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Curr Immunol Rev. Author manuscript; available in PMC 2010 October 28.
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Curr Immunol Rev. 2005 June ; 1(2): 119–137.

Interleukin-12: an update on its immunological activities,

signaling and regulation of gene expression

Jianguo Liu, Shanjin Cao, Sunjung Kim, Elaine Y. Chung, Yoichiro Homma, Xiuqin Guan,
Violeta Jimenez, and Xiaojing Ma*

Abstract
Interleukin-12 (IL-12) is a heterodimeric cytokine composed of the p35 and p40 subunits. It is
produced by antigen-presenting cells and plays a critical role in host defense against intracellular
microbial infection and control of malignancy via its ability to stimulate both innate and adaptive
immune effector cells. The potency of IL-12 renders itself to stringent regulation of the timing,
locality and magnitude of its production during an immune response. Subversion of the delicate
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control and balance frequently leads to immunologic disorders. In this article, we provide an update,
since our last review of the subject four years ago, on recent advances in: (1) uncovering of novel
activities of IL-12 and related molecules in various immunological settings and models; and (2)
dissection of the physiological pathways involved in the modulation of IL-12 production by
pathogens and immune regulators. The increased understanding of IL-12 immunobiology and
expression will likely benefit the development of therapeutic modalities to correct immune
dysfunctions.

Keywords
Interleukin-12; antigen-presenting cell; macrophage; dendritic cell; T helper cell; natural killer cell;
cytotoxic T lymphocyte

I. Interleukin-12, the Molecule

Interleukin-12 (IL-12) was identified in the late 1980s from the culture media of Epstein-Barr
virus (EBV)-transformed lymphoblastoid B cell lines, which was able to stimulate natural killer
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(NK) cell activity, to generate lymphokine-activated killer (LAK) cells, and to induce the
production of Interferon-γ (IFN-γ) from NK and T cells [1]. It was found to be a heterodimeric
molecule, and the genes encoding the two-subunit chains of IL-12, p35 and p40, were cloned
[2,3]. The heterodimeric IL-12 is often referred to as IL-12 p70. The two genes encoding IL-12
p40 and p35 are located on separate chromosomes (chromosomes 11 and 6, respectively, in
humans and mice, respectively) [4–7] and are not evolutionary duplicates. The p35 cDNA
encodes a 209 amino acid polypeptide corresponding to a mature protein of 27.5 kDa. It
contains seven cysteine residues and 3 potential N-glycosylation sites. The p40 cDNA
sequence encodes a 328 amino acid polypeptide with a 22 amino acid signal peptide which
generates a mature protein of 34.7 kDa. It contains ten cysteine residues and four potential N-
glycosylation sites, and one consensus heparin-binding site [2,8]. The p35 gene has some
homology to IL-6 and granulocyte colony-stimulating factor (G-CSF) [9] whereas the p40

*
Corresponding author: Xiaojing Ma, Ph.D., Associate Professor, Department of Microbiology and Immunology, Weill Medical College
of Cornell University, Immunology Graduate Program, Weill Graduate School of Medical Sciences of Cornell University, 1300 York
Avenue, New York, NY 10021, Tel: (212) 746-4404, Fax: (212) 746-4427, [email protected], website:
http://www.med.cornell.edu/research/xma/index.html.
 Liu et al. Page 2

chain is homologous to the extracellular portion of the α chain of ciliary neurotropic factor
(CNTF) receptor as well as to those of the IL-6 and G-CSF receptors [10,11]. The p40 chain
carries the hallmarks of the hematopoietin receptor family: one tryptophan and four cysteine
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residues in conserved positions and a WSEWAS sequence, similar to the consensus motif
WSXWS in the hematopoietin receptor family [12]. Most of the members of this
transmembrane receptor family can be released from the producer cells in soluble forms
containing the extracellular portion truncated after the WSXWS motif by either alternative
splicing of the mRNA transcripts or by proteolytic digestion of the receptor [12].

Heterodimers of p40 and p35 are formed via disulfide bonds and secreted, usually upon
stimulation of producer cells. However, IL-12 may also exist as a preformed membrane
associated molecule that can be quickly released (within minutes) from phagocytic cells upon
contact with intracellular microbes such as Leishmania in the absence of de novo transcription
[13]. This is in contrast to the production of IL-12 induced by bacterial products such as
lipopolysaccharide (LPS), which takes place with much slower kinetics (several hours) and
which depends on de novo transcription. Another study demonstrated that IL-12 production
by neutrophils, which mobilize rapidly to the site of infection by the protozoan pathogen
Toxoplamsa gondii, appear to be derived from prestored pools [14].

In addition to forming heterodimers with p35, both mouse and human p40 are secreted in large
excess as free p40 monomers and can also form homodimers (p402), which exhibit biological
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activities antagonistic to heterodimeric IL-12 p70 [15,16]. The production of

immunosuppressive IL-12p40 homodimers was also induced in DCs and macrophages exposed
to ultraviolet radiation [17]. Surprisingly, Jana et al. found that IL-12 p70, p402 (the p40
homodimer) and p40 (the p40 monomer) all induced the production of TNF-α in BV-2
microglial cells and in mouse primary microglia and peritoneal macrophages [18].

In 2000, Oppmann et al. reported a novel gene, p19, discovered in a computational screen of
genomic databases, as a p35 homologue and dimerization partner with p40. The resulting
cytokine, named IL-23, has biological activities both similar to and distinct from those of IL-12.
In particular, IL-23 can induce strong proliferation of mouse memory (CD4+CD45Rblow) T
cells [19], resulting in elevated IL-17 secretion [20], while IL-12 does not manifest such
activities. IL-23 also proved to be the critical cytokine for autoimmune inflammation in the
brain, rather than IL-12, which had long been suspected to be the main culprit [21]. Production
of natural IL-23 heterodimers has been shown both in mice and in humans. Although the full
spectrum of cell types producing IL-23 is not known yet, dendritic cells (DCs) that are potent
producers of IL-12 are also able to produce IL-23.

Homodimers of p35 have not been reported to date. However, p35, which is not secreted in
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the absence of a second chain, may heterodimerize and be secreted together with a second
cellular protein, EBV-induced gene 3 (EBI-3) with limited homology to IL-12 p40, although
no biological function of this novel heterodimer has yet been demonstrated [22]. Searching
sequence databases with a computationally derived profile of members of the IL-6 helical
cytokine family led to the identification of yet another novel hematopoietic cytokine, p28,
which is distantly related to IL-12 p35 [23]. IL-27 is an early product of activated antigen-
presenting cells (APCs). It drives rapid clonal expansion of naïve but not memory CD4+ T
cells [23], in contrast to IL-23.

II. Cell Types That Produce IL-12

II.1. B lymphocytes
Although IL-12 was originally identified and purified from EBV-transformed B cell lines,
normal B lymphocytes are poor producers of IL-12 even in the activated state. Schultze et al.

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demonstrated that a subset of human tonsillar B cells can be induced to secrete bioactive IL-12
mainly via CD40 ligation facilitated by activated Th1 cells [24]. Expression after CD40
activation is restricted to CD38−IgD±, non-GC B cells. IL-12 produced from these cells is
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postulated to provide a positive feedback during T-B interactions, thereby maintaining the
differentiation pattern of the T cells during amplification of the immune response [24]. Using
CpG oligodeoxynucleotides (ODN) conjugated with an Ag (ovalbumin), Shirota et al. showed
that murine B cells could serve as efficient APCs independently of surface Igs [25]. The B cells
cultured with CpG-conjugated Ag not only enhanced IFN-γ formation by Th1 cells, but also
induced Th1 differentiation from unprimed T cells. These effects were associated with an
increase in the expression of CD40, CD86, and class II molecules on B cells and the coordinated
production of IL-12 [25].

II.2. Macrophages
It has been now firmly established that the major physiological cell types that produce IL-12p70
are APCs such as monocytes/macrophages [26] and DCs [27]. Within macrophages, the so-
called classically activated and alternatively activated macrophage populations differ in their
ability to produce IL-12. For instance, chronic helminth infection induces alternatively
activated macrophages to express high levels of CCR5 and low levels of IL-12, associated with
a poor ability to induce antigen-specific proliferation of CD4+ T cells [28].

II.3. Dendritic cells

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Humans have two distinct types of DC precursors. Peripheral blood monocytes give rise to
immature myeloid DCs after culturing with granulocyte-macrophage colony-stimulating factor
(GM-CSF) and IL-4 [29,30] or after transmigration through endothelial cells and phagocytosis
[31]. These immature cells become mature myeloid DCs (designated DC1s) after stimulation
with CD40 ligand (CD40L) or endotoxin [32,33]. The CD4+CD3−CD11c− plasmacytoid cells
from blood or tonsils give rise to a distinct type of immature DC with features of the lymphoid
lineage after culture with IL-3 [34–36]. These cells differentiate into mature DCs (designated
DC2s) after CD40L stimulation [36]. Rissoan et al. demonstrated that DC2s produce low levels
of IL-12 and direct Th2 differentiation, whereas DC1s produce high levels of IL-12 and skew
T cell differentiation toward Th1 [37].

Chang et al. described a phenotypically and functionally novel monocyte-derived DC1 (mDC1)
subset that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of
Th0/Th2 cells. Like conventional mDC1s, this novel subset expressed high levels of CD11c,
CD40, CD80, CD86, and MHC class II molecules, but lacked expression of CD1a [38]. This
population could be matured into CD83+DCs in the presence of anti-CD40 mAbs and LPS
plus IFN-γ, but it remained CD1a− and lacked IL-12 production even upon maturation. The
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lack of IL-12 and CD1a expression by these cells did not affect their APC capacity. However,
while the conventional mDC1s strongly favored Th1 differentiation, the novel subset directed
differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells,
further indicating functional differences between the two subsets of mDC1 [38].

