By
From the * Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and
Infectious Diseases, National Institutes of Health; and the Laboratory of Molecular Growth
Regulation, National Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, Maryland
Mice lacking the transcription factor interferon consensus sequence binding protein (ICSBP), a
member of the interferon regulatory factor family of transcription proteins, were infected with
the intracellular protozoan, Toxoplasma gondii. ICSBP-deficient mice exhibited unchecked parasite replication in vivo and rapidly succumbed within 14 d after inoculation with an avirulent
Toxoplasma strain. In contrast, few intracellular parasites were observed in wild-type littermates
and these animals survived for at least 60 d after infection. Analysis of cytokine synthesis in
vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)- and
interleukin (IL)-12 p40 in the T. gondii exposed ICSBP
/
animals. In related experiments,
macrophages from uninfected ICSBP
/
mice were shown to display a selective impairment in
the mRNA expression of IL-12 p40 but not IL-1
, IL-1
, IL-1Ra, IL-6, IL-10, or TNF-
in
response to live parasites, parasite antigen, lipopolysaccharide, or Staphylococcus aureus. This selective defect in IL-12 p40 production was observed regardless of whether the macrophages
had been primed with IFN-
. We hypothesize that the impaired synthesis of IL-12 p40 in
ICSBP
/
animals is the primary lesion responsible for the loss in resistance to T. gondii because
IFN-
-induced parasite killing was unimpaired in vitro and, more importantly, administration
of exogenous IL-12 in vivo significantly prolonged survival of the infected mice. Together
these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-
-dependent host resistance.
ICSBP is a transcription factor belonging to the interferon
regulatory factor (IRF)1 family (1) which also includes
the IRF-1, IRF-2, IRF-3, interferon-stimulated gene factor (ISGF)3 Analyses of knockout (ko) mice carrying disrupted IRF
genes have begun to elucidate the distinct roles of ICSBP
and other IRF family proteins in host defense against various infectious agents (12). It was recently noted that
ICSBP-deficient mice display increased susceptibility to infection with vaccinia and lymphocytic choriomeningitis viruses but not vesicular stomatitis virus (13). This differential
susceptibility points to a defect in IFN- The IL-12 protein is a heterodimeric cytokine composed
of heavy (40 kD) and light (35 kD) chain subunits that are
encoded by two genes on separate chromosomes. Although
p35 expression is constitutive, p40 expression is induced
upon stimulation. Thus, p40 expression governs the production of the bioactive cytokine (reviewed in reference
21). The 70-kD IL-12 heterodimer is synthesized by macrophages, neutrophils, and dendritic cells and triggers effective immune responses indirectly by stimulating NK cells to produce IFN- In this paper, the role of ICSBP in regulating IL-12
function was examined using a model of Toxoplasma gondii
infection in which the cytokine is known to be critical for
host resistance (reviewed in reference 28). Infection with
this apicomplexan protozoan is characterized by a brief
acute stage during which rapidly dividing tachyzoites disseminate to peripheral host tissues followed by a chronic
stage in which the latent bradyzoite form persists in muscle
and CNS tissue. Studies of host immunity to the parasite
have identified IFN- In the present study, we show that ICSPB Experimental Animals
Mice with a targeted disruption of the ICSBP gene (ICSBP Parasites and Experimental Infection
Tachyzoites of the virulent RH strain were maintained in vitro
by infection of human foreskin fibroblasts and biweekly passage in
DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with
1% fetal calf serum (Hyclone Laboratories, Logan UT), penicillin
(100 U/ml), and streptomycin (100 µg/ml). Cysts of the avirulent ME49 strain (initially provided by Dr. J. Remington, Palo
Alto Research Foundation, CA) were harvested from the brains
of C57BL/6 mice that had been inoculated with ~20 cysts i.p. 1 mo
prior. For experimental infections, mice received 20 ME49 cysts by
the i.p. route. Soluble tachyzoite antigen (STAg) was prepared from
T. gondii (RH strain) tachyzoites by repeatedly sonicating the parasites, centrifuging the sonicate at 100,000 g, collecting the supernatant, and then determining the protein concentration.
