inflammation, 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 significantly 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
164
Journal of Leukocyte Biology Volume 75, February 2004
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 specificity 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-specific. 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 specificity 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 deficiencies 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].
http://www.jleukbio.org
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 efficient
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 deficiency 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 deficient
production of IFN- exhibited decreased neutrophil mobility
and NK cell activity, highlighting the importance of IFN- in
the inflammatory 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 inflammatory status caused by an IFN-
polymorphism, while not enough to significantly impact on the
individuals ability to clear infection, may prevent or defer
inflammation-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 significant 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 specific 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 first 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 verified that
the SH2 domain necessary for dimerization is also involved in
Schroder et al. Signals, mechanisms, and functions of IFN- 165
ST
AT
STA
1
T1
Cytosol
JAK2
JAK1
JAK1
JAK2
JAK2
JAK1
JAK1
JAK2
IFN
STAT1
STAT1
IRF-9
STAT2
STAT1
STA
T1
STA
T1
IRF-1
IRF-1
IRF-9
eg. ICAM-1
MIG
eg. IRF-2
iNOS
IFN
GAS
IFN-regulated gene
ISRE
IFN-regulated gene
STAT1
STAT1
IRF-1
P
IRF-E/GAS/IRF-E
GAS
IRF-1
Stat1
ST
AT
1
STAT1
STAT1
Nucleus
IRF-1
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 efficient
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:
166
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
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 sufficient 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, specificity
protein-1, and heat shock factor-1 have been reported (reviewed in ref. [119]) and are likely to influence specificity 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
insufficient 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 fluorescent 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 efficient 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 identified for Stat1, indicating an as-yet unidentified, 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 affinity 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 fibroblasts 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 significantly 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
126
to bacterial infection, viral infections took the normal clinical

course. This suggests that IFN--induced, physiologically significant, 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 specificity 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,
168
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 specific
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 inflammatory 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 specificity
The many cytokines that use Jak-Stat signaling direct transcription through specific but not necessarily unique combinations of Jaks and Stats. This raises the question of how signal
specificity is maintained when many cytokines use the same
signaling machinery.
Evidence for a devoted role of Jaks in signal transduction of
specific cytokines is controversial. Mutant cell lines lacking
Jak1 or Jak2 have demonstrated deficiency in types I and II
Receptor
expression level
SOCS-3
SOCS-1
PTPs/SOCS limit
receptor and Jak
phosphorylation
STAT1
STAT1
STA
T1
IRF-9
STAT2
STAT1
STA
T1
STA
T1
IRF-1
STA
T1
Cytosol
SHP-2
ST
AT
STA
1
T1
JAK2
JAK1
JAK1
JAK2
JAK2
JAK1
JAK1
JAK2
IFN
IRF-1
IRF-9
Nucleus
STAT1
Nuclear PTPs
dephosphorylate
Stat1 causing
inactivation and
nuclear export
STAT1
STA
T1
STA
T1
PTP
GAS
IFN-regulated gene
eg.
ISRE
IRF-1
IRF-2
IFN-regulated gene
Transcriptional
repression by IRF-2
competition for
ISRE/IRF-E sites
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 deficiencies are reflected in the Jak1 and Jak2 KO mice,
suggesting that these Jaks are functionally specific 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 deficits 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 specific range of cytokine receptors. Jak2
gene KO in mice is embryonic lethal as a result of deficiencies
in erythropoiesis [197, 198]. Other than suppression of IFN-

signaling, the cytokine response deficit 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 specific Stats appear to be dedicated to specific
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 specificity of response with respect to Stats include:
different thresholds/timeframes of Stat activation may be required for specific biological responses, and specific Stat com-
plexes induced by cytokines may give different biological

effects. It is widely agreed that signal specificity 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
specific 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-specific transcription
factors), which associate with Stats may contribute to the
specificity 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 fibroblasts 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 efficient 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 specificity of the immunoproteasome
may allow production of peptides better able to bind class I
MHC and thereby increase efficiency in the system. Peptide
diversity is thought to increase as a result of differences in
cleavage specificities 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 inflammation and thus avoid autoimmunity
without compromising appropriate T cell stimulation [216].
The efficiency 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 efficient generation of
TAP- and class I MHC-compatible peptides to increase overall
efficiency 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 efficient 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 efficient 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
Of the IFNs, IFN- alone can efficiently up-regulate the class

II antigen presenting pathway and thus promote peptide-specific 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.
IFN--Regulated Genes and Their Function in Producing IFN- Effects
Antigen Processing and Presentation

Class I antigen presentation pathway
Gene/protein up-regulated
by IFN-
LMP-2, LMP-7, MECL-1
PA28, PA28
TAP-1, TAP-2
Class I MHC heavy chain
2-microglobulin
Tapasin
Function
Refs.
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 specificity of peptides generated to increase
efficiency 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.
[210216]
[4, 217, 218, 219]