Martin et al. reported a new B220+ subpopulation of immature-like human DCs (B220+DCs)
with low levels of expression of major histocompatibility complex (MHC) and costimulatory
molecules and markedly reduced T-cell stimulatory potential, located in the thymus, bone
marrow, spleen, and lymph nodes. B220+ DCs display ultrastructural characteristics
resembling those of human plasmacytoid cells and accordingly produce IFN-α after virus
stimulation. B220+DCs acquired a strong APC capacity upon incubation with CpG ODN,
concomitant with a strong upregulation of MHC and costimulatory molecules and the
production of IL-12 and IL-10. The unstimulated B220+DCs may represent a subset of
physiological tolerogenic DCs endowed with the capacity to induce a nonanergic state of T-

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cell unresponsiveness, involving the differentiation of T regulatory cells capable of suppressing

antigen-specific T-cell proliferation [39].
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In mice, DCs express CD11c molecules on the cell surface. DCs had been previously
subdivided on the basis of CD4 and CD8α expression [40]. Several studies suggested that
CD8α+ DCs preferentially induce Th1 responses, whereas CD8− DCs induce Th2 development.
Using a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA), to assess
stimulation of murine DC subsets and the relationship between Ag burden and IL-12
production, Huang et al. showed that DCs were the sole producers of IL-12, although most
HKBA uptake was by splenic macrophages and granulocytes. However, more CD8α− than
CD8α+ DCs produced IL-12 after HKBA challenge, whereas only CD8α+ DCs produced IL-12
in response to challenge with another Th1-promoting antigen, soluble Toxoplasma gondii Ags
[41]. These findings challenge the notion that CD8α+ and CD8α− DCs are destined to
selectively induce Th1 or Th2 responses, respectively. They suggest that the nature of the
stimulating substance is important in determining which DC subsets are activated to produce
IL-12. Recently, murine DCs were re-categorized with respect to function, especially in terms
of cytokine production and in vitro T cell allo-proliferation activity. Similar to the functional
classification for human DCs, murine DCs are now classified as either DCs that possess high
T cell allostimulatory activity and produce high levels of IL-12 (similar to human DC1), or as
plasmacytoid DCs (pDCs) that have low T cell allostimulatory activity and produce high levels
of IFN-α or IL-12 following viral or CpG challenge [42,43].
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Henri et al. reported a hierarchy of susceptibility of murine splenic DC subsets to infection by

Leishmania major and an inverse relationship to IL-12 production [44]. CD4+ CD8α− DCs are
the most permissive host cells for L. major amastigotes, followed by CD4− CD8α− DCs;
CD4−CD8α+ DCs are the least permissive. However, the least susceptible CD4− CD8α+ DC
subset was the best IL-12 producer in response to L. major infection.

Three populations of DCs have been identified in the murine Peyer’s patch (PP). CD11b+
CD8α− (myeloid) DCs are localized in the subepithelial dome, CD11b− CD8α+ (lymphoid)
DCs in the interfollicular regions, and CD11b− CD8α− (double-negative; DN) DCs at both
sites [45]. Furthermore, Iwasaki and Kelsall described the presence of a novel population of
intraepithelial DN DCs within the follicle-associated epithelium and demonstrated a
predominance of DN DCs only in mucosal lymphoid tissues [46]. All DC subpopulations
maintain their surface phenotype upon maturation in vitro, and secrete a distinct pattern of
cytokines upon exposure to T cell and microbial stimuli. In an effort to understand the
functional relevance of the three DC subsets, these researchers purified DC from spleen and
PP, and stimulated them in vitro either through CD40 cross-linking (to mimic a mature T cell
stimulus) or with Staphylococcus aureus (a microbial stimulus). Only myeloid DCs from the
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PP produced high levels of IL-10 upon stimulation with soluble CD40L trimer, or
Staphylococcus aureus. In contrast, lymphoid and DN, but not myeloid DCs, produce IL-12
p70 following microbial stimulation, whereas no DC subset produces IL-12 p70 in response
to CD40 ligand trimer. These findings thus suggest that DC subsets within mucosal tissues
have unique immune inductive capacities.

Edwards et al demonstrated that microbial and T cell-derived stimuli can synergize to induce
production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets, but that the
choice of cytokine is dictated by the microbial pattern recognition receptor engaged [47].
Bacterial components such as CpG-containing DNA or extracts from Mycobacterium
tuberculosis predispose CD8α+ and CD8α− CD4− DCs to make IL-12 p70. In contrast,
exposing CD8α+, CD4+ and CD8α−CD4− DCs to heat-killed yeasts leads to production of
IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells,
which can be replaced by CD40L. Moreover, M. tuberculosis extracts promote IL-12

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production primarily via the Toll-like receptor 2 (TLR2)- and MyD88-dependent pathway,
whereas heat-killed yeasts activate DCs via a TLR2-, MyD88-, and Toll/IL-1R domain-
containing protein-independent pathway. This study suggests the notion that T cell feedback
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amplifies innate signals for cytokine production by DCs and that pattern recognition rather
than ontogeny determines the production of cytokines by individual DC subsets.

Murine thymic DC subsets are comprised of two subpopulations based on CD8α chain
expression (CD8α+ and CD8α− DCs). These thymic DCs synthesize IL-12 p70 when stimulated
with a combination of lipopolysaccharide (LPS), anti-CD40 monoclonal antibody (mAb), GM-
CSF, and IFN-γ [48]. Okada et al. further reported the identification of a heterogeneous murine
thymic cell subset expressing CD11c and B220 (CD45R), following the previous identification
of the population of CD11c+ B220− DCs [49]. The CD11c+ B220+ subset expresses
Ly6Chigh and MHC class IIlow in contrast with the CD11c+ B220− subset. Freshly isolated
thymic CD11c+ B220+ cells show typical plasmacytoid morphology and differentiate to mature
DCs in vitro upon stimulation with CpG ODN 2216 (TLR9 ligands); they are thus termed
thymic plasmacytoid DCs (pDCs) [49]. These thymic pDCs are highly sensitive to spontaneous
apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDCs express low
TLR2, TLR3 and TLR4 mRNA, and high TLR7 and TLR9 mRNA. Thymic pDCs also produce
high amounts of IFN-α following culture with CpG ODN 2216 as compared with the
CD11c+ B220− thymic DC lineage, which expresses low TLR9 mRNA and produces high
IL-12p40 with CpG ODN 2216.
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When monocytes differentiate into DCs, their ability to respond to different commensal
bacteria dramatically changes, and they become unresponsive to probiotic gram-positive
bacteria. Karlsson et al. demonstrated this by stimulating purified human monocytes and
monocyte-derived DCs with UV-inactivated Gram-positive (Lactobacillus plantarum and
Bifidobacterium adolescentis) and Gram-negative (Escherichia coli and Veillonella parvula)
bacterial strains that are normal gastrointestinal bacterial flora. Monocytes produced higher
levels of IL-12 p70 in response to L. plantarum than in response to E. coli and V. parvula. In
contrast, DCs secreted large amounts of IL-12 p70 in response to E. coli and V. parvula but
were practically unresponsive to L. plantarum and B. adolescentis. The lack of a response to
the Gram-positive strains correlated with lower surface expression of TLR2 on DCs than on
monocytes [50].

The immunobiology of IL-12 is briefly summarized in Fig 1.

III. IL-12 Receptors and Signaling

IL-12 receptors, IL-12Rβ1 (low affinity, Kd = 2–6 nM, 1000–5000 sites per cell) and β2 (high
affinity, Kd = 5–20 pM, 100–1000 sites per cell) chains, are primarily expressed on activated
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T and NK cells [51]. Both of these subunits have extensive homology with gp130, the common
receptor β chain of the IL-6-like cytokine superfamily. Using flow cytometry, Vogel et al.
showed that freshly isolated murine peritoneal B-1 and conventional B lymphocytes bound
IL-12, but splenic B cells failed to react unless first stimulated with LPS. All murine B cell
sources were found to express the IL-12Rβ1 subunit transcripts. IL-12 binding was also
detected on S. aureus/IL-2-stimulated B cell blasts but not on freshly isolated peripheral blood
lymphocytes [52]. In human primary B cells, Durali et al. found a functional IL-12 receptor
(IL-12R) that internalizes following IL-12 binding. IFN-γ and, to a lesser extent, IL-12
positively regulated the IL-12Rβ2 subunit but had no effect on IL-12Rβ1. IL-12 induced the
phosphorylation and nuclear translocation of STAT4 while having no direct effect on STAT1
activation or T-bet (T-box expressed in T cells) expression in primary B cells [53]. These
findings indicate that B cells represent another major target for IL-12 in addition to T and NK
cells, and that IL-12 can directly affect humoral immunity.

