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Cytokine & Growth Factor Reviews 20 (2009) 97–113

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Interferon-g: A historical perspective

Alfons Billiau *, Patrick Matthys
Rega Institute, University of Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium

Available online 5 March 2009

Abstract

This article reviews the main lines of thinking and exploration that have led to our current conception of the role of IFN-g in immune defense
and autoimmunity. In 1965 the first report appeared describing production of an interferon-like virus inhibitor in cultured human leukocytes
following exposure to the mitogen phytohemagglutinin. In the early 1970s the active principle became recognized as being distinct from classical
virus-induced interferons, leading to its designation as immune interferon or Type II interferon, and eventually IFN-g. Up to that point interest in
the factor had come almost exclusively from virologists, in particular those among them who were believers in interferon. Evidence first coming
forward in the 1980s that IFN-g is indistinguishable from macrophage-activating factor (MAF), then a prototype lymphokine, was the signal for
immunologists at large to become interested. Today IFN-g ranks among the most important endogenous regulators of immune responses.
# 2009 Published by Elsevier Ltd.

Keywords: Interferon-g; History

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
2. From the discovery to the cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
3. The belated recognition of IFN-g as a lymphokine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4. Macrophage activation by IFN-g and resulting anti-microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5. IFN-g and the helper effects of T cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
6. IFN-g produced by CD8+ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
7. IFN-g and NK cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
8. IFN-g, dendritic cells (DC) and antigen presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
9. IFN-g and the chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
10. IFN-g in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
10.1. Defence against infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
10.2. IFN-g and hypersensitivity reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
10.2.1. Delayed-type hypersensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
10.2.2. Hypersensitivity reactions elicited by endotoxin—the Shwartzman-type reactions . . . . . . . . . . . . . . . . . . . 106
10.2.3. IFN-g and autoimmune disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
11. Concluding thoughts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

1. Introduction
* Corresponding author. Tel.: +32 16 337341 (office)/622008 (home);
fax: +32 16 337340 (office)/503933 (home). If IFN-g were to be discovered today, the chances are
E-mail address: Alfons.Billiau@Rega.Kuleuven.be (A. Billiau). slim that it would be named an interferon. Its molecular

1359-6101/$ – see front matter # 2009 Published by Elsevier Ltd.

doi:10.1016/j.cytogfr.2009.02.004
98 A. Billiau, P. Matthys / Cytokine & Growth Factor Reviews 20 (2009) 97–113

structure is only superficially similar to that of other
interferons and its actions on cells are mediated by a receptor
different from that used by other interferons. Moreover, the
direct antiviral effect on cells would probably be deemed
insufficiently important, relative to the regulatory actions on
leukocytes, to justify creation of a second interferon
subfamily. Most probably the newly discovered cytokine
would be named interleukin-n, n representing a two-digit
number. That IFN-g was identified as an antiviral factor long
before it was recognized as an interleukin-type factor is
mainly due to the simplicity and extreme sensitivity of the
antiviral assay which preceded the more complex in vitro
assays by which other cytokines were discovered. However,
the early discovery as an antiviral factor made that many

Table 1
Chronology of IFN-g research.
IFN-g—general issues
1965 Induction of an IFN activity in human PBMC by phytohemagglutinin [12]
1966 Antigen-specific induction of IFN activity during virus infections [20]
1972 IFN induced by anti-lymphocyte serum differs physicochemically from classical IFN ! named ‘immune IFN’ [16]
1973 IFN produced during DTH reactions differs physicochemically from classical IFN ! named ‘Type II IFN’ [22]
1979 Progress in purification [28–31]
1980 Nomenclature Committee: definitive name ‘IFN-g’ [27]
1982 Dimeric structure of IFN-g suggested [31]
Cloning of IFN-g from cDNA [32,33]
IFN-g vs. DTH and chemokines
1968 Migration inhibitory factor (MIF) and skin-reactive factor (SRF) described [38]
Lymphotoxin (LT) described [40,197]
1969 Lymphocyte mitogenic factor(s) and the lymphokine concept [35]
1973 MIF co-induced with Type II IFN during DTH in mice [22]
1981 Chemotactic activity of PF4 (now CXCL4) recognized [91]
1985 g-IP10 (now CXCL10) described [89]
1992 IFN-g inhibits neutrophil-targeting chemokines, and . . . [98–104]
1993 IFN-g stimulates mononuclear cell-targeting chemokines [99,102,103,105–109]
IFN-g, the macrophage-activating factor (MAF)
1969 Lymphocyte-mediated activation of macrophages – MIF responsible? [43]
1979 Macrophage-activating factor (MAF) ! several MAF assays established [44]
1981 Mr of MAF = 45.000 [50]
1983 Anti-IFN-g antibody neutralizes MAF preparations [45]
1985 Cloned IFN-g possesses MAF activity [53]
IFN-g and the evolving T helper cell concept
1973 T cell-replacing factor (TRF, T cell help for B cells) described [58]
1977 Type II IFN inhibits antibody production in vitro [198]
1984 IFN-g proposed as a TRF [59,61–63]
1988 Th1 and Th2 clones described [64]
Role of IFN-g in Th1/Th2 paradigm [65]
2005 Th17 cell lineage defined in mice [68,181]
IFN-g inhibits differentiation of Th17 cells and IL-17 production by activated Th memory cells
IFN-g and natural killer (NK) cells
1980 IFN-g upregulates NK activity [77]
1983 IL-2 may induce IFN-g in NK cells [78]
1991 IL-12 induces IFN-g in NK cells [79]
1993 Novel IFN-g-coinducing factor ! IGIF/IL-18 [80]
1995 IL-18 induces IFN-g in NK cells [81]
IFN-g vs. antigen-presenting cells (APC) and dendritic cells (DC)
1982 IFN-g enhances MHC Class II expression in mononuclear phagocytes [46]
1984 IFN-g induces enzymatic breakdown of tryptophan [199]
1990 IFN-g induces IDO in vivo [200]
1996 IFN-g enhances MHC Class II expression in DCs [83]
2000 IFN-g optimizes IL-12 production by DCs [85]
2000 IFN-g-induced IDO conditions DCs to become tolerogenic [188–190]
IFN-g vs. CD4+CD25+ Treg cells
1995 CD4+CD25+ Treg cells defined [201]
2005 Defective functioning of Treg cells in IFN-gR KO mice with collagen-induced arthritis (CIA) [187]
2006 IFN-g can convert CD4+CD25 cells into CD4+ Treg cells able to suppress experimental autoimmune [185]
encephalomyelitis (EAE)
Release of IFN-g by Treg cells [186]
 A. Billiau, P. Matthys / Cytokine & Growth Factor Reviews 20 (2009) 97–113
aspects of the biology and biochemistry of IFN-g were
explored in close connection with those of the ‘real’
interferons, IFN-a and -b. These common aspects have been
touched upon in Parts I and II of this historical review [1,2].
Here I will discuss facets that deal specifically with the
history of IFN-g research. Milestones marking this history
are assembled chronologically in Table 1.
Over the years the status of our knowledge on IFN-g has
often been reviewed. The reader may be interested in having
the references to some of these reviews [3–11].