In Vivo Assessment of Acute Infection
Acute tachyzoite growth was assessed by evaluating cytocentrifuge smears of cells from infected animals. Samples were prepared from 1.5 × 105 peritoneal exudate cells in a Cytospin (Shandon
Lipshaw, Pittsburgh, PA) set for 5 min at 1,000 rpm. Slide preparations were fixed in absolute methanol for 5 min and then
stained with Diff-Quik (Baxter Healthcare Corporation, McGaw
Park, IL), a modified Wright-Giemsa stain, as specified by the
manufacturer. Differential analyses, including assessment of the number of infected cells, were performed on 400-500 cells using an
oil immersion (100× objective).
In Vitro Responses of Cells from Infected Animals
Single cell suspensions were prepared from spleen and PECs harvested at various time points after infection. PECs were cultured at 4 × 105 cells and spleen cells at 8 × 105 per well in a total volume of 200 µl in a medium consisting of RPMI-1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum,
penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine
(2 mM), Hepes (10 mM), and 2-mercaptoethanol (5 × 10 Serum Preparation
Blood was collected from mice at the time of death and allowed to clot at room temperature for 2 h. Sera were then separated from the individual samples after a 5-min centrifugation at
5,000 rpm and assayed for cytokine content.
Responses of Macrophage-Enriched Populations from
Uninfected Animals
Resident macrophages and inflammatory macrophages were
harvested from animals that were untreated or inoculated i.p. 4-5 d previously with 1.5 ml of 3% thioglycollate (Sigma Chemical Corporation, St. Louis, MO). Cells were harvested by injecting cold
RPMI into the peritoneal cavity and plated at 2 × 106 per ml in
24- and 96-well plates for 2 h in the presence or absence of
rMuIFN- Parasite killing was assessed in parallel cultures using a standard
T. gondii proliferation assay (34, 35). In brief, 96-well plates containing peritoneal cells and media alone or media and live tachyzoites were pulsed with Uracil-[5,6-3H] (ICN Pharmaceuticals,
Inc., Irvine CA) at 0.5 µCi/well from 24 to 36 h after culture
initiation. T. gondii growth was assessed by measuring the incorporation of radioactive uracil by the parasites. The percentage of
killing was determined by the following calculation (values are in
cpm):
[1 Cytokine Assays
ELISA Assay of Cytokine Proteins (IFN- RT-PCR Measurement of Cytokine mRNAs.
RNA was prepared from spleens dissolved in RNAzol (Tel-Test, Inc., Friendswood, TX) by phenol-chloroform extraction. cDNAs were synthesized from total RNA using Molony murine leukemia virus reverse transcriptase (Gibco BRL) in a reaction mixture containing 75 mM KCl, 3 mM MgCl2, 50 mM Tris-HCl, pH 8.3, 0.25 mM dNTPs, 0.8 U of RNasin, and random hexamer primers
(Promega, Madison, WI). cDNA was added to a solution containing 50 mM KCl, 3 mM MgCl2, 10 mM Tris-HCl, pH 9.0, 250 µM of dNTPs, and 1 U Taq DNA polymerase (Promega)
and 80 µg of sense and antisense primers in a total volume of 25 or 50 µl. For detection of IL-12 p40, the sense and antisense
primers 5 RNAse Protection Assay of Cytokine Gene Expression.
Peritoneal
macrophages were prepared from thioglycollate-elicited peritoneal
exudates (see above). Total RNA (3 to 5 µg) was subjected to
multi-probe RNAse protection assay by using a mouse cytokine/
chemokine template set (mck-2) supplied by PharMingen (San
Diego, CA) as a DNA template. The antisense riboprobes were synthesized with the T7 RNA polymerase to an average specific activity of 1.5 × 109 dpm/µg in an in vitro transcription system
provided by Ambion (Austin, TX). Total RNA (3-4 µg) was annealed to 3 × 105 dpm of riboprobes overnight at 55°C in 300 mM NaCl. The duplexes were digested with a cocktail containing 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 300 mM NaCl, 40 µg/ml RNase A, and 2 µg/ml RNase T1 (Boehringer Mannheim, Indianapolis, IN) for 1 h at 30°C. Samples were purified by
phenol-chloroform extraction and resolved on a 6% denaturing
polyacrylamide gel. The dried gels were autoradiographed on
XAR-5 (Eastman-Kodak, Rochester, NY) with an intensifying
screen (DuPont, Wilmington, DE).