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Signal transduction through IL-12R induces tyrosine phosphorylation of primarily the Janus
family kinases JAK2 and TYK2 [54], which in turn phosphorylate and activate STAT4 [55].
In addition to tyrosine phosphorylation, it has been demonstrated that STAT4 is phosphorylated
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on serine residue in response to IL-12 [56]. The IL-12-dependent STAT4 serine

phosphorylation is mediated by stimulation of p38 mitogen-activated protein kinase (MAPK)
through its upstream activators, MAPK kinase (MKK) 3/6, growth arrest and DNA damage
inducible (GADD)45-β and -γ [56,57]. It is not mediated by stimulation of extracellular signal-
regulated kinases (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Serine phosphorylation is
required for full transcriptional activity of STAT4 and IFN-γ production, but not for
proliferation. Moreover, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been
demonstrated to be activated by IL-12, and plays an important role in proliferation, but not in
IFN-γ production [58]. Using a yeast two-hybrid screening, Yoshimoto et al. identified mouse
sphingosine kinase 2 (SPHK2) as a molecule associated with the mouse IL-12Rβ1 cytoplasmic
region [59]. SPHK is a key enzyme catalyzing phosphorylation of sphingosine to form
sphingosine 1-phosphate (S1P), an important lipid messenger that is implicated in the
regulation of a wide variety of important cellular events, including cell growth, survival,
motility, cytoskeletal changes, and the release of calcium from intracellular stores [60].
Reciprocal mutagenesis analyses of the two interacting molecules revealed that the region
including the proline-rich domain in SPHK2 is probably responsible for the binding to
IL-12Rβ1, while the regions including the carboxyl terminus and Box II in the IL-12Rβ1
cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of
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wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated

transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone
significantly reduced IL-12-induced IFN-γ production, while that of wild-type SPHK2
enhanced it. In contrast, the expression of dominant-negative SPHK2 minimally affected
IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-γ production was
observed when dominant-negative SPHK2 was expressed in activated primary T cells using a
retroviral expression system [59].

During IL-12-induced signaling, activated STAT4 is recruited to phosphorylated Tyr-800 in

the human IL-12Rβ2. Yamamoto et al. demonstrated that suppressor of cytokine signaling-3
(SOCS-3) is also recruited to IL-12Rβ2 by the interaction involving the SOCS-3 SH2 domain
and phosphorylated Tyr-800 in IL-12Rβ2 [61]. Furthermore, SOCS-3, but not its SH2 domain-
defective mutant, inhibited the IL-12-induced activation of DNA-binding and transcriptional
activities of STAT4 [61]. These results suggest that SOCS-3, expressed at high levels in Th2
cells, plays an inhibitory role in STAT4-mediated IL-12 signaling by binding to the STAT4
docking site in IL-12Rβ2.

To note, the newly discovered IL-12-related cytokine IL-27 is reportedly able to set the early
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stage of Th1 differentiation by potently inducing the expression of the major Th1-specific
transcription factor T-bet and its downstream target IL-12Rβ2 independently of IFN-γ, thus
conferring upon the naïve T cells responsiveness to IL-12 [62].

IV. Immunological Activities of IL-12

IV.1. IL-12 in infectious diseases
IL-12 is produced by phagocytic APCs in response to intracellular bacterial and parasitic
infections. The importance of IL-12 in host resistance to infectious agents is underscored by
IL-12Rβ1 expression deficiency found in otherwise healthy individuals highly susceptible to
mycobacterial [63] and Salmonella [64] infections. IL-12 p40 expression deficiency in a young
female patient was associated with recurrent episodes of pneumococcal pneumonia with sepsis
and other infections in the absence of fevers [65]. Because of the shared cytokine chain of
IL-12 p40 and the β1 chain of the receptor between IL-12 and IL-23, it is difficult to discern

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the relative importance of IL-12 and IL-23 in host defense against intracellular microbes.
Although the relative roles of IL-12 and IFN-γ in Th1-cell priming may be to a significant
extent pathogen-dependent, in most infections IL-12 regulates the magnitude of the IFN-γ
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response at the initiation of infection, thus potentiating natural resistance, favoring Th1-cell
development, and inhibiting Th2 responses. Treatment of animals with IL-12, either alone or
as a vaccine adjuvant, has been shown to prevent diseases caused by many of the same
infectious agents, by stimulating innate resistance or promoting specific reactivity [66].

IV.2. IL-12 in malignant diseases

Recombinant human IL-12 has been studied as a single agent for systemic treatment of various
types of cancer in patients. Following the first phase II clinical trial that unexpected resulted
in severe toxicity and deaths [67], improvements have been made with respect to the insertion
of a treatment-free period of one week after the first administration of IL-12, conforming with
most phase I studies, which reduced toxicity of subsequent injections [68,69]. However, two
recent phase II studies performed in patients with advanced renal cell and ovarian cancer
yielded disappointing results, with overall response rates of only 7% and 4%, respectively
[70,71]. The lack of efficacy in these studies is postulated to be due to endogenous IL-10
production which occurs at relatively high dose levels of IL-12, countering the biological
effects of IL-12. However, a strong CTL response was observed in patients with advanced
melanoma after IL-12 administration. The number of tumor-specific CTL increased in the
circulation, and influx of specific memory CD8+ T cells into metastasized lesions was
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demonstrated [72].

Alternatively, greater efficacies have been achieved using IL-12 in different types of cancer
vaccines as an adjuvant. Many animal studies have shown that IL-12 plus tumor vaccine was
more effective and less toxic than either component alone [73–79]. The effects of IL-12 as a
vaccine adjuvant are believed to be related to its ability to induce multiple inflammatory
cytokines such as GM-CSF, TNF-α, IL-8, IL-6, IL-15 and IL-18 [80,81], and to activate NK
cells [82], to enhance the function of DC such as their maturation and antigen presentation
[83], and to prime naïve T cells. The use of IL-12 can also induce tumor-specific humoral
immunity. In evaluating the efficacy of DC-based and/or IL-12 gene-based therapy in the
treatment of 38C13 B cell lymphoma using a hydrodynamic transfection-based technique to
deliver a high and persistent level of IL-12 from a plasmid encoding IL-12 (pIL-12), it was
found that either treatment alone was ineffective. However, a combined treatment induced
100% long-term survival [84]. Furthermore, a long-lasting anti-tumor immunity was induced
in the mice which resisted further tumor challenge 58 days after initial inoculation. The
surviving mice showed a strong IFN-γ-producing Th cell response and humoral antibody
response, without detectable CTLs. The antibody from the immune sera mediated a
complement-dependent lysis of tumor cells that was tumor specific. Furthermore,
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immunization of mice with DC-based vaccine and pIL-12 treatment elicited higher levels of
anti-idiotype IgG titer and an enhanced IgG2a response which increased the efficacy in
mediating 38C13 tumor lysis [84].

A phase II human study of immunization with Melan-A peptide-pulsed PBMC plus

recombinant human IL-12 was conducted in 20 patients with advanced melanoma (who had
received prior therapy and had visceral metastases). Two patients achieved a complete
response, five patients achieved a minor or mixed response, and four patients had stable disease.
There were no grade 3 or 4 toxicities. There was a significant increase after vaccination in
Melan-A-specific CD8+ T-cell responses by IFN-γ production. There was a correlation
between the magnitude of the increase in Melan-A-specific T cells and clinical response [85].
Evidence that IL-12 may increase the immune response was also obtained in a phase I study
involving 48 patients with high-risk resected stage III or IV melanoma, who were immunized

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with melanoma-specific peptides with or without a low dose of IL-12 [86]. These data suggest
that even in advanced cancer patients, IL-12 can stimulate antigen-specific immune responses
and supporting further development of IL-12 as a vaccine adjuvant.
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IFN-γ mediates most of the well-known immunological activities of IL-12. Shi et al. recently
reported IFN-γ-independent activities induced by therapeutic application of recombinant IL-12
in restricting tumor growth and metastasis in the 4T1 murine mammary carcinoma model
[87]. IFN-γ-deficient mice carrying 4T1 tumor exhibited no gross defect in the number of
tumor-infiltrating lymphocytes but have exaggerated angiogenesis in the tumor.
Administration of IL-12 significantly restricted neoangiogenesis in the tumor in the absence
of IFN-γ, and retained certain therapeutic efficacy even when applied late during tumor
progression. IL-12 exposure in vivo did not irreversibly modulate the immunogenicity of the
tumor. A global gene expression analysis of primary tumors revealed interesting IL-12-induced
molecular patterns and changes, implicating a number of novel genes potentially important for
IFN-γ-independent immune responses against the tumor, as IL-12-mediated anti-proliferation,
anti-metastasis, and anti-angiogenesis activities [87].

IV.3. IL-12 in autoimmune diseases

The property of IL-12 to strongly promote the development of Th1 cells is a double-edged
sword. It endows IL-12 with the ability to orchestrate host defense against intracellular
infectious agents and malignant growth on the one hand, and the possibility to cause or
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exacerbate inflammatory cell-mediated diseases such as autoimmune disorders on the other.

IL-12-mediated inflammatory pathogenesis has been reported in such autoimmune diseases as
inflammatory bowel disease (IBD), collagen-induced arthritis (CIA), and insulin-dependent
diabetes mellitus (IDDM). IL-12 was for many years thought to be responsible for the T cell-
mediated pathogenesis in experimental autoimmune encephalomyelitis (EAE) until recently
when IL-23 was shown to be the “culprit” [21]. IBD, including Crohn’s disease (CD) and
ulcerative colitis (UC), is a chronic inflammatory disease that causes destruction of
gastrointestinal tissue. It is characterized by an exaggerated immune response. In CD, IL-12
plays a pivotal role in driving increased expression of Th1 cytokines and the associated
pathology [88].

Mutations in CARD15, which encodes nucleotide-binding oligomerization domain 2 (NOD2),

underlie the occurrence of intestinal inflammatory disease in a substantial subgroup of patients
with Crohn’s disease [89]. NOD2 is a member of the NOD-leucine-rich repeat (LRR) protein
family, whose members share a tripartite domain structure consisting of a C-terminal peptide
recognition (LRR) domain, a central NOD domain, and an N terminus made up of protein-
protein interaction domains, such as caspase recruitment domains (CARDs) or pyrin domains
[90]. NOD2 is expressed intracellularly in APCs [91] and through its C-terminal LRR it allows
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these cells to recognize and react to a component of bacterial peptidoglycan (PGN), muramyl
dipeptide (MDP) [92,93]. Watanabe et al. used Card15−/− mice to show that intact NOD2
signaling inhibited TLR2-driven activation of NFκB, particularly of the NFκB subunit c-Rel.
Moreover, NOD2 deficiency or the presence of a CD-like Card15 mutation increased Toll-like
receptor TLR2-mediated activation of NFκB-c-Rel, IL-12 production, and enhanced Th1
responses [94].