2. From the discovery to the cloning
In 1965, 8 years after the discovery of interferon induction
by viruses, Wheelock [12] reported on the appearance of
interferon-like antiviral activity in supernatant fluid of
cultures of fresh human leukocytes following incubation
with the plant lectin phytohemagglutinin (PHA). At the time,
like today, PHA was much in use by cytogeneticists to induce
blastogenesis and mitosis in human peripheral lymphocytes,
so as to enable chromosomes to be visualized. Like virus-
induced interferon, the PHA-induced inhibitor was macro-
molecular (non-dialysable) but soluble (non-sedimentable)
and acted on human cells only. However, its activity was less
resistant to heat and to acid, two criteria that, besides others,
would remain in use up to the present day to differentiate IFN-
g from other interferons.
Wheelock’s observation came at a time when reports on
induction of interferon-like activity by various natural
products were legion. Most notable were the reports that
intravenous injection of endotoxin in chicks, mice or rabbits
induced appearance of interferon-like activity in the blood
stream. The possibility was considered that the PHA-induced
human interferon was the homologue of these animal
interferons induced in vivo by endotoxin. Endotoxin-induced
interferon in rabbits [13] or in cultured rabbit macrophages
[14] had indeed been found to be relatively acid- and heat-
sensitive. Moreover, endotoxins, like PHA, had been shown to
possess mitogenic potential for certain lymphocytes. Simila-
rities in molecular weight were also invoked, though the
values estimated at the time were at best tentative. As we know
today, circulating interferon induced by endotoxin in mice is a
mixture of Type I and Type II interferons, the former ones
produced by myeloid cells [15] and the latter one by NK cells
and some populations of T lymphocytes.
Another question debated at this early time was whether
interferon production following induction by PHA was linked
to the blastogenic response. PHA preparations were hetero-
geneous and the possibility was considered that the two
effects were triggered by different components. However, as
would soon become evident, other blastogenic and/or mitotic
agents can also induce the acid-labile interferon. The early list
of such agents included other plant lectins (concanavalin A,
pokeweed mitogen) but also the bacterial toxin streptolysin-O
and antilymphocyte serum (ALS) [16]. In the 1970s and 1980s
99
the list would still grow to include monoclonal anti-CD3
antibody, additional bacterial proteins (e.g. staphylococcal
enterotoxins and staphylococcal protein A) later to be grouped
with others as the super-antigens, and certain enzymes (e.g.
galactose oxidase) (for review see Refs. [17–19]). Induction
of IFN-g by these stimulants would appear to be due to
binding and cross-linking of receptor-like glycoproteins on
the cell membranes of lymphocytes resulting in polyclonal
activation.
Polyclonal activation of lymphocytes was not yet a
current concept at the end of the 1960s, and the analogy
between stimulation by these mitogens and that by antigens
was not yet generally recognized. In fact, antigen-specific
induction of interferon was discovered quite independently.
In 1966, Glasgow [20] hypothesized that interferon
production might take place and play an important role
not only during primary infection by viruses, but also
following re-infection. To make his point, he immunized
mice against Chikungunya virus and tested interferon
production by their cultured peritoneal leukocytes following
in vitro challenge with the same virus. Leukocytes from
immunized mice produced up to four times as much
interferon-like antiviral activity as cells from naı̈ve mice.
The interferon activity was stated to be acid-resistant,
though a parallel comparison with virus-induced interferon
was not undertaken. Therefore, in retrospect, it cannot be
ascertained if and to what extent IFN-g accounted for this
observation. However, in discussing the data, the author
argued that interferon production by immune-sensitized
leukocytes following re-exposure to the viral antigen could
play an important role in ‘cellular or tissue immunity’ (as
opposed to antibody-mediated immunity). Of note, at the
time, the concept of cellular immunity against virus
infection, as we know it today, was poorly developed.
Few years later (1969), these findings in mice and mouse
cells exposed to live virus were extended so as to apply to
humans and human leukocytes following sensitization with
non-viral antigens: Green et al. [21] demonstrated interferon
production by blood lymphocytes from humans sensitized to
tetanus toxoid, diphtheria toxoid or tubercular PPD when such
leukocytes were exposed in vitro to the respective antigens. In
this study the amounts of interferon and the size of the
blastogenic response were compared with those in blood
leukocytes exposed to PHA, and both were found to be only a
fraction of the PHA-induced response. The possibility that
interferon induced by these agents might be different from
classical virus-induced interferon was not considered. In
particular, acid-lability, though explicitly mentioned by
Wheelock in 1965 as a peculiarity, was not tested for.
Reviewing the literature on ‘non-viral inducers of interferon’
in 1972–1973, Merigan [17] did discuss the biological
significance of induction by mitogens and antigens, including
Glasgow’s hypothesis. He also cited unpublished data
indicating that ‘The molecular weight of interferon induced
by PHA in white blood cell cultures is 18,000, the same as that
induced by Newcastle disease virus in the same cells.’
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Thus, by the end of the 1960s the concept of ‘immune
induction’ of interferon was rather well established. The
possibility that such interferon might reside in different
molecular forms was entertained, but available evidence was
considered to weak. Then, two milestone studies appeared in
1972 and 1973, confirming that immune-induced interferon
differed physicochemically and biologically from ‘classical’
virus-induced interferon. Rebecca Falcoff characterized
interferon induced by anti-lymphocyte immunoglobulin in
cultured peripheral human lymphocytes and showed it to be
acid-labile and to migrate differently from virus-induced
interferon on gel filtration and on DEAE-cellulose
chromatography [16]. In fact, her estimate of a molecular
mass of 50,000, as opposed to 25,000 for virus-induced
interferon, was as close to reality as was possible to estimate
with the available reagents of the time. On the basis of this
evidence she concluded: ‘. . . synthesis of interferon by
immunocompetent cells under sets of circumstances which
certainly represent true immune responses, suggests that this
interferon can be called immune interferon’. In another
milestone study, Youngner and Salvin [22] showed that
BCG-infected mice inoculated intravenously with old
tuberculin (OT) release a viral inhibitor into the circulation
(up to 8000 units/ml). This inhibitor also had all basic
properties of interferon, but differed from virus-induced
mouse interferon by being less stable at pH 2 and at 56 8C,
by a different host range specificity and by resisting
neutralization by an antiserum prepared against highly
purified virus-induce mouse interferon. On the basis of this
evidence, the authors concluded: ‘. . . the interferon elicited
by OT in BCG-infected or control mice is . . . a distinct
molecular species of viral inhibitor and is designated ‘‘Type
II interferon’’.’ They objected against the term immune
interferon arguing that ‘. . .definition of interferon based on
the cell of origin or on the stimulus does not take into
account the characterization of the inhibitor molecules and
the mechanism of their induction’. This debate continued for
several years. In their comprehensive review of 1975 Ho and
Armstrong [18] refrained from using the terms Type I and
Type II to designate molecular entities or even families of
molecules, as they rather adhered to the terms Type I and
Type II induction. Typically, whereas Youngner and Salvin
[22] considered endotoxin-induced interferon as Type I, Ho
and Armstrong, in their review [18] catalogued it under
‘Type II induction’. Also, in his exhaustive monography
published in 1979, W.S. Stewart II [19] (pages 145–149)
contested the adequacy of the terms Type I and Type II
interferons. Nevertheless, the later 1970s saw the appear-
ance of more reports of interferon induction in leukocytes by
mitogens or antigens all confirming the observations of the
two aforementioned studies (reviewed by Stewart [19] and
by Epstein [3]). Furthermore, in these years, purification and
characterization of the classical mouse and human inter-
ferons took giant leaps forward. In the course of 1979 the
amino-terminal sequences of pure human leukocyte- and
fibroblast interferons were revealed [23–26], and there was
evidence for the existence of homologous interferon
molecular species in mice. Thus, the need developed for an
adequate nomenclature for these proteins. In the spring of
1980 a Committee of experts [27] agreed on proposing the
terms IFN-a and IFN-b for the ‘classical interferons’ both of
which had been obtained in pure form and appeared to be
homogenous. Although the immune-induced interferon had
not yet been obtained in pure form, the Committee recognized
that its main component differed from IFN-a and IFN-b, and
gave it the name IFN-g, thus settling the debate on the choice
between ‘Type II interferon’ and ‘Immune interferon’.