Statistical Analyses
Statistical determinations of the difference between means of
experimental groups was determined using an unpaired, two-tailed Student's t test.
The observation of decreased constitutive levels of
IL-12 p40 and IFN-
The rapid development of fulminant infection in ICSBP
Since IL-12 triggers early IFN-
To evaluate whether the lack of
IL-12 p40 production in infected ICSBP
We also assayed expression of IL-4, a signature cytokine
for Th2 responses. Before infection, IL-4 mRNA was undetectable in both ICSBP Having demonstrated
that mice deficient in ICSBP express negligible amounts of
IL-12 p40 after exposure to T. gondii in vivo, we next
asked whether cells from these animals are also defective in
their IL-12 response to other stimuli both in the absence
and presence of an IFN-
Adherent, thioglycollate-elicited cell populations produce significant levels of IFN-, and Pip/IRF4 proteins (2). The gene for interferon consensus sequence binding protein (ICSBP) has
been sequenced in both human and mouse, and in the latter species has been localized to chromosome 8 (7). Unlike
the other members of the IRF family, ICSBP expression is
limited to cells of the immune system including resting B
cells (8) and activated macrophages (9) and T cells (8). Proteins of the IRF family, including ICSBP, bind to the interferon-stimulated response element (ISRE) and control
activities of promoters carrying this element, which is
present in many IFN-
/
-inducible genes (10). IRF-1 and
ISGF3 are activators of IFN-
/
-inducible genes (2, 11),
while IRF-2 and ICSBP are repressors of the same set of
promoters (3, 8). Although the above observations link
ICSBP to IFN-
/
-regulated gene activation, ICSBP expression is also stimulated by IFN-
suggesting that the
transcription factor is a component of the gamma-activated
site signal transducer and activator of transcription pathway
of transcription (GAS-STAT).
- rather than IFN-
/
-dependent defense, since protection against vesicular
stomatitis virus is primarily associated with type I interferon,
whereas control of vaccinia virus and lymphocytic choriomeningitis viruses involves cooperation between both type I
and II IFNs (19). Similarly, ICSBP-deficient mice were
shown to be highly susceptible to infection with Listeria
monocytogenes, a bacterium that is also controlled by IFN-
-
dependent host resistance mechanisms (16, 20). Nevertheless, the above studies did not address the possibility that induction of IFN-
itself might be impaired in these animals,
a defect that would be consistent with the observation that
constitutive expression of mRNA for IFN-
as well as IL-12 p40 is diminished in splenic tissue from naive ICSBP
/
mice (13). Since IL-12 plays a critical role in IFN-
induction, the impaired IFN-
-dependent resistance displayed by
ICSBP
/
animals could thus result from a lesion in IL-12
responsiveness.
and by inducing naive T cells to develop
into IFN-
-producing CD4+ T cells (Th1) (22). IFN-
,
in turn, induces expression of effector molecules that elicit
potent antiviral, antibacterial, and antiparasitic activities (reviewed in reference 25). The IL-12 p40 gene is highly sensitive to IFN-
priming, and this effect results in a positive
feedback loop on IL-12 p40 mRNA and protein synthesis (26, 27).
as the key cytokine that restricts T. gondii expansion early in infection and prevents reactivation of dormant parasite stages (reviewed in reference 29). Nonetheless, neither T nor NK cells are directly stimulated by
the parasite to synthesize IFN-
; rather, T. gondii triggers
synthesis of IL-12 by cells belonging to the macrophage/
dendritic cell lineage, which in turn drives IFN-
production
by the former cell populations (reviewed in reference 28).