CIA is an experimental autoimmune model for human rheumatoid arthritis. Administration of

an anti-IL-12 antibody significantly reduces the symptoms of arthritis and abrogates the
humoral and Th1 cytokine response to the autoantigen collagen type II [95]. Paradoxically,
these effects are observed in IFN-γ receptor-null mice [95], suggesting that endogenous IL-12
can promote Th1 and disease pathology through a pathway independent of IFN-γ production.
In the insulin-dependent diabetes mellitus (IDDM) model, endogenous IL-12 appears to be
inessential for spontaneous development of the disease, since IL-12 p40-null non-obese

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diabetic (NOD) mice developed IDDM and insulitis as well as wild type mice [96]. However,
Th1 development in peripheral tissues, but not in the inflamed pancreas, was impaired in IL-12
p40-knockout NOD mice. Moreover, the pancreas-infiltrating T cells in NOD mice treated
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with IL-12 antagonists were skewed to a Th2 phenotype and the treated mice were protected
from IDDM [96]. Interestingly, a similar pattern of pathogenic and protective effects of IFN-
γ was seen in this model [97].

V. Receptor-mediated Regulation of IL-12 Gene Expression

V1. Induction of IL-12 production via Toll-like receptors (TLRs)
Microbial recognition and differentiation are mediated in part by pattern recognition receptors
(PRRs). The Toll-like receptor (TLR) family is the best characterized class of PRRs in
mammals. There are an estimated 10–15 TLRs in different mammalian species [98]. TLRs
detect multiple pathogen-associated molecular patterns (PAMPs), including LPS (by TLR4),
bacterial lipoproteins and lipoteichoic acids (LTA) (by TLR2), flagellin (by TLR5), the
unmethylated CpG DNA of bacteria and viruses (by TLR9), double-stranded RNA (by TLR3),
and single-stranded viral RNA (by TLR7) [99–101]. Qi et al. evaluated the role of TLR4 and
TLR2 in the induction of IL-12 and IL-10 by their respective ligands, LPS, PGN and yeast
zymosan, respectively, in bone marrow-derived mouse DCs. LPS induced low-levels of IL-10
but high-levels IL-12 p70 production. In contrast, DCs exposed to PGN produced low levels
of IL-12 but high levels of IL-10. Zymosan-exposed DCs produced high levels of both IL-10
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and IL-12 [102]. This observation suggests that LPS, PGN, and zymosan have inherently
distinct abilities to induce DC IL-10 and IL-12 production. Alternatively, this phenomenon
may reflect different sensitivities of DCs to these microbial stimuli. In addition, TLR2 functions
by heterodimerizing with TLR1 and TLR6 in order to recognize ligands [103]. Lore et al.
analyzed the effects of different TLR ligands to enhance immune responses induced by human
APCs, including CD123+ plasmacytoid DCs (PDCs), CD11c+ myeloid DCs (MDCs),
monocytes, and B cells. PDCs, which express TLR7 and TLR9, responded to
imidazoquinolines (imiquimod and R-848, synthetic ligands for TLR7) and to CpG-ODN (for
TLR9) stimulation, resulting in enhancement in expression of costimulatory molecules and
induction of IFN-α and IL-12 p70. In contrast, MDCs, which express TLR3, TLR4, and TLR7,
responded to poly(I:C), LPS, and imidazoquinolines with phenotypic maturation and high
production of IL-12 p70 without producing detectable IFN-α [104]. Agrawal and coworkers
demonstrated that E. coli flagellin, which engages TLR5, triggers immature human monocyte-
derived DCs to stimulate Th1 responses via IL-12 p70 production in a manner that depends on
the phosphorylation of p38 and JNK 1/2 [105].

V2. Induction of IL-12 production viaCD40

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In addition to the innate pathway of induction of its synthesis through TLRs, IL-12 production
by macrophages or DCs can also be induced during a memory immune response via contact
with activated T cells by the CD40/CD40L interaction. Kennedy et al. first showed that the
expression of CD40L by activated T cells is critical for T cell-dependent IL-12 production by
mouse macrophages [106]. However, Padigel et al. recently reported that CD40L−/− mice could
control Leishmania major infection when inoculated with low numbers of parasites and that
cells from these mice produce IL-12 [107,108]. Moreover, in vivo treatment with a TNF-related
activation-induced cytokine (TRANCE, also known as RANK-L)-receptor fusion protein in
CD40L−/− mice results in a decrease in the number of IL-12-producing cells as well as a shift
from a dominant Th1 to Th2 type response in infected mice [109]. The interaction of TRANCE
and its receptor RANK (TRANCE-R) is important for bone remodeling and essential in the
development and activation of osteoclasts [110]. TRANCE, expressed on activated T cells, can
induce IL-12 production via its interaction on Dcs with RANK, and also enhance DC survival
[111]. These results demonstrate that in CD40L−/− mice the TRANCE-RANK costimulatory

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pathway is alternatively used to promote IL-12 production and the activation of a protective
Th1 type response. Yu et al. studied the role of three signaling pathways, p38MAPK, ERK,
and PI3K, in CD40L-induced monocyte-derived DC activation, survival, and expansion of
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virus-specific CD8+ T cell responses. The study showed that the p38MAPK pathway was
critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation, and that
CD40L-induced monocyte-derived DC IL-12 production is mediated by both the p38MAPK
and PI3K pathways [112]. Paradoxically, IL-10 is also induced by the CD40/CD40L interaction
in macrophages. How CD40 signaling regulates the secretion of these counteractive cytokines
is the subject of another study, which showed that weak CD40 signals induce ERK-1/2-
dependent IL-10 expression, whereas stronger signals induce p38MAPK-dependent IL-12
production [113].

V3. CCR5-mediated induction of IL-12 production in dendritic cells

The activation of DCs to produce IL-12 is thought to be a key step in the initiation of cell-
mediated immunity to intracellular pathogens. Aliberti et al. first showed that chemokines were
rapidly induced in the spleen of mice injected with soluble toxoplasma antigen (STAg) of
tachyzoites of Toxoplasma gondii [114]. Ligation of the C-C chemokine receptor (CCR) 5 can
provide a major signal for the induction of IL-12 synthesis by the CD8+ subset of DC by T.
gondii and this pathway appears to be important in establishing interferon-dependent resistance
to the parasite. Purification of the STAg extract showed that cyclophilin-18 (C-18) was its
principal component [115]. Antibodies generated against recombinant C-18 inhibited STAg-
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induced synthesis of IL-12. Recombinant C-18 showed high affinity for and triggered cell
signaling through CCR5. These findings suggest that the unusual potency of T. gondii in
inducing IL-12 from DCs results from its synthesis of a unique chemokine mimic that signals
through CCR5. The ability to generate this strong protective response may benefit parasite
transmission by preventing the protozoan from overwhelming its intermediate hosts.

However, a seemingly opposite role of CCR5 in the regulation of IL-12 was observed in an
oral tolerance experimental autoimmune encephalomyelitis (EAE) mouse model [116].
DePaolo et al. showed that two CCR5 ligands, CCL4 and CCL5, are expressed in gut tissues
after feeding of the antigen myelin oligodendrocyte glycoprotein (MOG). CCR5−/− mice were
unable to be tolerized by feeding a high dose of MOG and were not protected from developing
EAE. Moreover, CCR5−/− mice fed with MOG displayed higher IL-12 production in the
intestinal mucosa compared to the wild type mice. A selective CCR5 antagonist, methionine
(Met)-RANTES inhibited CCL2 expression, resulted in enhanced IL-12 production and the
inability for mice treated with Met-RANTES to become orally tolerized [116]. This study
suggests that CCR5 ligands may function inhibit IL-12 levels in the gut after Ag feeding,
promoting a cellular environment that favors tolerance rather than immunity.
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V4. IFN-γ priming for IL-12 production

IL-12 production in primary monocytes is strongly dependent on the activation state of the
cells. IFN-γ provides a powerful stimulation signal for monocytes to become activated
macrophages and bactericidal with a much enhanced potential to produce IL-12, although it
alone does not induce IL-12 p40 gene expression. This is referred to as the “priming” effect of
IFN-γ on monocytes or monocytic cell lines [117]. IFN-γ’s enhancing effect is likely mediated
through activation of some members of the Interferon Regulatory Factor (IRF) family induced
by IFN-γ, particularly IRF-1 and Interferon Consensus Sequence Binding Protein (ICSBP or
IRF-8) that interact directly with the IL-12 p40 and p35 promoters in a synergistic manner
[118,119]. The priming effect of IFN-γ for augmented IL-12 production may represent a
mechanism by which a robust IL-12-induced Th1 response is invoked and sustained in vivo.

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V5. Complement receptor-mediated inhibition of IL-12 production

Marth and Kelsall reported that cross-linking of complement receptor (CR) 3 (also known as
CD11b or Mac-1) with antibody or certain particulate ligands (including particles coated with
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the complement C3 activation product iC3b) inhibits IL-12 production by both murine and
human monocytes/macrophages with little if any down regulation of the production of other
proinflammatory cytokines or chemokines [120]. On the other hand, ligation of iC3b to CR3
on antigen-presenting cells leads to the sequential production of TGF-β2 and IL-10, which is
essential for the induction of tolerance in an immune-privileged site after intraocular antigen
injection [121]. IL-10, and possibly TGF-β, can inhibit IL-12 production in an autocrine
manner. The ability of CR ligation to specifically inhibit IL-12 production suggests that
complement activation products can directly regulate the type of immune response through
interaction with APCs.