Ironically, the terms Type I and Type II interferon have
survived up to the present day, the only reason being that
‘Type I interferon’ conveniently allows to collectively
designate the biologically related IFN-a and IFN-b.
Difficulties encountered in purifying IFN-g to homo-
geneity included low yields in production systems and
irreversible loss of biological activity during purification.
Between 1979 and 1982 these difficulties were overcome in
several laboratories [28–31], such that in 1982 it had become
clear that native human IFN-g had a Mr of 45 kDa, being
composed of glycopeptides of 20 to 25 kDa. In the same
year, two laboratories [32,33] succeeded in cloning the
cDNA of human IFN-g. Cloning and analysis of the genomic
DNA revealed that, contrary to the intronless genes of IFN-a
and IFN-b, the IFN-g gene contained three introns [34].
3. The belated recognition of IFN-g as a lymphokine
In 1969 Dumonde et al. [35] proposed the term
‘lymphokines’ to designate ‘non-antibody mediators of
cellular immunity generated by lymphocyte activation’. This
readily accepted proposal, was based on four biological
activities of lymphocyte/macrophage supernatants, and
marked a turning point in the years-long efforts of many
research groups to find an in vitro correlate for cellular
immunity. At the time, the preferred experimental animal
model for studying cellular immunity was delayed-type
hypersensitivity (DTH) in the guinea pig. Typically, dermal
reactions were elicited in animals infected with BCG or
immunized with Freund’s adjuvant, and challenged intra-
dermally with PPD. It had become evident that not only
lymphocytes but also macrophages play an important role in
this local skin reaction (reviewed in Ref. [36]). Accordingly,
in their attempts to develop an in vitro test, researchers
focused on these cells. Peritoneal exsudate of guinea pigs
contains ca. 75% lymphocytes and 15% macrophages. So, it
was logical to culture peritoneal exsudate cells of sensitized
animals and to observe their reaction towards exposure to the
antigen. An ‘avant la lettre’ observation of 1932 inspired the
researchers to use cell migration as read-out: Rich and Lewis
had discovered that ‘migration of cells from splenic explants
from hypersensitive guinea pigs was specifically inhibited by
antigen’ (loc. cit. [37]). This led to the development of the
classical macrophage migration inhibition assay in which
 A. Billiau, P. Matthys / Cytokine & Growth Factor Reviews 20 (2009) 97–113
macrophages, with or without lymphocytes, are cultured in
open-ended capillary tubes lying on a horizontal glass
surface. Without exposure to antigen, the macrophages
emigrate from the tubes and form monolayers on the glass.
In the presence of sensitized lymphocytes and antigen, this
migration is inhibited. Working with this assay, Bloom and
Bennett [37] established that ‘the sensitized lymphocytes,
upon interaction with the antigen in vitro, elaborate into the
medium a soluble material capable of inhibiting migration
of normal exudate cells’. In a further study, the same authors
[38] called the soluble material ‘migration inhibitory factor’
(MIF) and showed that, after partial purification, this
proteinaceous material was also able to elicit a skin reaction
upon intradermal injection, later leading to the designation
of skin-reactive factor (SRF). In hindsight, it seems plausible
that the activities of these ‘factors’ probably corresponded to
a mixture of several cytokines and chemokines. In 1967,
exposure of lymphocytes from sensitized guinea pigs to an
immunizing antigen (e.g. a hapten-carrier complex) had
been shown to also result in blast formation [39], and in his
1969 study Dumonde et al. [35], using peritoneal exudate
cells of BCG-sensitized guinea pigs, demonstrated that this
mitogenic activity is associated to the appearance of (a)
soluble blastogenesis-inducing factor(s) in the stimulated
cultures. Thus, lymphocyte growth factors became seen as a
third type of lymphokine.
A fourth soluble biological activity included by
Dumonde et al. [35] in their definition of lymphokines
was cytotoxicity. That lymphocytes could under certain
circumstances exert toxic effects on other cells had been
noted in the earlier 1960s. Release of a soluble cytotoxic
factor (lymphotoxin) by sensitized lymphocytes following
exposure to antigen and the link of this phenomenon to DTH
were established in 1968 [40,41].
Thus, by 1970, the ‘lymphokine’ concept was fairly well
documented. However, neither the interferonologists nor the
lymphokinologists considered interferon as a lymphokine.
The first ones, having their minds set on defence against
virus infections, were still uncertain about the nature of the
peculiar interferons induced by such non-viral substances as
endotoxin and PHA. The second ones, being concerned with
DTH as a marker of cellular immunity against mycobacteria
had little interest in anti-virus defence but were more alert
for the role of DTH in rejection of tumors and tissue
transplants. Moreover, interferon workers and DTH workers
formed separate scientific communities, with only few
occasions of mutual interaction.
Yet, in the debate on the nature of Type II and immune
interferon, Salvin et al. in 1973 [42] came close to the point
of considering immune-induced interferon as a lymphokine
as they showed the appearance of both MIF and Type II
interferon in the circulation of mice during DTH reactions.
Only towards the end of the 1970s would reviewers, for
instance Epstein [3], consider IFN-g as a bona fide
‘lymphokine’. In his 1984 review on IFN-g, Kirchner [4]
remarked: ‘. . .(IFN-g) was the first lymphokine the
101
molecular structure of which has been identified’. An
important factor in settling the debate was the definition,
around 1980, of one additional lymphokine activity, i.e.
macrophage-activating factor (MAF).
4. Macrophage activation by IFN-g and resulting
anti-microbial activity
Today it is common knowledge that, during host defense
against microorganisms, mononuclear phagocytes become
activated by lymphokines and thereby acquire the capability
to efficiently kill intracellular pathogens and to attract
additional cellular immune components into the inflamma-
tory focus. This concept of macrophage activation owes
much to a series of studies conducted by Mackaness and
collaborators in the course of the 1960s. One particular study
from 1969 [43] deserves special attention. It addressed the
observation that BCG-infected mice mount a degree of a-
specific resistance against Listeria. It was shown that
lymphocytes of such mice can transfer this resistance to
naı̈ve recipients provided they are injected together with an
eliciting dose of BCG. Furthermore, it was demonstrated
that ‘The peritoneal macrophages of animals so treated
develop the morphology and microbicidal features of
activated macrophages’. From these observations, the
authors ‘. . . inferred that acquired resistance depends upon
the activation of host macrophages through a product
resulting from specific interaction between sensitized
lymphoid cells and the organism or its antigenic products’.
Considering various possible mechanisms for the interaction
between lymphoid cells and macrophages, the author also
speculated that MIF, the mediator described by Bloom and
Bennett [37], ‘could fulfill the role of the hypothetical
humoral mediator which influences macrophage function’.