/
mice fail
to develop early resistance to Toxoplasma infection due to a
selective defect in IL-12 p40 production. We further demonstrate that ICSBP
/
cells display a generalized deficiency in IL-12 p40 mRNA and protein responses when
stimulated with IFN-
and parasitic or bacterial products.
We conclude that ICSBP plays an essential role in the activation of the IL-12 gene and, as such, this transcription factor can directly regulate IFN-
-dependent host defense.
/
)
were generated as previously described (13). In brief, a neomycin resistance gene (PGK-neo) was inserted into the second exon, and a
reading frame disruption was introduced by multiple stop codons. ICSBP
/
animals were backcrossed six times to the C57BL/6
strain, and wild-type (wt), heterozygous and homozygous mice were
generated by brother-sister mating of heterozygote mice. Young
ICSBP
/
mice (6-12 wk old) were used in all experiments as
they do not display the CML-like syndrome which can develop in
aged animals. Breeding pairs of IL-12 p40
/
mice (30) were kindly
provided by Dr. Jeanne Magram (Hoffman-LaRoche, Nutley, NJ).
Animals carrying the IL-12 p40 mutant allele were backcrossed
five times to the C57BL/6 genetic background followed by intercross of the heterozygotes to generate mice homozygous for the
targeted mutation. Breeding pairs of mice with a targeted disruption of the IFN-
gene were originally provided by Dyana Dalton and T. Stewart (Genentech, San Bruno, CA) (31). The IFN-
ko mice used were at the seventh generation of backcrossing to the C57BL/6 strain. Animals were housed in specific pathogen-free conditions, and both male and female mice were used for experiments at 5-12 wk of age.
5 M)
in the presence or absence of STAg (5 µg/ml). Supernatants were
harvested 72 h later for IFN-
and IL-12 p40 measurements (see
Cytokine Analysis below).
200 U/ml (generously provided by Genentech, Inc., South San Francisco, CA). In the experiments indicated, cells were
incubated for 2 h at 37°C, and nonadherent cells were removed before the addition of IFN-
. Cultures were then incubated in the presence of medium alone, STAg (5 µg/ml), RH tachyzoites
(0.2 or 1.0 per cell), LPS from Escherichia coli K235 (200 ng/ml;
Sigma) or Staphylococcus aureus Cowan strain 1 (SAC) (Pansorbin;
Calbiochem-Gehring, La Jolla, CA; 0.0075%). In selected experiments, neutralizing mAb specific for IL-4 (32) and IL-10 (33)
(Genzyme Diagnostics, Cambridge, MA) were included to assess
the effects of these cytokines on the induction of monokine synthesis by cell cultures. 24-well plates were harvested 6 h after
stimulation. A second set of cultures was incubated for 48 h and
the supernatants from these cells used to measure the induction of
IL-12 p40 protein and of nitrite (NO2).
({[IFN-
(+parasites)]
[IFN-
(
parasites)]}
{[media(+parasites)]
[media(
parasites)]})] × 100%
, IL-12 p40).
Levels of
IFN-
and IL-12 p40 protein were assayed by two-site ELISA as
previously described (36, 37). Cytokine levels were quantitated
by reference to standard curves generated with rIFN-
(donated
by Genentech) or rIL-12 (provided by Genetics Institute, Cambridge, MA).
-GTGAAGCACCAAATTACTCCGG-3
and 5
-GCTTCATCATCTGCAAGTTCTTGGG-3
were used and probed
with 5
-CAGTGTCCTGCCAGGAGGATGT-3
. For IL-12 p35,
primers 5
-GGCTACTAGAGAGACTTCTTCC-3
and 5
-GTGAAGCAGGATGCAGAGCTTC-3
were used and probed with
5
-GATGACCAGACAGAGTTCCAG-3
. Oligomers for HPRT,
IL-4, IL-10, and TNF-
have been described (38). Reactions for
spleen RNAs was initiated by denaturation at 95°C for 4 min followed by 28 cycles of amplification reactions at 94°C for 1 min,
55°C for 1 min, and 72°C for 1.5 min for IL-12 p40, IL-12 p35,
IL-4, IL-10, and TNF-
. Reactions for HPRT had 22 cycles. Reactions under these conditions were within the linear range of
amplification (39). Samples were then separated on 1 or 1.5% agarose gel and analyzed by Southern blot hybridization with
[32P]-labeled oligonucleotide probes.