V6. Fcγ receptor-mediated inhibition of IL-12 production

Ligating Fcγ receptors (which bind to the Fc portion of IgG often complexed with an
immunogen) on macrophages by immune complexes (IC) results in profound and selective
suppression of pathogen-activated IL-12 production [122–124]. Cross-linking FcγRI, -II, and
-III inhibited IL-12 p70 production in monocytes, whether stimulated by Staphylococcal
enterotoxin B or LPS. Inhibition of IL-12 by FcγR cross-linking was not mediated by TNF-
α, as the presence of an anti-TNF-α Ab could not restore the reduced IL-12 production [125].
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However, the inhibitory effect of ICs on IL-12 p40 production can be converted into a
stimulatory one when heat-inactivated normal human serum (NHS) devoid of a functionally
intact complement system was used [126]. The effect was seen only for IL-12 p40, as
production of IL-6 and IL-10 is stimulated by immune aggregates (IA), consisting of heat-
aggregated gamma globulin (HAGG) as model IC, in the context of native NHS, whereas the
effect was abolished in heat-inactivated NHS [126]. IA-induced IL-12p40 production in a C4
deficient serum was lowered by addition of C4, and addition of the peptide compstatin, which
blocks C3 activation and mimicked the effects of heat inactivation on IL-12p40 levels. IA-
induced production of IL-10 was partially blocked by anti-Fcγ RII antibodies, whereas Fcγ R
or CR blockade had no effect on IL-12p40 production [126]. Since IC and local or systemic
complement activation characterize rheumatoid arthritis, systemic lupus erythematosus and
many malignancies, different and complement-dependent effects on the production of IL-10
and IL-12 could be of importance in these diseases, where control of the complement system
might be a way to direct IC-induced cytokine production in either a type 1 or type 2 direction.

V7. CD47-mediated inhibition of IL-12 production

Thrombospondin 1 (TSP) elicits potent anti-inflammatory activities in vivo, as evidenced by
persistent, multi-organ inflammation in TSP-null mice. TSP is believed to be the natural ligand
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of CD47 antigen, also named integrin-associated protein (IAP) [127], which transiently
accumulates at the inflammatory site. Engagement of CD47 by anti-CD47 monoclonal
antibodies, by TSP, or by 4N1K, a peptide of the COOH-terminal domain of TSP selectively
binding CD47, inhibits IL-12 release by monocytes and DCs [128,129]. Furthermore, CD47
ligation selectively inhibits the development of human naive T cells into Th1 effectors in the
presence of exogenous IL-12, suggesting that it also interferes with IL-12 downstream
signaling [130]. Human monocyte-derived immature DCs spontaneously produce TSP, which
is strongly enhanced by prostaglandin (PG)E2 and to a lesser extent by TGF-β, two soluble
mediators secreted by macrophages after engulfment of damaged tissues [131]. Activation of
DCs by microbial stimuli increases TSP production. The endogenous TSP produced during
early DC activation negatively regulates IL-12, TNF-α, and IL-10 release through its
interactions with CD47 and CD36 [131]. DC-derived TSP thus may serve as a negative

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regulator that contributes to arrest of cytokine production, active resolution of inflammation,

and maintenance of homeostasis.
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V8. IL-10-mediated inhibition of IL-12 production

IL-10 is a major macrophage-deactivating and immunosuppressive cytokine. It is a critical
component in the maintenance of the fine balance between swift and potent immune responses
against invading pathogens on the one hand, and the control of detrimental systemic
inflammation on the other. IL-10 is a potent inhibitor of IL-12 production in accessory cells,
which occurs primarily at the level of transcription of the IL-12 p40 gene [132]. However, the
transcriptional mechanism whereby IL-10 inhibits IL-12 p40 expression has remained un-
established. Recently, Cao et al. reported that IL-10 and c-musculoaponeurotic fibrosarcoma
(Maf) induce their mutual expression in inflammatory macrophages [133]. They demonstrated
that c-Maf, an essential transcription factor for development [134] and IL-4 gene expression
in Th2 differentiation [135], is also a physiological mediator of IL-10’s immunosuppressive
activities in macrophages. When overexpressed, c-Maf selectively inhibits transcriptional
activation of IL-12 p40 and p35 genes while potently activating IL-10 and IL-4 expression,
potentially contributing to the development of a state of anti-inflammation and dichotomy of
immunologic polarization [133]. c-Maf induces changes in nuclear DNA-binding activities at
multiple sites including the ets (E26), GA-12, NF-κB, C/EBP, and AP-1 elements. Nonetheless,
the essential c-Maf-responsive element appears to be located elsewhere. Inhibition of IL-12
p40 gene expression by c-Maf requires the N-terminal transactivation domain, suggesting an
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indirect mechanism of transcriptional inhibition involving the induction of an unidentified

repressor. In c-Maf-deficient murine macrophages, IL-10 production is impaired. However,
IL-10-mediated inhibition of IL-12 production remains intact, indicating the existence of
alternative mediators in the absence of c-Maf [133].

V9. Effects of type I interferons on IL-12 production

Type I interferons (IFN-α/β) are potent antiviral and immunoregulatory cytokines. Although
first noted for their ability to inhibit viral replication, type I interferons are also known to exert
multiple immunoregulatory effects on NK and T cells [136], with some of the functions
overlapping those of IL-12. Certain viral infections induce IL-12 to elicit NK cell IFN-γ
production and antiviral mechanisms. However, high levels of IFN-α/β are dominant in the
context of viral infections and act to regulate other innate responses, including induction of
NK cell proliferation in vivo and overall negative regulation of IL-12 production [137]. Byrnes
et al. showed that type I IFNs are potent inhibitors of IL-12 production by pathogen-activated
human monocytes/macrophages. The underlying mechanism involves transcriptional
inhibition of the IL-12 p40 gene, marked by downregulation of PU.1 binding activity at the
upstream Ets site of the IL-12p40 promoter [138]. However, in a separate study, Heystek et al.
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investigated the direct effect of IFN-α/β on monocyte-derived DCs at different stages of

development, and found that IFN-α/β enhanced IL-12 p70 production by immature DCs but
inhibited IL-12p70 production by mature DCs [139]. Interestingly, IFN-α/β strongly
counteracted the IL-12-enhancing effect of IFN-γ on DCs regardless of their maturation status
[139]. The differential modulatory effect of IFN-α/β on the IL-12-producing capacity of DCs
and their cross-regulatory effect on IFN-γ may reduce inflammatory processes. The IL-12-
enhancing effect of IFN-α on DCs was corroborated in an independent study where double-
stranded RNA was used to induce DC maturation and cytokine production. IFN-α enhanced
the production of IL-12 and TNF-α induced by double-stranded RNA but had no effect on
IL-10 production [140].

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V10. Adrenoceptors-mediated regulation of IL-12 production

Catecholamines are a class of endogenous mediators that may potentially direct the
responsiveness of macrophages through α- or β-adrenoceptors. Expression of α2- and β-
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adrenoceptors on macrophages can be activated by the endogenous ligand norepinephrine and

by adrenergic drugs frequently used in clinical practice [141,142]. Stimulation of these
receptors affects lymphocyte trafficking [143], migration [144] and proliferation [145]. They
also modulate cytokine production and the functional activity of different lymphoid cells
[146]. Accumulative evidence indicates that agents such as catecholamines that stimulate the
β2-adrendoreceptor-cAMP-protein A pathway inhibit the production of type 1/
proinflammatory cytokines such as IL-12, TNF-α and IFN-γ by APCs or Th1 cells [147–
149]. In contrast, the same compounds stimulate the production of type 2/anti-inflammatory
cytokines such as IL-10 and TGF-β [150]. Stimulation of cells via the α2-adrenoceptor by
agonists such as clonidine, guanfacine, and oxymetazoline, however, significantly induced
IL-12 p40 and p70 production by macrophages in a PKC- and p38MAPK-dependent manner
[151].

V11. Apoptotic cell-mediated inhibition of IL-12 production during macrophage

phagocytosis
The elimination of apoptotic cells and cell bodies by phagocytes represents an evolutionarily
conserved means to prevent exposure of surrounding tissue to potentially cytotoxic,
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immunogenic, or inflammatory cellular contents [152]. Resolution of inflammation depends

not only on the effective removal of apoptotic cells but also on active suppression of
inflammatory mediator production. Aberrations in either mechanism are associated with
chronic inflammatory conditions and autoimmune disorders [153]. Cytokines play significant
roles in the etiology and pathology of many autoimmune diseases. The uptake of apoptotic
cells by phagocytes is thought to suppress autoimmune responses in part through the release
of IL-10, TGF-β, platelet activating factor (PAF), and PGE2, and inhibition of TNF-α, GM-
CSF, IL-12, IL-1β, and IL-18 production [154]. Suppression of the production of inflammatory
cytokines such as IL-12 during the clearance of dead cells by professional phagocytes is a
critical mechanism to generate a tolerant state in the immune system to autoantigens [155].
Recently, Kim et al. explored how apoptotic cell-derived signals regulate IL-12 gene
expression [156]. They demonstrated that cell-cell contact with apoptotic cells is sufficient to
induce profound inhibition of IL-12 production by activated macrophages. The cell membrane
lipid molecule phosphatidylserine (PS), which becomes externalized during apoptosis and
which serves as a critical recognition molecule on apoptotic cells for clearance by phagocytes,
could mimic the inhibitory effect. The inhibition does not involve autocrine or paracrine actions
of IL-10 and TGF-β. These researchers identified a novel zinc finger nuclear factor, named
GC-binding protein (GC-BP), that is induced following phagocytosis of apoptotic cells by
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macrophages or by treatment with PS. GC-BP selectively inhibits IL-12 p35 gene transcription
by binding to its promoter in vitro and in vivo, thus decreasing IL-12 production. GC-BP itself
undergoes functionally significant tyrosine dephosphorylation in response to apoptotic cells.
These findings significantly enhance the understanding of an essential physiological process
in which cytokine responses are tightly regulated, with implications in the development and
pathogenesis of inflammatory and autoimmune diseases.