In the years that followed, in vitro evidence for this concept
was collected. In particular, various tests for macrophage
activation were developed. Also, from 1979 onwards, the term
‘macrophage-activating factor’ (MAF) first appears in the
literature, referring to the ability of supernatants of mitogen-
or antigen-challenged mononuclear cell cultures to augment
various biological activities of macrophages. The MAF
bioassays variably relied on intracellular killing of ingested
microorganisms, increased oxidative metabolism [44,45],
enhanced expression of Class II antigens [46] or enhanced
tumor cell killing [47,48]. In vitro studies in the early 1980s
demonstrated that exposure of macrophages to lymphokine
preparations results in enhancement of their potential to kill
ingested various microorganisms [49–51]. Cultured mouse
peritoneal cells, which supported proliferation of Leishmania
tropica, were shown to kill the intraphagolysosomal organ-
isms following exposure to crude lymphokine [50]. Fractio-
nation of the lymphokine by gel filtration indicated that a
45 kDa protein component accounted for this macrophage-
activating effect. The fraction also produced a cytocidal effect
for tumor cells and for extracellular Schistosomula, whereas a
102 A. Billiau, P. Matthys / Cytokine & Growth Factor Reviews 20 (2009) 97–113
smaller fraction (23 kDa) provided only activity against the
extracellular targets [52]. The authors’ presumption that the
45 kDa fraction represented IFN-g was confirmed by a later
study employing cloned IFN-g [53].
Meanwhile, availability of pure IFN-g and specific
antibodies made it possible to demonstrate that IFN-g by
itself could augment bactericidal activity of macrophages
against Toxoplasma in mouse macrophages [45] and
Chlamydia in human monocyte-derived macrophages
[54]. Furthermore, characterization with monoclonal anti-
IFN-g antibodies revealed that IFN-g is an essential
component of MAF in crude lymphokine preparations
[45]. In some assay systems for MAF, e.g. hydrogen
peroxide production elicited by phorbol ester, IFN-g is
sufficient for activation to take place, but in others,
additional cytokines such as TNF-a are required. This
seems to be particularly so in the case of bactericidal effects
against Mycobacteria [53] or Listeria [55–57].
5. IFN-g and the helper effects of T cells
That antibody responses require help from T cells was well
known already in the 1960s. In the early 1970s work in several
laboratories indicated that antigen-nonspecific soluble factors
secreted by the T cells could at least in part account for this
help. The term ‘T cell-replacing factor’ (TRF) to designate the
ensemble of these factors came in use around 1973 [58]. In the
years that followed, assay systems for TRF were multiplied
and refined, allowing to further underpin the distinction
between different stages in B cell biology and to define the
corresponding factors present in TRF. At first, the source of
TRF consisted of freshly harvested normal thymocytes or
splenocytes stimulated with mitogens. Later on, thanks to the
discovery of the T cell growth factor (TCGF) that eventually
became IL-2, cloned T cell lines capable of producing TRFs
became available, and these facilitated purification and
characterization work. Though interferonologists were using
quite similar settings to produce IFN-g, the question of its
possible TRF activity could not be tackled until 1982 when
pure IFN-g and specific anti-IFN-g antibody became
available. In 1984 cloned murine IFN-g was found to induce
Ig secretion by resting B cells or by a B-cell tumour line [59].
A second study [60] demonstrated identity of cloned IFN-g
with a T cell-line-derived factor required for purified B mouse
lymphocytes to produce an antibody response to sheep red
blood cells when stimulated by IL-1 and IL-2. Subsequently,
an inhibitory effect of IFN-g on B cell proliferation was
shown [61,62]. Together, these observations suggested that
one role of IFN-g might consist in redirecting B cells from
proliferation towards differentiation.
As TRF activities from various sources were further
analysed, certain clonal murine T cell lines appeared to be
highly specialized in production of particular types of helper
factors. One such clone (D9.1) was found to produce help
that allowed LPS-stimulated B cells to selectively produce
IgE and IgG1. Remarkably, this selective induction was
counteracted by addition of IFN-g [63], suggesting hat IFN-
g played a role in directing isotype switch. Follow-up of
these findings soon led to the classification of murine helper
T cell clones into T-helper-1 (Th1) and -2 (Th2) categories
[64] and to the division of in vivo immune responses in Th1
and Th2 responses. Both were characterized by particular
cytokine production profiles, whereby production of IFN-g
is the Th1 hallmark by excellence. Thus, it appeared that
IFN-g indeed qualifies as an important component of T cell
helper cytokines, as it critically co-directs the isotype
switch. Evidently the question also rose as to whether IFN-g
would play a role in the proliferation and differentiation of T
helper cells. As far as murine T cell clones are concerned,
IFN-g was found to inhibit proliferation of Th2 but not Th1
clones [65]. Follow up of this observation would later lead to
the finding that the b-chain of the IFN-g receptor is not
expressed in Th1 clones [66].
From the foregoing it should be evident that studies
focusing on IFN-g have been of paramount importance in
establishing the Th1/Th2 paradigm, which holds that IFN-g
(1) favours development of Th1 over Th2 cells, (2) provides
help from Th1 cells to macrophages (activation) and (3)
provides help from Th1 cells to B cells (isotype switch).
Over almost two decades the Th1/Th2 concept has inspired
numerous studies clarifying fundamental aspects of both
normal and pathological immune responses. However, the
Th1/Th2 concept failed to accommodate several observa-
tions, in particular in connection with the role of IFN-g in
experimental models of autoimmune disease.
The discovery, in the early 2000s, of an additional T cell
lineage (Th17) characterized by production of IL-17 (for
review, see Ref. [67]) finally paved the way for explaining
these long-standing unsolved questions. Most importantly,
IFN-g produced by Th1 cells was shown to counteract
development of Th17 cells [68]. Differentiation of naı̈ve T
cells towards IL-17-producing cells was greatly enhanced
when the medium was supplemented with neutralizing anti-
IFN-g antibodies or when the T cells were derived from IFN-
g- or IFN-gR knock-out mice.
6. IFN-g produced by CD8+ cells
In the early 1980s reports appeared demonstrating the
ability of cytolytic T cells to produce IFN-g, alongside with
other cytokines. These studies used alloreactive murine T
cells, either freshly harvested [69] or obtained as clonal lines
[70,71]. It would take several years for the idea to permeate
that this might represent an accessory mechanism by which
cytolytic CD8+ cells eliminate virus from infected tissues: in
1990 Fong and Mosmann [72] noted that the cytokine
production spectrum of murine alloreactive cytolytic clones
closely resembles that of Th1 cells, and postulated the
existence in viral antigen-specific CD8+ clones of ‘cytokine-
mediated mechanisms or functions that may be especially
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important . . .in combating viruses, . . ..’ In fact, over the
ensuing years, studies devoted to this question did confirm
production of IFN-g by virus-specific CD8+ cells, but also
indicated that the role of IFN-g is particularly evident in
models of virus infections where the cytolytic capacity of
CD8+ cells is not expressed. Non-cytolytic viral clearance
that depends both on CD8+ cells and on IFN-g has been
reported in models of persistent nervous system infection
with viruses such herpes virus [73], measles virus [74] or
borna virus [75]. In fact, working with LCM-infected mice,
Oldstone et al. [76] had already noted that the central
nervous system possesses a mechanism to avoid cytolysis
and inflammation which in other tissues such as liver or
lungs accompanies viral clearance by CD8+ cells. In tissues
with a restricted potential for tissue renewal the ‘usual’ form
of viral clearance by elimination of virus-infected cells may
not be an optimal strategy.