ICSBP/
Mice Succumb to Acute Infection with T. gondii.
mRNA in spleen cells from
ICSBP
/
mice (13) suggested that these mice might display increased susceptibility to T. gondii infection. However, we and others have described an association between
neutrophil responses and control of infection (40, 41). Since
ICSBP
/
animals display a predilection toward neutrophilia (13), the mutation could potentially result in increased rather than decreased resistance to parasite exposure. To assess their susceptibility to T. gondii, ICSBP
/
mice were infected with 20 cysts of the ME49 strain and
their survival compared with IFN-
/
, IL-12 p40
/
, and
ICSBP wt littermates. In agreement with previous reported findings (27, 40), the IFN-
/
and IL-12 p40
/
strains
succumbed to the infection within 14 d of parasite exposure, while the wt animals survived throughout the 30-d
duration of the experiment (Fig. 1, A and B). In contrast to
the wt controls, ICSBP
/
mice exhibited an acute mortality pattern comparable to that of IFN-
/
and IL-12 p40
/
mice. Enumeration of infected cells in cytospin smears of
PECs taken 5 d after parasite exposure revealed numerous
cells containing parasites in ICSBP
/
but not wt animals
(> 35 versus < 1%, Fig. 1 C). PEC populations from wt
controls, IFN-
/
, IL-12p40
/
, and ICSBP
/
mice were
all similar in composition before infection consisting of 70-
80% large mononuclear cells (LMc), 20-30% small mononuclear cells (SMc), 1-3% PMN, 2-4% eosinophils (Eos),
and 1-3% mast cells. At 5 d after infection the number of
cells recovered increased from two- to fivefold and the composition of the exudate was significantly different amongst
the groups of mice (Fig. 1 C). As compared with the wt
animals, exudates from infected ICSBP
/
mice contained
a lower percentage of LMc and SMc and the difference was
largely attributable to a corresponding increase in PMNs. However, cells from infected IL-12 p40
/
and IFN-
/
mice were comparable and contained a greater percentage
of SMc and Eos than the exudates from ICSBP
/
animals.
Fig. 1.
Progression of T. gondii infection in ICSBP/
versus control
animals. Mice were infected by the i.p. route of infection with 20 ME49
cysts and cumulative mortality monitored. The data shown in (A and B)
are pooled from three independent experiments and involve a total of 14 ICSBP
/
, 15 ICSBP+/+, 14 IL-12 p40 ko and 19 IFN-
ko animals. (C)
Qualitative analysis of the PEC present in wt (dark grey bars), ICSBP
/
(black bars), IFN-
ko (pale grey bars) and IL-12 p40 ko (hatched bars) animals at 5 d after infection with ME49. Cytospin smears of PEC were prepared and stained with Diff-Quik reagent, as described in Materials and
Methods. The percentage of infected cells, large mononuclear cells (LMc;
macrophages, monocytes, and blasting lymphocytes), small mononuclear
cells (SMc; resting lymphocytes), polymorphonuclear cells (PMN) and
eosinophils (Eos) were calculated. Uninfected mice of each of the four
strains did not differ significantly in the composition of their PECs. Values shown are the mean ± SE of 6-12 infected animals per group. Statistically significant differences between the values observed in ICSBP
/
and wt (*) or ICSBP
/
and both IFN-
ko
/
and IL-12 p40 ko (**)
samples are indicated (P
0.05).
[View Larger Version of this Image (26K GIF file)]
/
Mice Fail to Develop
IFN-
and IL-12 p40 Responses to the Parasite.