[4, 215, 220223]
[4, 224231]
[222, 230, 232]
[4, 233]
Class II antigen presentation pathway

by IFN-
1, 2, 1, 2 MHC II
chains
Ii chain
DMA, DMB
Cathepsins B, H, L
CIITA
Function
Refs.
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 traffics 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.
[234237]
Function
Refs.
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-specific 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.
[154, 242247]
[237239]
[235, 237]
[240, 241]
[234]
Antiviral Effect
by IFN-
PKR
ADAR
GBP1, GBP2
[248]
[61, 249, 250]
Schroder et al. Signals, mechanisms, and functions of IFN-
171
TABLE 1.
(Continued)
Antiproliferative Effect
by IFN-
PKR
p21, p27
Rb
p202
Mad1
Gene/protein downregulated by IFN-

c-myc
Function
Refs.
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 specificity 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.
[154, 242247]
[251]
Function
Refs.
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.
[263]
[264]
[154]
[251]
[265]
[259]
Function
Refs.
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 defined mechanisms.
Mediates IFN-, Fas/apolipoprotein, and IFN--induced cell death by poorly
defined 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.
[165, 266, 267]

[267269]
[252]
[253256]
[257]
[155, 258]
[259262]
Apoptotic Effect
by IFN-
IRF-1
Caspase 1 (IL-1-converting
enzyme)
PKR
DAPs
Cathepsin D
Fas/Fas ligand
TNF- receptor
[267269]
[270272]
[273276]
[277]
[278, 279]
[280]
Activation of Microbial Effector Functions

Production of NO intermediates
by IFN-
iNOS/NOS2
Argininosuccinate synthetase
GTP-cyclohydroxylase I
172
Function
Refs.
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.
[281]
[282284]
[285, 286]
TABLE 1.
(Continued)
Production of reactive oxygen species (ROS)

by IFN-
phox
gp91
gp67phox
Function
Refs.
phox
phox
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.
[287, 288]
[289]
Other antimicrobial mechanisms

by IFN-
NRAMP1
FcRI
C2, C4, Factor B
Complement receptor CR3
(Mac-1)
Function
Refs.
The natural resistance-associated macrophage protein (NRAMP1) confers

resistance to macrophage intracellular pathogens by largely unknown
mechanisms.
Expression of the high-affinity 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 fibroblasts 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.
[4, 290]
[291]
[292294]
[295]
Immunomodulation, th Development, and Leukocyte Trafficking

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
Refs.
NK cell activator and differentiation factor driving CD4 cell development to a

Th1 phenotype.
Chemoattractant for monocytes and T cells.
[296, 297]
Chemoattractant for monocyte/macrophages.
[299]
[155]
Chemoattractant for T cells.

Chemoattractant for CD4, CD8, and memory T cells.
[155, 300]
[301]
[155]
[155, 302]
Chemoattractant for memory CD4 T cells and monocyte/macrophages.
[155, 298]
Adhesion molecule-binding to lymphocyte function-associated antigen-1 and

Mac1.
Adhesion molecule-binding to very late antigen-4.
[303]
Surface molecule on APCs that provide costimulus for antigen-specific T cell

activation.
[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].
[304]
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), specific cytotoxic immunity (via T cell:APC interactions), and macrophage activation (discussed below) [4].
IFN--induced, specific cytotoxic immunity is promoted by
direct and indirect mechanisms. IFN- promotes specific cytotoxic immunity by indirect mechanisms, such as growth
inhibition of Th2 populations and up-regulation of antigen
processing, presentation, and APC costimulatory molecules,
Schroder et al. Signals, mechanisms, and functions of IFN-
173
thereby increasing CD4 differentiation. IFN- also influences nave CD4 cell differentiation toward a Th1 phenotype
more directly. The phenotype adopted by a naive T cell during
T cell activation is heavily influenced 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 specific 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 amplification 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 defining
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].
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 specific cellular
substrates [333]. One of these substrates is the eIF-2 subunit,
a rate-limiting factor in the normal cellular translational ma174
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 reflect 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- influences
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-specific 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 sufficient 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 inflict injury. The importance of these molecules in host defense is highlighted by the
highly pathogen-susceptible phenotype of mice doubly deficient 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, nonspecific inflammatory response [362]. Production of RNI also
inhibits replication of a range of viruses, through ill-defined
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 nonspecific cytotoxic response. The
superoxide anion, O2, is generated by the multicomponent
flavocytochrome enzyme phagocyte oxidase during a process
called respiratory burst. This enzyme is composed of two
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-affinity 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].
Immunomodulation and leukocyte trafficking