7. IFN-g and NK cells
NK cells are both targets of IFN-g action and producers of
IFN-g. Following the discovery that Type I interferons can up-
regulate NK cell activity in vitro, a similar effect for IFN-g
was reported [77]. The ability of NK cells to produce IFN-g
was suggested in 1983 by Handa et al. [78] who showed IFN-g
production by mouse splenocytes enriched in NK cells by
growth in IL-2. Later studies confirmed that NK cell-enriched
lymphocyte populations produce IFN-g in response to various
exogenous and endogenous stimuli, including the NK-
stimulating factors IL-12 [79] and IL-18. In fact, IL-18
was discovered by virtue of its ability to act as a co-stimulator
for IFN-g induction in resting T cells or NK cells by IL-2 or
anti-CD3 antibody. The factor was first observed in serum of
P. acnes-primed and LPS-challenged mice [80]. Subse-
quently, it was isolated from liver tissue of the mice [81] and
found to qualify as an IFN-g-inducing factor (IGIF).
8. IFN-g, dendritic cells (DC) and antigen
presentation
DCs are the most potent antigen-presenting cells (APC).
Exploration of their diversity, maturation and function has
made fast progress in recent years. Insight into the role of
IFN-g in their biology is of recent date and still evolving.
Human peripheral blood monocytes differentiate into
antigen-presenting DCs by cultivation in GM-CSF + IL-4;
the alternative differentiation pathway leading to a macro-
phage phenotype is favoured by M-CSF, IL-6 and IL-10.
When added during differentiation of DCs, IFN-g switches
the differentiation pathway towards macrophages [82],
partly due to endogenous production of M-CSF and IL-6. In
mice with a subnormal expression of MHC Class II
molecules on DC, treatment with IFN-g was found to
correct the defect [83]. In cultured human blood-derived DC
103
IFN-g also augmented expression of MHC Class II
molecules, but inhibited expression of other membrane
receptor molecules that are also important in antigen
presentation [84]. Production of IL-12 is an important
marker of DC activation and there is evidence that, under
certain circumstances, exposure to IFN-g is a prerequisite
for optimal production of this cytokine [85]. That IFN-g may
crucially affect DC function appears from experiments
showing that injections of DC that have been exposed to
IFN-g suppress autoimmune diabetes in NOD mice [86] and
reduce auto-antibody production and disease severity in
mice with autoimmune myasthenia gravis [87].
9. IFN-g and the chemokines
Chemokines came of age in the late 1980s with the
discovery of IL-8 (now also termed CXC chemokine ligand
8 or CXCL8). At that time, immunologists still considered
IFN-g mainly as a stimulant of immune defense against
viruses and ‘intracellular’ bacteria. The concept of it being
an inflammation-regulating cytokine was poorly developed
(for a contemporary review, see [88]). Nevertheless, as a
prologue to the chemokine era, in 1985 a report appeared
demonstrating induction by IFN-g in human cells of a
transcript encoding a protein named gIP-10 (g-IFN induced
peptide-10, now also named CXCL10) with sequence
homology to platelet factor 4 (PF4) [89]. Human PF4 (now
also designated as CXCL4), had already been extensively
studied by haemostasis researchers, who described it in 1964
as an antiheparin factor [90]. Evidence for its chemotactic
potential would come forward as late as 1981 [91]. This 17
years-long delay testifies to the fact that haemostasis
researchers had until then manifested little interest in the
relation between blood clotting and inflammation.
In 1987 cDNAs of PF4 and gIP-10 were cloned and found
to share extensive sequence homology [92]. Also around that
time, interest for secretion of chemotactic cytokines had
been growing in several laboratories of leukocyte biology
such that in 1987 three teams independently reported the
release by such cells of granulocyte chemotactic factor(s)
[93–96]. Originally the factors were named differently in
each laboratory. Because in each instance the purified
protein appeared indistinguishable from the others, and
because the general impression was that it represented the
next interleukin to be discovered, it was rather hastily named
interleukin-8 (Larsen et al., 1989; International Meeting of
Novel Neutrophil Stimulating Peptides: Source, Structure
and Role in Inflammation, London, 1988, loc. cit. Ref. [97]).
In hindsight, this was not a wise decision, as both this new
factor and PF4 would soon appear to belong to a large family
of proteins with a molecular structure quite different from
that of all interleukins isolated up to that point. This episode
signified the start of an avalanche of revelations on novel
chemotactic factors, produced by various cell types and also
affecting movement of various leukocyte categories. Today,
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the numerous chemokines and the cellular receptors targeted
by them are organized as molecular families and sub-
families. Synthesis and release of chemokines by leukocytes
as well as other cells can be constitutive or inducible by
exogenous or endogenous stimuli. Chemokines not only
regulate leukocyte migration and homing; some of them also
induce degranulation of leukocytes and regulate angiogen-
esis. Production of chemokines and expression of their
receptors is under regulatory control by cytokines. As to IFN-
g, studies have documented either positive or negative effects
depending on the particular chemokine and cell system
considered. The general trend is for induction of neutrophil-
chemotactic chemokines to be inhibited by IFN-g [98–104],
and for induction of mononuclear cell-chemotactic chemo-
kines to be stimulated [99,102,103,105–109]. But according
to some reports, exceptions to this trend may exist [110].
Moreover, in the whole animal, or in in vitro systems
consisting of more than a single cell type, IFN-g is more likely
to act in an indirect fashion by affecting production of other
cytokines (e.g. TNF-a) that may counteract the primary effect
of IFN-g [111].
These data are indicative of an important role for IFN-g
in chemokine-controlled processes, such as traffic and
degranulation of leukocytes and angiogenesis. Whereas an
interpretation for stimulatory effects is usually easy to
conceive in terms of well-established pro-inflammatory
effects of IFN-g, an interpretation of inhibitory effects is less
evident. In vague terms, inhibition of chemokine production
by IFN-g has been invoked to provide an explanation for the
sometimes puzzling anti-inflammatory roles of IFN-g in
certain murine models of autoimmune diseases. However,
detailed mechanistic schemes are so far mostly lacking.
Clearly, more in depth analysis of the IFN-g/chemokine
relation in defined in vivo situations is needed. One example
is the demonstration in collagen-induced arthritis (CIA) in
mice that ablation of IFN-g results in an excessive
proportion of neutrophils in the inflamed joint synovia, as
opposed to predominance of mononuclears in IFN-g-
competent mice, a difference that was associated with
higher levels of GCP-2/CXCL-6 [104]. Another example is
the demonstration of transient IFN-g-dependent inhibition
of the expression of two homeostatic chemokines (SLC and
BCA-1/BLC) in the lymphoid tissues of mice after infection
with LCM (lymphocytic choreomeningitis) virus. This
modulation was associated with altered distribution of
lymphocytes and dendritic cells in these tissues and with a
transiently impaired immune response to a second primary
challenge with a different pathogen [112].
10. IFN-g in vivo
10.1. Defence against infection
The specific activating effect of IFN-g on macrophages,
established around 1980, was seen as an indication that its
most important in vivo role would consist in mounting an
effective response towards pathogens (bacteria, molds,
protozoans) whose elimination from the body depends on
phagocytosis and intracellular killing. Today, our conception
of the immune response to such agents holds that, early after
entry of these agents, NK cells produce IFN-g which primes
mononuclear phagocytes for production of monokines,
including TNF-a and IL-12. IFN-g and TNF-a then act in
concert to augment the bacteriostatic potential of the
phagocytes. Later on, guided by IL-12, a Th1 response is
mounted resulting in additional IFN-g production by
activated CD4+ and CD8+ T cells.