/
mice and
their subsequent mortality within the first 2 wk of parasite exposure suggested that induction of IFN-
synthesis might
be defective in these animals. To investigate this hypothesis,
IFN-
levels were compared in ICSBP
/
and ICSBP+/+
mice at different times during the first week of T. gondii infection. Wt mice displayed a characteristic elevation in IFN-
protein as measured directly in the serum and in cultures of
cells from the peritoneum and spleen (Fig. 2 A). In contrast, IFN-
levels did not increase in ICSBP
/
mice at
any of the time points analyzed (Fig. 2 A), and addition of
STAg to the cultures failed to amplify the production of
cytokine (data not shown).
Fig. 2.
Kinetics of cytokine
synthesis in infected wt (open
squares) and ko (solid circle) animals. Serum, PECs, and spleen
tissue were harvested from
ICSBP/
and ICSBP+/+ mice
on days 0, 3, 5, and 7 after i.p.
infection with ME49. IFN-
(A)
and IL-12 p40 (B) levels were
measured by ELISA in diluted sera or in 72-h culture supernatants of single-cell suspensions as
described in Materials and Methods.
Data points are the mean ± SE
for four individual mice. An asterisk indicates a statistically significant difference (P <0.05 by
Student's t test) between the values observed in ICSBP
/
and
ICSBP+/+ samples. The experiment shown is representative of
three performed.
[View Larger Version of this Image (23K GIF file)]
synthesis in T. gondii-infected mice (reviewed in reference 28), we asked whether
the diminished IFN-
expression might result from defective IL-12 activity in the ICSBP
/
mice. Whereas IL-12
p40 increased dramatically in the serum and in cultures of
peritoneal and spleen cells from wt mice, these responses
were essentially absent in the mutant animals (Fig. 2 B).
Levels of IL-12 p40 protein were also two- to threefold lower in serum and splenic cell cultures from naive ICSBP
/
compared with ICSBP+/+ mice. Notably, however, spleen
cells from ICSBP
/
mice produced substantial amounts of
IFN-
when stimulated with concanavalin A, a response
which is IL-12 p40 independent (Fig. 3).
Fig. 3.
Con A-induced IFN- production by spleen cells from uninfected ICSBP+/+, ICSBP
/
, and IL-12 p40
/
mice. Spleen cells were
cultured with media alone (white bars) or Con A (black bars) at 5 µg/ml
and the supernatants assayed for IFN-
72 h later. Cytokine synthesis
(mean ± SE) is shown for four individual animals per group.
[View Larger Version of this Image (13K GIF file)]
/
Splenic Tissue.
/
mice is attributable to a deficiency in the mRNA expression, semiquantitative RT-PCR was performed to measure levels of transcripts for IL-12 p40 and p35 during parasite infection (Fig.
4). After parasite inoculation, IL-12 p40 mRNA levels remained low in ICSBP
/
mice throughout the 7 d of the
experiment, with only minor variation among animals. In
contrast, IL-12 p40 mRNA levels were markedly increased
in wt mice after infection, reaching maximum levels on
days 3 and 5 and declining by day 7. Of note, the levels of mRNA encoding the constitutively expressed IL-12 p35
subunit were comparable between ICSBP
/
and ICSBP+/+
mice before parasite exposure and did not significantly change for 7 d after infection. Consistent with the reduced production of IFN-
protein (Fig. 2), the IFN-
transcript
levels remained low in the ICSBP
/
spleens, while clearly
increasing in tissues from +/+ animals.
Fig. 4.
Kinetics of cytokine mRNA expression in splenic tissue from T. gondii-infected ICSBP+/+ and ICSBP/
mice. (A) Splenic tissue was harvested from 4 individual mice at days 0, 3, 5, and 7 after parasite exposure from the same animals studied in Fig. 2. Extracted RNAs were subjected to
quantitative RT-PCR analysis using primers specific for IL-12 p40, IL-12 p35, IFN-
, IL-10, IL-4, and HPRT genes. (B) RNA from four individual mice
were pooled and levels of TNF
expression were determined by RT-PCR as performed in (A). Similar results were attained in a second experiment.