V12. Other G protein coupled receptor-mediated inhibition of IL-12 production

PGE2 is one of the major immunosuppressive factors derived from many cell types including
macrophages and some tumors. Mitsuhashi et al. reported that murine mammary carcinoma-
derived PGE2 potently inhibits the production of endogenous IL-12 at the level of protein
secretion, mRNA synthesis, and transcription of the constituent p40 and p35 genes. The
inhibition can be reversed by NS-398, an inhibitor of the enzymatic activity of cyclooxygenase

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2 in PGE2 synthesis. Moreover, PGE2-mediated inhibition of IL-12 production requires the

functional cooperation of AP-1, and AP-1 strongly suppresses IL-12 p40 transcription. This
study reveals a molecular mechanism underlying the interaction between a progressive
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malignancy and the immune defense apparatus. PGE2 or IL-4 treatment of IFN-γ and LPS-
activated primary human monocytes has been shown to induce a novel binding activity to a
repressor element, a purine-rich sequence at −155 termed GA-12 (GATA sequence in the IL-12
promoter) [157].

Prostaglandin D2 (PGD2) and its metabolites are known to be important mediators during acute
and chronic inflammation. Faveeuw et al. showed that PGD2 inhibits the CD40- and LPS-
induced secretion of IL-12 by murine splenic DCs [158]. The inhibition of IL-12 production
is mediated only partially by the cell surface Gαs protein-coupled D prostanoid receptor (DP1)
but not by the Gαi protein-coupled DP receptor, DP2. Recruitment of DP1 in DC results in the
activation of a cyclic AMP/protein kinase A pathway which is in part responsible for the
inhibition of IL-12 production [158].

VI. Intracellular Signaling in IL-12 Gene Expression

Hacker and coworkers [159] analyzed the MAPK pathways triggered by CpG-DNA and their
significance for cytokine production in macrophages and DCs, and found that CpG-DNA
induced ERK activity in macrophages in a classic MAPK/ERK kinase (MEK)-dependent way.
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This pathway upregulated TNF-α production, but downregulated IL-12 production. However,
in DCs, CpG-DNA and LPS failed to induce ERK activity. Consistent with a specific negative
regulatory role for ERK in macrophages, chemical activation of this pathway in DCs
suppressed CpG-DNA-induced IL-12 production. These results suggest that differential
activation of MAP kinase pathways may be a basic mechanism by which distinct subsets of
innate immune cells regulate their effector functions.

In a study to define distinct signaling mechanisms that regulate LPS-mediated induction of

IL-12 p40 and p35 in macrophages, Goodridge et al. reported differential regulation of IL-12
p40 and p35 induction via ERK MAPK-dependent and -independent mechanisms [160]. While
LPS-induced p38 MAPK activation is required for the induction of both p40 and p35 subunits,
ERK MAPK signaling mediates negative feedback regulation of p40, but not p35, production.
Such ERK activation is downstream of calcium influx and targets LPS-induced IL-12 p40
transcription by suppressing the synthesis of IRF-1. In contrast, negative regulation of the p35
subunit of IL-12 occurs via a calcium-dependent, but ERK-independent, mechanism likely to
involve NFκB signaling [160].

Sugimoto et al. recently identified a serine/threonine kinase, Cot/Tpl2, as a modulator of

bacterial DNA-induced IL-12 production and Th cell differentiation [161]. Cot/Tpl2 is
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indispensable for ERK activation and production of TNF-α and PGE2 in LPS-stimulated
macrophages, but is not essential for bacterial CpG-DNA-mediated ERK activation. Peritoneal
macrophages and bone marrow-derived DCs from Cot/Tpl2−/− mice produced significantly
more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production
in Cot/Tpl2−/− macrophages is at least partly regulated at the transcriptional level, and the
elevated IL-12 p40 mRNA level in Cot/Tpl2−/− macrophages is accompanied by decreased
amounts of IL-12 p40 transcription repressors, such as c-Maf and GATA sequence in the IL-12
promoter-binding protein (GA-12-binding protein; GAP-12). Consistently, Cot/Tpl2−/− mice
showed Th1-skewed antigen-specific immune responses upon OVA immunization and
Leishmania major infection in vivo [161]. This work identifies a new negative regulator of
IL-12 gene expression. It is yet another example that supports the notion that many oncogenes
do not simply promote cell survival and proliferation but are directly involved in suppression
of cell-mediated immunity against malignant growth.

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The TLR2/MyD88 pathway is important for the production of IL-12 in response to the parasite
Toxoplasma gondii in NFκB-dependent and independent manners [162]. The adaptor molecule
TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases
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involved in the activation of both NFκB and MAPK. Mason et al. investigated the role of
TRAF6 in the intracellular signaling pathways involved in the production of IL-12 in response
to soluble toxoplama antigens (STAg). TRAF6−/− mice and macrophages failed to produce
IL-12 p40 in response to STAg. It was also demonstrated that TRAF6-dependent activation of
p38 MAPK is required for the production of IL-12 p40 in macrophages in response to
toxoplasma antigen. Furthermore, toxoplasma antigen also activates ERK, which leads to the
inhibition of IL-12 p40 production, and this may represent a strategy of the parasite to evade
early host immune responses [163]. Nevertheless, T. gondii possesses molecules that
themselves induce eventual IL-12 synthesis through both MyD88- and CCR5-dependent
pathways. The balance between activation and interference with proinflammatory signaling is
likely to reflect the need to achieve an appropriate level of immunity that allows the host and
parasite to maintain a stable interaction [164].

Utsugi et al. investigated the role of JNK in IL-12 production by glutathione redox, which is
the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG) [165]. They
found that LPS induced IL-12 p40 protein and mRNA in PMA-treated THP-1 human
macrophage cell line, and that it activated JNK and p38MAPK, but not ERK, in PMA-treated
THP-1 cells. Inhibition of JNK activation using SP600125 enhanced both LPS-induced IL-12
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p40 production from THP-1 cells and p70 production by human monocytes. Antisense JNK
oligonucleotide augmented IL-12 p40 protein production and mRNA expression. The increase
in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose
dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-
OEt augmented p38MAPK activation, but suppressed the JNK activation induced by LPS.
These findings indicate that JNK negatively affects LPS-induced IL-12 production from human
macrophages, and that glutathione redox regulates LPS-induced IL-12 production through its
differential control of JNK and p38MAPK activation.

However, in a separate study, Ma et al. investigated the role of JNK in IL-12 p40 gene
expression in LPS-stimulated promonocytic THP-1 cell line stably transfected with CD14,
treated also with dexamethasone (DXM), an anti-inflammatory glucocorticoid. A role for JNK
in LPS-induced IL-12p40 regulation was demonstrated by using specific inhibitors of JNK
activation: SP600125 and a dominant-negative ERK-1 mutant. The study suggests that DXM
may inhibit IL-12p40 production in LPS-stimulated human monocytic cells by downregulating
the activation of JNK, AP-1, and NFκB transcription factors [166]. The bases for the differences
in the role of JNK activation in IL-12 p40 gene expression demonstrated by the above two
studies are not understood. They may reflect differences in how the THP-1 cells were activated
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in the two studies.

Recent studies have shown that PI3K is an endogenous suppressor of IL-12 production
triggered by TLR signaling and limits excessive Th1 polarization [167]. Fukao et al. found that
numerous stimuli that induced IL-12 production concomitantly elicited PI3K activation in DCs,
but both PI3K−/− and PI3K inhibitor-treated DCs showed increased IL-12 production.
Consistent with enhanced IL-12 production, an augmented Th1 response was observed upon
Leishmania major infection in PI3K−/− mice [168]. These findings indicate that a negative
feedback mechanism exists that regulates IL-12 production during DC activation and may help
prevent the excessive Th1 polarization that causes undesirable immune responses. This study
was also supported by the investigation of Martin et al. into the role of the PI3K-Akt pathway
in regulating Porphyromonas gingivalis LPS-induced production of IL-10, IL-12 p40, and
IL-12 p70 by human monocytes [169]. P. gingivalis LPS selectively activates the PI3K-Akt
pathway via TLR2, and inhibition of this pathway results in an abrogation of ERK1/2

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phosphorylation, whereas the activation of p38 MAPK and JNK 1/2 kinases were unaffected.
Inhibition of the PI3K pathway resulted in suppressed IL-10 production and enhanced IL-12
production, respectively, accompanied by a pronounced augmentation of NFκB p65 that was
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independent of IκB-α degradation. Furthermore, the ability of the PI3K-Akt pathway to

modulate IL-10 and IL-12 production appears to be mediated by the selective suppression of
ERK1/2 activity, as the MEK1 inhibitor PD98059 closely mimicked the effects of wortmannin
and LY294002 to differentially regulate IL-10 and IL-12 production by P. gingivalis LPS-
stimulated monocytes [169].

VII. Transcription Factors That Directly Regulate IL-12 Gene Expression

The study of the regulation of IL-12 gene expression is complicated by the necessity to analyze
the coordination of expression of the p40 and p35 genes, which are encoded on different
chromosomes. While expression of the p40 gene is restricted to cells that produce IL-12 p70,
the p35 gene is more ubiquitously expressed. Many studies have demonstrated that the primary
regulatory step for the expression of both IL-12 p40 and p35 genes is at the level of
transcription. Thus, this chapter deals exclusively with transcription factors that directly
participate in the regulation of IL-12 p40 and p35 genes in macrophages and DCs.