Experimental work leading to these insights started
when, also around 1980, pure mouse IFN-g and specific
neutralizing (monoclonal) antibodies were made avail-
able. More refined analysis followed when gene knock-out
mice with defective expression of IFN-g or its receptor
became available from 1993 onwards. Application of
these tools in studies with animal models revealed a
crucial role of IFN-g in defence against infections with
the most diverse bacteria. Beyond confirming the
importance of IFN-g-mediated macrophage activation,
these experiments also revealed additional mechanisms by
which IFN-g influences the pathogenesis of infections. In
Listeria infection, administration of IFN-g was found to
augment normal resistance [113] or restore compromised
resistance [114,115]. Also, treatment with neutralizing
antibodies against IFN-g abrogated resistance [116,117].
Importantly, however, IFN-g production during the first 2
days of infection was shown to be critical for development
of protective antigen-specific T cells [118]. Thus, it
appeared that IFN-g not only optimizes effector cell
function, but also acts as a regulator of the adaptive
immune response to the bacterial agent.
As to the role of IFN-g in Mycobacterial infections, an
important early observation was that repeated percutaneous
application of IFN-g on skin lesions of lepromatous leprosy
patients caused increased infiltration with lymphocytes and
reduction in the local bacterial load [119]. In the early 1990s,
as gene knock-out mice became available, mice deficient in
IFN-g production proved unable to control otherwise
sublethal doses M. tuberculosis [120,121] or M. bovis
[122]. Bacteria multiplied more extensively and caused
more widespread damage in affected tissues. Likewise, mice
with a disrupted gene for the IFN-g receptor were found to
fail controlling infection with M. bovis [123]. In these IFN-
g-ablated mice not only innate resistance in the early phase
of infection, but also later development of protective
immunity was compromised. Although some DTH did
develop, the mice failed to recover from infection suggesting
that IFN-g, while dispensable for DTH, is essential for
protective immunity [120]. Along this line of thinking, IL-12
production was found to be absent in IFN-gR KO mice
infected with M. bovis [124], indicating that early
production of IFN-g (by NK cells) is an important factor
in directing the immune response towards the Th1 pathway.
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These observations were also seen as an indication that IFN-
g might prove to be useful as vaccine adjuvant. However,
attempts to obtain protective immunity by adding IFN-g to
killed vaccine preparations for Listeria or Mycobacteria
have remained unsuccessful.
In further work with IFN-g-ablated mice, a role for IFN-g
in immunopathological changes during mycobacterial infec-
tions became evident. Splenomegaly and altered splenic
architecture that accompanies such infections was shown to
be enhanced in IFN-gR KO mice infected with M. bovis,
indicating that IFN-g mitigates excessive extramedullary
myelopoiesis [125]. IFN-g was also found responsible for
apoptosis of CD4+ T cells taking place in the later phases of
the immune response to mycobacteria [126], and for the
appearance of ‘immunosuppressive’ macrophages [127].
10.2. IFN-g and hypersensitivity reactions
10.2.1. Delayed-type hypersensitivity
The delayed-type hypersensitivity (DTH) cutaneous
reaction, first defined in the context of the immune response
to mycobacteria, is universally considered as the prototype of
T-cell-mediated, antigen-specific inflammation. DTH inflam-
matory reactions are crucial for defense against intracellular
pathogens, but also represent the cellular mechanism under-
lying pathologic responses to allergens, e.g. in contact
dermatitis, and autoantigens, e.g. in autoimmune diseases.
Despite recognition, around 1980, of the close association of
IFN-g with the host response to intracellular pathogens, and
although 7 years earlier Youngner and Salvin [22] had
described production of acid-labile interferon following PPD
challenge in BCG-infected mice, the possibility that IFN-g
might somehow control DTH-associated inflammation was
not considered until the mid 1980s. This hesitation is
remarkable if one considers that in 1975 De Maeyer had
described inhibitory effects of Type I interferon on DTH
reactions in mice [128,129]. In his review on ‘Gamma
Interferon’ of 1984, Kirchner wrote: ‘Based upon the well-
known effects of [Type I] interferon on immune reactivities, a
major role was postulated for IFN-g as an immunoregulatory
molecule in reactions in which it is produced. However, such a
role is far from being proven’. A simple explanation may be
that the tools to examine the in vivo role of IFN-g, pure IFN-g
and monospecific neutralizing antibodies, were still lacking.
When these tools became available in the mid-1980s, their
application to several animal models of DTH revealed the
existence of both stimulatory and inhibitory IFN-g-dependent
circuits of which one or the other appeared to predominate
depending on the model system under study. This finding went
against the expectation that IFN-g would play a positive role
in DTH, mainly because, at the time, DTH hypersensitivity
had become seen as a typical Th1-type immune response,
involving mainly IFN-g-producing CD4+ cells.
In a DTH induction model using sheep erythrocytes as an
antigen in mice, exogenous IFN-g was found to reverse
inhibition of the response by anti-CD4 or anti-IL-2R
105
antibodies [130], supporting the concept that production of
IFN-g by TH1 cells is essential for the reaction. Using a
local adoptive transfer assay with Ag-specific mouse TH1
clones, administration of anti-IFN-g antibodies was found to
inhibit responses in some but not all instances [131],
indicating that the positive contribution of IFN-g to the DTH
effector phase is real but can vary perhaps with specificity of
the clone or with mouse strain. In the rat, administration of
IFN-g-neutralizing monoclonal antibodies inhibited lym-
phocyte recruitment in DTH sites induced with a
haemocyanin [132]. Transgenic mice with ectopic expres-
sion of IFN-g in the photoreceptors of the retina, were found
to develop a DTH state, suggesting that intraocular IFN-g
production can overcome the so-called anterior chamber-
associated immune deviation [133].
In a passive transfer model in mice allowing to study a
population of T cells that suppresses haptene (dinitrofluor-
obenzene – DNFB) contact sensitivity responsiveness,
exogenous IFN-g was found to antagonize suppressive
cells, suggesting the existence of a pathway by which IFN-g
potentiates contact sensitivity [134]. However, in a DNFB
contact sensitivity model in rats [135] IFN-g was found to
act as a counter-regulator of the inflammatory changes:
systemic administration of anti-IFN-g augmented ear
swelling towards haptene challenge in sensitized animals.
This effect was accompanied by reduced MHC Class II
antigen expression on keratinocytes. Administration of IFN-
g in this same model inhibited ear swelling if given prior to
challenge, but not if given later [136]. The authors
considered that MHC Class II antigen-expressing keratino-
cytes are instrumental in the counterregulatory effect of IFN-
g. Such cells had indeed been shown to be able to inhibit T
cell responses [137]. In disagreement with these studies in
rats, IFN-g receptor-deficient mice were found to display
reduced hypersensitivity to haptens, as evident from reduced
cellular infiltrates [138].
More recent studies have indicated that IFN-g present in
DTH inflammatory sites originates mainly in CD8+ Tc1
cells [139,140], while another CD8+ subpopulation releases
IL-17 [141], which acts in a pro-inflammatory fashion.
Furthermore, some of the CD4+ cells were identified as
CD25+ Treg cells [142,143]. Involvement of IL-15 was
evident from a study using IL-15 receptor-deficient mice, as
these mice displayed reduced hapten contact hypersensi-
tivity together with reduced infiltration by CD8+ cells,
diminished levels of IFN-g and chemokines CCL5/
RANTES and CXCL10/IP-10 [144].