[View Larger Versions of these Images (75 + 51K GIF file)]
/
and ICSBP+/+ mice. On days
5 and 7, IL-4 transcripts were induced in all ICSBP
/
mice and, with the exception of one sample, these animals
displayed uniformly higher expression of IL-4 mRNA than
did wt mice. IL-10 mRNA was also detected in both wt
and ICSBP
/
mice before and after infection. On days 3 and 5, the levels were somewhat higher in ICSBP+/+ mice
than in ICSBP
/
mice, although they became similar by
day 7. Levels of HPRT mRNA, measured as a housekeeping gene control, were comparable for all samples tested.
Expression of TNF-
transcripts was also investigated since
IL-12 and TNF-
are co-induced in macrophages exposed
to T. gondii (42). As shown in Fig. 4 B, TNF-
mRNA
was clearly induced in both wt and ICSBP
/
mice at comparable levels.
/
Mice Exhibit a Global Impairment in IL-12 p40 Responses.
priming signal. The ability of adherent, thioglycollate-elicited peritoneal macrophages from
uninfected mice to produce IL-12 p40 protein after stimulation in vitro was assessed. In both +/+ and
/
cultures,
>85% of the adherent population were macrophages as determined by their expression of the Mac-1 but not GR-1
cell surface markers (data not shown). Exposure of wt cells
to live tachyzoites, STAg, LPS, or heat-killed SAC resulted in increased IL-12 p40 levels. In contrast, while cells from
ICSBP
/
mice also responded to these stimuli, the relative
increase in IL-12 p40 production was markedly less (Fig. 5
A, left). In the four individual experiments performed, the
most dramatic reduction in IL-12 synthesis resulting from
the ICSBP mutation was observed in the response to live
tachyzoites or STAg (always >80%), followed by LPS
(>70%) and then SAC (>50%). This hierarchy was consistently observed in each experiment.
Fig. 5.
Defective IL-12 p40 protein and mRNA response in
thioglycollate-elicited macrophage-enriched populations from ICSBP/
and ICSBP+/+ mice. (A) IL-12 p40 protein synthesis in cultures of
/
(black bars) and +/+ (white bars) cells. (B) IL-12 p40 mRNA induction in
peritoneal macrophages stimulated in vitro. PECs were collected from
five mice that had been injected with thioglycollate 5 d earlier. Pooled
adherent cells (macrophage enriched) were cultured in triplicate in the
presence of SAC (1:1,000), LPS (200 ng/ml), RH (0.2 tachyzoites/cell),
STAg (5 µg/ml), or media alone. Where indicated, cells were incubated
with IFN-
(200 U/ml) and/or anti-IL-4/10 mAbs (10 µg/ml each) for
2 h before the addition of stimuli and throughout the culture period.
Cells were collected at 6 h for RNase protection assay and supernatants at
48 h for protein measurement by ELISA. Three µg of RNA were subjected to RNase protection assay to detect transcripts for IL-12 p40, IL-1
,
IL-1
, IL-1Ra, and GAPDH. The experiment shown is representative of
three performed.
[View Larger Versions of these Images (16 + 57K GIF file)]
upon stimulation with STAg
(Sher, A., unpublished findings) and thus the influence of
ICSBP on IL-12 p40 production by these cells could reflect an
indirect effect on IFN-
priming. To rule out this possibility,
we evaluated the response of spleen cells from uninfected
mice to STAg stimulation, a system in which IL-12 p40 responses are induced de novo in the absence of endogenous
IFN-
(55). Spleen cells from wt and IFN-
/
animals
produced high levels of IL-12 p40 when exposed to STAg in vitro (Table 1). The latter response was totally absent in the ICSBP
/
cultures. Moreover, addition of an excess of
neutralizing mAb against IFN-
failed to reduce IL-12 p40
production by the wt cells, confirming the IFN-
-independent nature of that response.
Activation of IL-12 p40 gene transcription is greatly enhanced when macrophages are preexposed to IFN- (26).