VII1. NFκB
An “NFκB half site” at −132/−122 (TAAAATTCCCC) was initially described in the mouse
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IL-12 p40 promoter [170], which is well conserved in the human counterpart [171]. This site
binds p50/p65 and p50/c-Rel heterodimers induced by LPS [170–172]. The two heterodimers
bind to this site with comparable affinities and exhibit equivalent transcriptional activities in
in vitro assays. However, in vivo, c-Rel plays a more crucial role than p65 in the regulation of
IL-12 p40 gene transcription [173]. Grumont et al. showed that, in contrast to macrophages
which require c-Rel for microbe-stimulated p40 transcription, in mouse CD11c+ DCs, the
induced expression of p40 by inactivated S. aureus, CpG-DNA, or LPS is c-Rel independent
[174]. On the other hand, expression of the IL-12 p35 gene is dependent on and regulated
directly by c-Rel complexes binding to its promoter.

VII2. C/EBPβ
The transcription factor C/EBPβ is believed to play a fundamental role in regulating activated
macrophage functions. Plevy et al. first reported that C/EBPβ plays a crucial and direct role in
the transcriptional regulation of mouse IL-12 p40 gene [172]. However, this finding was not
corroborated by another study. Gorgoni et al. showed that in immortalized macrophage-like
cell lines from C/EBPβ-deficient mice, though IFNγ/LPS-dependent induction of IL-6,
IL-1β, TNF-α, inducible NO synthase, and plasminogen activator inhibitor-1 mRNA
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expression was variably impaired, IL-12 p40, RANTES and macrophage inflammatory
protein-1β mRNA expression was upregulated in the absence of C/EBPβ [175]. The differential
mRNA expression correlated with differential transcription levels of the corresponding genes,
and was in most cases confirmed in primary macrophage populations. Moreover, in sharp
contrast to the enhanced induction of IL-12 p40 mRNA, C/EBPβ−/− primary macrophages
derived from both the bone marrow and the peritoneal cavity displayed totally defective
expression of IL-12 p35 mRNA. Therefore, the IL-12 p35 gene may represent a novel
obligatory target for C/EBPβ in macrophages and this may explain the defective production
of bioactive IL-12 and the impaired Th1 responses of C/EBPβ-deficient mice to Candida
albicans infection [175]. Another study also found that IFN-γ, TNF-α, and IL-12 p40 mRNA
expression was within the normal range in C/EBPβ−/− mice infected with Mycobacterium
tuberculosis strains [176].

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VII3. PU.1
PU.1 belongs to the ets family of DNA binding proteins [177,178]. It is expressed
predominantly in macrophages, B-cells, and erythroid cells [179,180]. PU.1 plays important
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roles in the development of hematopoietic cells. Ma et al. first reported the binding of PU.1 to
the human IL-12 p40 promoter constitutively at two sites: immediately upstream of the NFκB
half site at −117/−110 [171], and at the ets site located at −211/−207[181]. Use of a dominant
negative mutant of PU.1 [178] cotransfected with the human IL-12 p40 promoter-luciferase
gene into RAW264.7 cells (a murine macrophage cell line) abolished both the reporter activity
as well as the endogenous IL-12 p40 protein secretion, suggesting that PU.1 is an obligatory
factor for the transcriptional activation of human IL-12 p40 [124]. Type I IFNs are potent
inhibitors of IL-12 production by human monocytes/macrophages. The underlying mechanism
involves transcriptional inhibition of the IL-12p40 gene, marked by down-regulation of PU.1
binding activity at the upstream Ets site of the IL-12p40 promoter [138]. However, its
importance is not confirmed in the mouse IL-12 p40 gene transcription in transient systems
[172].

VII4. IRF-1 and ICSBP

Coordinated expression of the two constituent IL-12 genes, p40 and p35, is crucial for
appropriate immune responses in timing, location, and magnitude. IFN-γ priming of IL-12
production by macrophages and DCs represents an important physiological process in vivo for
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escalated cellular response to microbial infections. Liu et al. showed that IRF-1-deficient
macrophages have a selective impairment in mRNA synthesis of IL-12 p35 but not the p40
gene, and a strong deficiency in the production of IL-12 p70 but not p40 [182]. They further
demonstrated that IRF-1 plays a major role in the transcriptional activation of the IL-12 p35
gene by physically interacting with an inverted IRF element within the IL-12 p35 promoter
upon IFN-γ activation. Moreover, IRF-1-mediated transcriptional activation of the p35
promoter requires the cooperation of two adjacent Sp1 elements [182]. Thus, IRF-1 acts as a
critical component of IFN-γ signaling in the selective activation of IL-12 p35 transcription in
synergy with LPS-mediated events.

The lack of a strong deficiency in IL-12 p40 mRNA expression in IRF-1−/− macrophages shown
in this study is in apparent disagreement with the results of two previous studies [183,184].
The possible reasons for this discrepancy were investigated in this study, which indicates that
prolonged exposure of macrophages to IFN-γ before LPS stimulation is able to rescue the
deficiency in IL-12 p40 production in IRF-1−/− cells via an alternative, uncharacterized
mechanism. This unknown alternative pathway is unlikely to play a role in IL-12 p35 and p70
production, because they are not rescued by the IFN-γ pretreatment. The rescue effect of IFN-
γ pretreatment on IL-12 p40 expression in IRF-1−/− macrophages was also observed in a
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previous study by Salkowski et al. [185]. The differential impact of the length of IFN-γ
pretreatment on p40 but not on p70 production supports the notion that IRF-1 contributes to
the transcriptional regulation of IL-12 p40 and p35 genes through different mechanisms
[182].

Recently, Liu et al. reported that ICSBP-deficient macrophages are highly defective in the
production of IL-12 [118]. The defect is also observed at the level of IL-12 p40 and p35 mRNA
expression. Transcriptional analyses reveal that ICSBP is a potent activator of the IL-12 p35
gene. It acts through a site localized to −226 to −219, named ICSBP-response element (ICSBP-
RE), in the human IL-12 p35 promoter through physical association with IRF-1 both in vitro
and in vivo. Coexpression of ICSBP and IRF-1 synergistically stimulates the IL-12 p35
promoter activity. Mutations at the ICSBP-RE results in the loss of protein binding as well as
transcriptional activation by ICSBP alone, or together with IRF-1 [118]. This study provides

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novel mechanistic information regarding how signals initiated during innate and adaptive
immune responses synergize to yield greater IL-12 production and sustained cellular immunity.
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In a study by Zhu et al., ICSBP was found to be associated with NFAT in the absence of DNA,
as detected by co-immunoprecipitation of endogenous proteins. A composite NFAT/ICSBP
binding site at −68 to −54 was identified which is functionally important for mouse IL-12 p40
promoter activation by LPS and LPS plus IFN-γ. DNA binding of NFAT and ICSBP is
demonstrated on the endogenous promoter by chromatin immunoprecipitation. NFAT is
required for ICSBP binding to this region [186].

VII5. AP-1
The activation protein-1 (AP-1) transcription factors are immediate early response genes
involved in a diverse set of transcriptional regulatory processes [187]. The AP-1 complex
consists of a heterodimer of a Fos family member and a Jun family member. This complex
binds the consensus DNA sequence (TGAGTCA) sites found in a variety of promoters [188,
189]. The Fos family contains four proteins (c-Fos, Fos B, Fra-1, Fra-2) [190–192], while the
Jun family is composed of three (c-Jun, Jun-B, and Jun-D) [193–196]. Fos and Jun are members
of the basic leucine-zipper family of sequence-specific dimeric DNA-binding proteins [197].
AP-1 has been shown to be important for the initiation of cell growth [194,197].

The potential link between AP-1 and IL-12 goes back to early observations of a profound effect
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of trauma and sepsis on IL-12 production. After burn trauma, splenocytes from mice
demonstrate aspects of impaired cellular immunity along with diminished production of IL-2,
IL-12, and IFN-γ, and increased IL-4 and IL-10 synthesis, which would be consistent with a
Th2 phenotype [198]. Importantly, IL-12 treatment after burn injury restores normal resistance
to bacterial challenge [198]. Similarly, studies from humans after major injuries demonstrate
predominance of the Th2 phenotype and diminished IL-12 and IFN-γ production [199,200].
The murine IL-12 p40 promoter is noted to contain sites for AP-1, GATA, AP3, and PU.1
[6]. The initial in vitro studies, however, generated data that did not support an inhibitory role
of AP-1 in IL-12 p40 transcription. For example, using a strategy to demonstrate functional
activity in a minimal promoter context, Zhu et al. identified a functional AP-1 element in the
mouse IL-12 p40 promoter activation at −79 to −74. Mutations at this site significantly reduced
LPS-induced promoter activity. Electrophoretic mobility shift assays demonstrate binding of
AP-1 family members to this region. Spacing between the previously identified upstream
element C/EBP and the AP-1 site is important for promoter activation, suggesting cooperativity
between these elements. In this system, overexpressed c-Jun activated the mouse IL-12 p40
promoter and synergistically activated the promoter when co-expressed with C/EBPβ [201].