Most likely, the ambiguity in reports analyzing the role of
IFN-g in DTH reactions reflects the pathogenic complexity
of the systems under study. The effects may differ depending
on the antigen used (protein or hapten), the route of exposure
(injection or contact with the skin) and the time point during
the reaction (during the sensitization or the elicitation
phase). Furthermore, DTH reactions rely on both natural and
acquired immune response mechanisms and, again, IFN-g
may act differently on these components.
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10.2.2. Hypersensitivity reactions elicited by
endotoxin—the Shwartzman-type reactions
First described in the late 1920s in rabbits, the Shwartz-
man reaction exists in a localized and a generalized variant
(for review, see Ref. [145]). Both are two-stage phenomena,
requiring a preparative (sensitizing) injection of endotoxin
followed after about 24 h by an eliciting (provoking)
injection. Classically, to obtain a local Shwartzman reaction
in the rabbit, the preparative dose is given in the skin, and the
eliciting dose is given intravenously. Within a few hours a
thrombohemorrhagic reaction appears at the prepared skin
site. When both doses are given intravenously, a generalized
reaction can occur, which often leads to death. This reaction
resembles the thrombohemorrhagic shock that sometimes
occurs in humans affected by meningococcal sepsis. The
hypothesis of involvement of endogenous ‘mediators’ (e.g.
complement, serotonin and prostglandins) in the Swartzman
reaction already prevailed when in the early 1980s
interleukin-1 and TNF/cachectin were characterized bio-
chemically. Both these cytokines were conceptually linked
to the biological effects of endotoxin, IL-1 as the proposed
‘endogenous pyrogen’ responsible for fever induction by
endotoxin, and TNF/cachectin as the mediator of the tumor
necrosis effect of endotoxin. No wonder that, in the early
1980s, investigators of the Shwartzman phenomenon turned
to these two cytokines as the possible endogenous mediators
of their phenomenon [146].
Contrastingly, the question as to a possible involvement
of IFN-g did not immediately come up. The reason for this
may have been that, at the time, evidence for specific
induction of IFN-g by endotoxin, or for any role of IFN-g-
producing cells (T cells and NK cells) in endotoxin
pathology was minimal. In fact, the first evidence that
IFN-g does indeed play a crucial role in the Shwartzman
phenomenon came from a separate line of investigation in
which mouse footpad reactions elicited by endotoxin were
used as an assay to detect anti-inflammatory mediators
present in various biological fluids [147]. Pre-treatment with
neutralizing anti-IFN-g completely protected the mice
against this reaction [148,149]. The essence of this
observation was confirmed by studies using several other
variants of endotoxin-induced lethal reactions in mice [150–
155]. One of these studies compared the protective
effectiveness of anti-IFN-g antibody with that of anti-
TNF-a antibody and found the latter one to be ineffective
[154], suggesting that in some forms of endotoxin-mediated
lethality, endogenous IFN-g may be more important than
endogenous TNF-a.
Injection of the anti-IFN-g antibody at different time
points allowed to situate the action of IFN-g in the interval
between the preparative and the eliciting dose. Furthermore,
mice treated with anti-IFN-g antibody showed reduced
production of circulating TNF following the eliciting dose
[149]. Together, these data supported a model according to
which the preparative dose relies on IFN-g in order to
sensitize TNF-producing cells (mononuclear phagocytes)
and possibly also TNF’s target cells (endothelial cells and
various leukocytes). Several studies were undertaken to
identify the cellular source of IFN-g during Shwartzman-
type reactions [156–158]. Together these studies pointed
mainly at NK and NKT cells.
10.2.3. IFN-g and autoimmune disease
In the mid-1980s recombinant IFN-g and neutralizing
monoclonal antibodies became available for in vivo experi-
ments on the role of endogenous IFN-g in experimental
diseases. At the time, the major lead regarding pathogenesis of
autoimmune diseases was the association with MHC genes,
and IFN-g had just become known as a prominent stimulator
of expression of MHC Class II genes. Most notably, IFN-g
was found able to induce MHC Class II gene expression by
cells that are normally Class II-negative [159,160], and it was
proposed that such ‘ectopic’ expression MHC Class II
antigens might result in cross-presentation of auto-antigens,
leading to breakdown of peripheral tolerance and develop-
ment of autoimmune disease. In support of this view,
transgenic mouse strains harboring the MHC Class II or IFN-
g genes linked to the insulin promoter were shown to develop
insulin-dependent diabetes mellitus [161]. Furthermore, a
disease-promoting role of IFN-g in autoimmune diseases was
confirmed in other animal models of autoimmune diabetes
[162,163] as well as in lupus-like nephritis [164] and
autoimmune neuritis [165].
The concept of a possible role of IFN-g in autoimmune
pathogenesis became more specific with the then emerging
Th1/Th2 paradigm [166] which heralded IFN-g as the
hallmark of the Th1-type immune response. Auto-immune
encephalomyelitis (EAE) and similar autoimmune diseases
that had previously been classified as ‘cell-mediated’, now
came to be seen as typically Th1-mediated. Accordingly,
ablation of endogenous IFN-g was presumed to inhibit such
diseases. Surprisingly, however, experiments with neutraliz-
ing anti-IFN-g antibodies had already been yielding
evidence to the contrary. Most notably, administration of
anti-IFN-g antibody to mice was found to cause aggravation
of EAE [167], an observation many times confirmed
subsequently [168–170] and complemented by evidence
for a similar protective effect of endogenous IFN-g in other
models of T cell-mediated autoimmune disease, notably
experimental autoimmune uveitis (EAU) [171] and CIA
[172–175].
Over the 20 years since the first report on these
counterintuitive effects of IFN-g, several possible underlying
mechanisms have been proposed (for reviews, see Ref. [176–
180]). Though each of these may play a certain role at some
stage, their integration into a comprehensive explanatory
model has only recently become possible, thanks to
recognition of important novel cell lineages, in particular
Th17 cells, Treg cells and tolerogenic DCs. The identification
of the Th17 cell lineage in 2005 [68,181] has necessitated
revision of the Th1/Th2 paradigm (for review, see Ref. [67]).
The experimental autoimmune diseases, EAE, EAU and CIA,
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formerly considered as Th1-driven conditions, are now seen
as Th17-driven. Indeed, deficient production or action of IL-
17 is associated with loss of susceptibility to these
experimental autoimmune diseases [182,183]. Naı̈ve T cells
differentiate into Th17 cells when their cytokine environment,
created by antigen-presenting cells, is dominated by IL-6 and
IL-23. However, conditions for optimal in vitro induction of
IL-17-producing T cells by IL-23 were found to include
neutralization of endogenous IFN-g [68]. Also, during in vivo
infection with mycobacteria, ablation of IFN-g resulted in
increased numbers of IL-17-producing T cells [184]. This led
to the conclusion that endogenous IFN-g inhibits differentia-
tion of Th17 cells. Accordingly it is not more than normal that
functional ablation of endogenous IFN-g should boost disease
in EAE and CIA.
Another significant development of recent date has been
the re-emergence of the concept that T cell autoreactivity is
kept in check by ‘‘suppressor’’ T cells. Best studied so far are
the thymus-derived CD4+CD25+ natural regulatory T cells
(Treg cells). Their importance in controlling autoimmunity
is most clearly illustrated by the development of auto-
immune disease in athymic nude mice upon transfer of Treg-
depleted CD4+ T cells. In addition, many studies have
documented blocking of autoimmune diseases by injection
of syngeneic Treg cells. Two important aspects concern the
role of IFN-g in Treg functioning. In vitro treatment of
CD4+CD25 cells with IFN-g was shown to cause their
conversion into CD4+ Treg cells, with increased expression
of FoxP3, a specific marker for Treg cells, and acquisition of
full regulatory properties as evident by their ability to
suppress EAE by adoptive transfer [185]. Furthermore,
evidence exists for induced Treg cells to rapidly release IFN-
g, that may be important for their suppressive activity [186].