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 trafficking of specific immune cells to
sites of inflammation 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 inflammation cause local
dilation of the blood vessels, thereby decreasing the local blood
flow rate and causing gathering of blood in leaky vessels.
Specific leukocyte subsets are instructed by the cytokine/
176
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 proinflammatory cytokines (e.g., TNF-,
IL-1, IL-6). LPS:macrophage interaction can be beneficial or
deleterious to the host. Macrophages sense and are activated to
fight and clear bacterial infection through LPS recognition, a
mechanism obviously advantageous to the host; however, high
doses of LPS that trigger excessive production of inflammatory
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 identified (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 inflammatory 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 inflammatory 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 significance of
LBP
MD-2
LPS
CD14
TLR4
1
Cytosol
MyD88
MyD88-dependent
pathway
IRAK
TRAF6
IRF-3
NIK
MyD88-independent
pathway
TRIF?
p38 MAPK
JNK/SAPK
ERK
IKK
Rapid activation of
NFB and AP-1
Slow activation of
NFB and AP-1
4
NF-B
5
AP-1
Transcription of IFN/
Transcription of target genes
Transcription of target genes
eg. IL-1, IL-6, TNF
eg. GARG16, IRG-1, IP-10
Nucleus
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-defined
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 proinflammatory 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 figure): (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- influences LPS-dependent signaling capabilities by
promoting ligand-receptor interactions as well as downstream
signaling machinery. Efficient 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
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 significance 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 specific 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 sufficient 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 identified 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 significance
of autocrine/paracrine functions of type I IFN in mediating
LPS-induced gene expression been acknowledged [391, 415,
178
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-defined, 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 amplification 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,
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.
19.
20.
21.
22.
23.
ACKNOWLEDGMENT
24.
The authors thank Katharine Irvine for critical reading of the

manuscript and helpful suggestions.
25.
26.
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Interferon-: an overview of signals, mechanisms

Macrophages respond to a range of different cell products

Journal of Leukocyte Biology Volume 75, February 2004 163

as B cells, NKT cells, and professional antigen-presenting cells

THE IFN- RECEPTOR

Journal of Leukocyte Biology Volume 75, February 2004

IFN- SYSTEM DYSFUNCTION

Schroder et al. Signals, mechanisms, and functions of IFN- 165

DNA binding. The C-terminal transactivation domain contains

Journal of Leukocyte Biology Volume 75, February 2004

Stat2:IRF-9 complex (known as ISGF3) was thought to be the

platelet-derived growth factor [110 112, 123, 124]. This may

sequence very similar to the Simian virus-40 T antigen NLS,

Schroder et al. Signals, mechanisms, and functions of IFN- 167

to bacterial infection, viral infections took the normal clinical

Negative regulation of IFN- signaling

Journal of Leukocyte Biology Volume 75, February 2004

IFN signaling (Jak1) or IFN- signaling (Jak2) [194, 195].

in erythropoiesis [197, 198]. Other than suppression of IFN-

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

plexes induced by cytokines may give different biological

Cross-talk between IFN-/ and IFN- pathways

IFN- stimulation induces a replacement of the constitutive

Class II antigen presentation pathway

Journal of Leukocyte Biology Volume 75, February 2004

Of the IFNs, IFN- alone can efficiently up-regulate the class

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

Antigen Processing and Presentation

Class I MHC heavy chain

IFN--inducible, enzymatic proteasome subunits, which replace the constitutive

[4, 217, 218, 219]

[222, 230, 232]

Class II antigen presentation pathway

Protein kinase dsRNA-regulated (PKR) is a serine/threonine kinase activated

[61, 249, 250]

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

Gene/protein downregulated by IFN-

PKR is an antiviral enzyme, which functions as a serine/threonine kinase when activated

c-myc controls G1/S transition by activating cyclin:CDK complexes and inducing

IRF-1 is a tumor-suppressor gene required for the induction of apoptosis by

[165, 266, 267]

Activation of Microbial Effector Functions

Journal of Leukocyte Biology Volume 75, February 2004

Production of reactive oxygen species (ROS)

Other antimicrobial mechanisms

The natural resistance-associated macrophage protein (NRAMP1) confers

Immunomodulation, th Development, and Leukocyte Trafficking

NK cell activator and differentiation factor driving CD4 cell development to a

Chemoattractant for monocyte/macrophages.

Chemoattractant for T cells.

Chemoattractant for memory CD4 T cells and monocyte/macrophages.

Adhesion molecule-binding to lymphocyte function-associated antigen-1 and

Surface molecule on APCs that provide costimulus for antigen-specific T cell

sins B, H, and L, lysosomal proteases implicated in production

IFN- and development of Th1 response

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

The IFN-induced antiviral state

Journal of Leukocyte Biology Volume 75, February 2004

chinery. Phosphorylation by PKR prevents recycling of eIF-2

Cell cycle, growth, and apoptosis

Activation of microbicidal effector functions

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

Immunomodulation and leukocyte trafficking

Journal of Leukocyte Biology Volume 75, February 2004