Despite the general properties of AP-1 as a transcriptional activator, overexpression of c-Fos

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has been shown to have an inhibitory effect on the transcription of several genes [202,203].
Barke et al showed that transcription of the hepatic mitochondrial enzyme carnitine
palmitoyltransferase (CPT, the rate-limiting step in long chain fatty acid oxidation) is inhibited
after peritoneal sepsis [204], and this inhibition is associated with increased c-Fos mRNA
expression and nuclear protein binding to the AP-1 DNA regulatory element in the CPT
promoter [202,204]. These observations led to the hypothesis that after LPS stimulation,
induction of macrophage c-Fos expression provides inhibitory control of IL-12 p40 and p35
transcription, and removal of c-Fos-mediated transcriptional inhibition will permit increase of
macrophage IL-12 p40 and p35 transcription, resulting in elevated IL-12 p70 protein synthesis.
This hypothesis was tested in a murine homozygous c-Fos knockout model, which revealed a
significant increase in IL-12 p70 protein, p40 mRNA, and transcription rate in peritoneal
macrophages stimulated with LPS [205]. Moreover, the priming-induced enhancing effects on
IL-12 production by IFN-γ and IL-4 have both been attributed at least in part to the
downregulation of c-Fos by these two cytokines during the priming phase [205,206]. Similarly

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to the mouse gene, the human IL-12 p40 promoter activity stimulated by IFN-γ and LPS is
strongly inhibited by overexpression of c-Fos and c-Jun in RAW264.7 cells. Conversely,
blocking AP-1 activity using a dominant negative mutant dramatically increases IL-12 p40
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transcription and protein synthesis in macrophages [207].

VII6. Erythroid Kruppel-like factor (EKLF)

Recognition and binding to CACCC sequences have been shown to be mediated predominantly
by a family of proteins referred to as Kruppel-like factors (KLFs) [208–210]. KLFs have been
reported as both activators [211] and repressors [212,213] of gene expression, depending on
the type of KLF, cell type, and other transcription factors with which they may interact. For
example, erythroid Kruppel-like factors (EKLFs) activate β-globin gene expression when
binding to the CACCC element of its promoter [214], but can repress gene expression by
recruiting co-repressors such as histone deacetylases [215]. KLFs have a broad biological
functions such as cellular proliferation/differentiation, apoptosis, angiogenesis, and
tumorigenesis as reviewed recently [216]. Recent evidence suggests that KLFs regulate the
promoter activities of complement C4 [217] and iNOS [218], implicating a role of KLFs in
immune system due to the gene-regulatory role via the CACCC cis-element. Luo et al.
described the expression of EKLF in human primary macrophages and identified a role of
EKLF in IL-12 p40 expression [219]. EKLF-specific binding to the CACCC element (−224
to −220) on the human IL-12 p40 promoter was observed in resting human primary
macrophages. Functional analysis of the CACCC element revealed a dependent role for EKLF
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binding in activating IL-12 p40 transcription in resting RAW264.7 cells, whereas EKLF
overexpression in the presence or absence of this element repressed IL-12 p40 transcription in
IFNγ/LPS-stimulated RAW264.7 cells. Murine endogenous IL-12 p40 mRNA was induced
by overexpressed EKLF in resting RAW264.7 cells, whereas EKLF suppressed IL-12 p40
expression in activated RAW264.7 cells. The bi-functional control of IL-12 p40 by EKLF and
its modulation of NFκB support a potential function for this factor in regulating homeostatic
IL-12 p40 production in macrophages [219].

VIII. Endotoxin Tolerance-Mediated Inhibition of IL-12

Endotoxin tolerance, the deactivation of a subset of endotoxin-driven responses after an initial
exposure to endotoxin, may provide protection from uncontrolled immunological activation
of acute endotoxic shock. On the other hand, the inhibition of monocyte/macrophage functions
associated with endotoxin tolerance can lead to an inability to respond appropriately to
secondary infections in survivors of endotoxic shock, a phenomenon known as “immunological
paralysis”. IL-12 plays an important role in pathological responses to endotoxin. Karp et al.
[220] examined the regulation of IL-12 during endotoxin tolerance and found that pre-exposure
of human monocytes to small doses of LPS (priming) ablates IL-12 production induced by
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secondary challenge with LPS. This suppression of IL-12 production is primarily

transcriptional. Decreased IL-12 production in vivo is multifactorial, involving both loss of
CD11c(high) DCs as well as alterations in the responsiveness of macrophages and remaining
splenic DCs [221]. No demonstrable mechanistic role was found for B or T lymphocytes, the
soluble mediators IL-10, TNF-α, IFN-αβ, nitric oxide, or the NFκB family members p50, p52,
and RelB [221]. Recently, Kobayashi and colleagues showed that a novel intracellular
molecule, IRAK-M, is induced upon TLR stimulation and negatively regulates TLR signaling.
IRAK-M prevented dissociation of IRAK and IRAK-4 from MyD88 and formation of IRAK-
TRAF6 complexes [222]. IRAK-M−/− cells exhibited increased cytokine production upon
TLR/IL-1 stimulation and bacterial challenge, and IRAK-M−/− mice showed increased
inflammatory responses to bacterial infection. Endotoxin tolerance was significantly reduced
in IRAK-M−/− cells [222]. How this relates to the transcriptional suppression of IL-12
expression induced by endotoxin tolerance remains to be further explored.

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IX. Chromatin Remodeling in Induction of IL-12 Gene Expression

Nucleosome positioning, remodeling, and transcription factor binding at inducible mammalian
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promoters are important for gene regulation. Weinmann et al. first analyzed the chromatin
remodeling of the mouse IL-12 p40 promoter induced in macrophages by bacterial products
[223]. High-resolution micrococcal nuclease analyses revealed that a positioned nucleosome,
nucleosome 1, spans the promoter, with three positioned nucleosomes further upstream. Upon
activation, nucleosome 1 was rapidly and selectively remodeled in a protein synthesis-
dependent manner. In primary macrophages, IFNγ synergistically enhanced IL-12 p40
expression, but little effect on remodeling or promoter occupancy was observed. These results
suggest that remodeling complexes are selectively targeted to a single, promoter-encompassing
nucleosome and that IFNγ influences an event that is independent or downstream of remodeling
[223].

Albrecht et al. showed that in macrophages and DCs, stimulation by selective TLR ligands
CpG-DNA (TLR9), LPS (TLR4) and LTA (TLR2), resulted in striking differences in
expression of IL-12, while stimulating similar amounts of TNF-α. Although an IL-12p40
promoter reporter construct was activated equally by CpG-DNA, LPS and LTA, differences
in nucleosome remodeling around the endogenous IL-12p40 promoter contributed to the
differential IL-12 induction. Upon stimulation, nucleosome architecture was changed to
provide increased access to the IL-12p40 promoter [224].
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Goriely et al. determined the positioning of nucleosomes within the IL-12(p35) promoter using
an indirect end-labeling technique in the THP-1 monocytic cell line [225]. Stimulation with
LPS and IFN-γ resulted in hypersensitivity to digestion with DNase I, micrococcal nuclease,
and specific restriction enzymes in the region encompassing nucleotides (nt) −310 to −160,
indicating selective inducible chromatin remodeling involving disruption of a single
nucleosome (named nuc-2). Promoter deletion mutants and reporter gene assays led to the
identification of 2 Sp1-binding sites, which acted as key regulatory elements for both basal
and LPS/IFN-γ-inducible p35 gene expression: Sp1#1 lies within the remodeled nuc-2 region
and Sp1#2 is located in the nucleosome-free region immediately upstream of nuc-2. The same
nucleosomal organization and remodeling were observed also in DCs derived from human
monocytes. Moreover, in DCs, LPS and IFN-γ synergized in the induction of nucleosomal
remodeling and chromatin remodeling at the IL-12 p35 locus immediately preceded its
transcription [225].

X. Future Prospects
In the last four years since we reviewed on the subject of IL-12 immunolobiology and gene
expression, significant progress has been made with respect to discovery of new IL-12-like
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cytokines and regulatory mechanisms by which IL-12 gene expression is controlled. Important
challenges still lie ahead. First, we need to extensively explore the immunological activities of
the newly identified IL-12-related molecules, p19, p23, and EBI3, in their various combinations
in the context of their functional relationship to IL-12. Secondly, greater efforts should be taken
to explore the adjuvant activities of IL-12 and IL-12-related cytokines in immunotherapy of
various infectious and malignant diseases, both in animal models and human clinical
applications. This approach looks increasingly promising and necessary given what we already
know about IL-12’s “non-specific” effects. Last but not least, we need to intensify
investigations into the coordination and disassociation of the expression of the individual
constituent genes of IL-12 and IL-12-related cytokines. These will include their cell type
distribution, kinetics, and magnitude of expression, and the involvement of the innate TLR
pathways that lead to their expression, as well as the combinatorial usage of the limited number
of transcription factors that control their expression in a spatial and temporal manner. The

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potential of IL-12-related cytokines is tremendous, which can be fully and prudently realized
only through on an intimate and comprehensive understanding of their immunobiology.
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Fig 1. Schematic representation of the immunobiology of IL-12

IL-12 is produced primarily by monocytes/macrophages, DCs, and B cells, typically in
response to recognition of intracellular pathogens through by various TLRs. The principal cell
types targeted by IL-12 are NK/NKT, T (both CD4+ and CD8+), and B cells. IL-12-stimulated
NK cells proliferate, produce IFN-γ and exhibit potent cytotoxicity. CD4+T cells, upon IL-12
stimulation, undergo differentiation to become Th1 effectors at the expense of Th2
differentiation, which is promoted by IL-4/IL-13. IL-12 can also directly activate CD8+ T cells
and enhance their cytolytic potential. B cells can respond to IL-12 directly or indirectly through
IFN-γ production stimulated by IL-12 to produce cytotoxic immunoglobulins such as IgG2a
in mice. Mφ, macrophage; Mo, monocyte; DC, dendritic cell; CTL, cytotoxic T lymphocyte.
The color of the arrows indicates stimulation (green) or inhibition (red). The thickness of the
arrows is proportional to the potency of the stimulus.
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