Increased severity of CIA in IFN-gR KO mice was found to
be associated with reduced Treg activity. In vitro analysis by
co-culturing Treg with Teff cells from both IFN-gR KO and
wild-type mice suggested that a third population of
accessory cells participates in the stimulatory effect of
IFN-g on Treg cells in wild-type mice [187].
Thirdly, studies on the role of IFN-g in regulating diversity
and function of DCs have revealed pathways by which IFN-g
can safeguard peripheral tolerance. One such pathway
concerns release by certain DCs of the enzyme indoleamino
2,3-dioxygenase (IDO) [188,189] (for review, see Ref.
[190,191]). Among cytokines IFN-g is the most potent
inducer of IDO. The enzyme degrades the indole moiety of
tryptophan, serotonin and melatonin, and produces kynur-
enines, some of which elicit apoptosis of T cells. By depleting
tryptophan, IDO also contributes to innate host resistance
against those pathogens that depend metabolically on
exogenous supply of the amino acid. However, local depletion
of tryptophan also inhibits proliferation of T cells. Similar
effects on T cell proliferation and induction of apoptosis have
been described for the IFN-g-induced production of nitric
oxide. IFN-g may also control peripheral tolerance by up-
regulating expression of B7-H1 and B7-H2 on APCs and
107
some other cells [192]. B7-H1 and B7-H2 are two recently
discovered members of the B7 family that negatively regulate
T cell activation by interaction with the ‘programmed death 1’
(PD1) molecule on T cells.
Whereas these models of IFN-g-mediated control of
autoimmune disease are focused on antigen-specific T cells
as taking the central position in gain or loss of peripheral
tolerance, it should be recognized that the protective effect
of IFN-g in experimental autoimmune disease may derive
from effects on non-antigen-specific stages. Intriguing is the
observation that the protective effect of IFN-g seems to be
limited to models that use CFA as an adjuvant to immunize
animals with the (auto)antigen. In the case of collagen-
induced arthritis, omission of CFA from the experimental
protocol was found to reverse the effect of endogenous IFN-
g from being protective to disease-promoting. Further
analysis of this system revealed the possibility that IFN-g
might act by inhibiting extramedullary myelopoiesis
brought about by the immunoadjuvant [193–195]. Even
more striking was the finding that injection of IFN-gR KO
mice with only CFA (without a joint-specific antigen)
resulted in the development of a full erosive arthritis [196].
These observations are highly suggestive of an important
contribution of innate immune mechanisms in these model
autoimmune diseases. Since CFA contains killed myco-
bacteria, toll-like receptors (TLR) come to mind as possible
players in autoimmune pathogenesis. Recognition of
microbial stimuli by TLRs on myeloid cells is essential
for the regulation of inflammatory reactions and adaptive
immunity but imbalances in the feedback control of TLR-
activated innate immune cells, by non-functioning of one
single cytokine such as IFN-g, may lead to autoimmunity,
and this will be an interesting field for further research.
11. Concluding thoughts
Almost 5 decades separate us from the first report on the
release by leukocyte cultures of an interferon-like activity
that would later appear to represent IFN-g. Since then, the
number and variety of biological activities assigned to IFN-g
have steadily increased. In immune networks IFN-g operates
at multiple successive levels. Moreover, actions at one level
may counteract effects at others. Though such pleotypic
effects and negative feedback loops are usual features of
cytokines in general, in the case of IFN-g, the long history of
research has made that we have a fairly detailed conception
of the resulting network and its impact on in vivo situations.
Today IFN-g, perhaps more than any other cytokine, figures
high in many of our pathogenesis schemes of anti-infectious
host defense, of inflammatory conditions and of auto-
immune disease. A remarkable feature in the history of IFN-
g research has been the evolution of its conceived place in
the immune system from a merely antiviral factor to a broad-
spectrum antimicrobial agent, and finally to an important
player in overall inflammatory responses to exogenous as
108 A. Billiau, P. Matthys / Cytokine & Growth Factor Reviews 20 (2009) 97–113
well as endogenous agents. As with other cytokines,
neutralizing antibodies and gene knock-out mice have been
foremost important tools to reveal these roles. However,
more than with other cytokines, the experimental results
have confronted researchers with difficult-to-resolve para-
doxes. Whereas multiple immunoregulatory pathways have
been identified by which these paradoxes can possibly be
resolved, the relative importance of each pathway in each
particular experimental situation remains to be determined.
A topic not covered in this historical review is the clinical
application of IFN-g. In contrast to Type I interferons, IFN-g
has not reached the stage of a clinically useful drug. While
pilot trials with exogenous administration of IFN-g have been
indicative of beneficial effects in some clinical conditions, in
particular visceral leishmaniasis and chronic granulomatous
disease, trials establishing applicability of IFN-g in major
human pathologies are not available. As evident from this
review, IFN-g has been demonstrated to play a critical role in
models of acute and chronic inflammatory diseases. Whereas
this role is comparable to that of TNF-a, antibodies against
IFN-g, unlike those against TNF-a, have so far not found their
way into the clinic.
In conclusion, two important further paths of discovery
still lie ahead: (1) annotating the network of interactions of
IFN-g such that it becomes increasingly suitable to interpret
its role in natural diseases, and (2) expanding the
applicability of IFN-g (or IFN-g antagonists) in the clinic.
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Alfons Billiau (January 20th 1937) is emeritus
professor of microbiology and immunology at
the University of Leuven (Katholieke Universi-
teit Leuven, KU Leuven). He formerly headed
the Laboratory of Immunobiology at the Rega
Institute for Medical Research, affiliated to this
University. This laboratory has a long-standing
tradition of pioneering work in the field of inter-
ferons, interleukins and other cytokines, as well
as chemokines and matrix metalloproteinases. In
the 1970s Billiau was particularly involved in pre-industrial production of
natural human fibroblast interferon (Interferon-b). Expertise of his labora-
tory in this domain seamlessly led to the isolation and characterization, in
the 1980s, of interleukin-1 as an interferon-b-inducing cytokine and of
interleukin-6 as a hybridoma growth factor co-produced with interferon-b.
Billiau’s name is also connected with studies on the peculiar mode of action
of interferon against retroviruses. More recent work under his leadership
bears on the role of interferon-g in animal models of inflammation and
autoimmune disease, in particular experimental autoimmune encephalo-
myelitis and collagen-induced arthritis.
Patrick Matthys (September 19th 1963) is Pro-
fessor in immunology at the University of Leu-
ven and a research director at the Rega Institute.
He earned his PhD in 1993 with experimental
work demonstrating the key role of IFN-g in
eliciting tumor-associated cachexia, a wasting
syndrome that had until then been attributed
mainly to the production of tumor necrosis fac-
tor/cachectin. As a postdoctoral fellow of the
Fund for Scientific Research Flanders, Matthys
continues conducting research at the Rega Institute, focusing on the
pathogenesis of autoimmune diseases, in particular autoimmune arthritis
and related pathology. The complex role of IFN-g in pathogenesis remains a
preferred subject of his investigations, however also with in-depth analysis
of interactions with other cytokines, chemokines and other inflammatory
mediators.