To assess the role of ICSBP in this second level of IL-12
triggering, we measured its synthesis in IFN-
-primed,
thioglycollate-elicited macrophage populations (Fig. 5 A,
middle). Addition of rIFN-
to the wt cell cultures significantly increased IL-12 p40 levels in response to STAg, live
tachyzoites, LPS, or SAC. In striking contrast, in 4 of 4 experiments, the level of IL-12 p40 synthesis by ICSBP
/
cells was not affected by the addition of rIFN-
to the cultures. Similar results were obtained in experiments in which
IFN-
-primed resident PEC populations from ICSBP
/
or ICSBP+/+ mice were used (data not shown).
Since IL-12 p40 synthesis by macrophages is known to
be suppressed by IL-10 or IL-4 (43, 44), the decreased IL-12
p40 mRNA expression in infected ICSBP/
as compared
with ICSBP+/+ mice could conceivably result from the effects of these downregulatory cytokines. Indeed, as noted
earlier (Fig. 4), overproduction of IL-4 mRNA was observed in spleen cells from infected ICSBP
/
animals. Addition of neutralizing mAbs against both IL-4 and IL-10 resulted in a two- to threefold increase in the IL-12 p40 response in both ICSBP
/
and ICSBP+/+ cultures (Fig.
5A, right). Nonetheless, even in cultures containing the
anti-IL-4/10 mAbs, the IL-12 p40 mRNA and protein
levels in ICSBP
/
cells were 20-40-fold less than those of
comparably treated wt cells.
To confirm that the observed
impairment of IL-12 p40 expression occurs at the RNA
level, RNAse protection and RT-PCR assays were performed on thioglycollate elicited macrophage populations exposed to T. gondii or bacterial stimuli (Fig. 5 B and 6).
The issue of whether the above stimuli induce other cytokine genes in ICSBP/
macrophages was also addressed.
RNAse protection results (Fig. 5 B) demonstrate that IFN-
primed ICSBP+/+ macrophages express IL-12 p40 transcripts when stimulated by SAC, STAg or LPS. In contrast,
none of these agents induced detectable IL-12 p40 mRNA
in ICSBP
/
macrophages. Although IFN-
alone did not
stimulate IL-12 p40 even in the wt cells, it stimulated expression of IL-1 receptor antagonist (IL-1Ra) transcripts in
cells from both mouse strains. Furthermore, ICSBP
/
macrophages expressed IL-1
and IL-1
transcripts when stimulated by SAC, STAg, or LPS at levels comparable to
ICSBP+/+ cells.
To further address the selectivity of the ICSBP macrophage deficiency the expression of three additional cytokines, TNF, IL-6, and IL-10, was analyzed by RT-PCR
(Fig. 6). IL-12 p40 mRNA induction was negligible in
ICSBP
/
macrophages in response to SAC, LPS, or STAg
even after priming with IFN-
. Addition of anti-IL-4 and
anti-IL-10 antibodies failed to reveal IL-12 p40 mRNA
expression in SAC stimulated ICSBP macrophages suggesting that the defect observed is not due to IL-4
/
IL-10-
mediated suppression. In contrast to the impaired IL-12 p40 mRNA induction, ICSBP
/
macrophages expressed
TNF
mRNA in response to IFN-
, STAg and LPS at
levels comparable with ICSBP+/+ cells. Nevertheless, TNF
mRNAs induced by SAC alone, but not SAC + IFN-
,
were reduced in macrophages from ICSBP
/
mice. Macrophages from both strains of mice also synthesized comparable levels of IL-6 transcripts in response to all of the agents tested. Interestingly, ICSBP
/
macrophages constitutively expressed IL-10 mRNA and higher levels of this
cytokine transcript were induced in ICSBP
/
as compared
with ICSBP+/+ macrophages in response to most of the
stimuli. Quantitative analysis of the cytokine transcript data
in Fig. 6 A is provided in Fig. 6 B.
ICSBP
To determine
whether the ICSBP/
lesion also influences effector functions downstream from the defect in IL-12 p40 induction, we assessed the ability of IFN-
-activated macrophage
populations from ICSBP mutant animals to restrict the
growth of T. gondii in vitro as measured by incorporation
of [3H]uracil, a nucleotide preferentially used by the parasite. Unactivated cells from either wt or ICSBP
/
mice
exhibited a 50-80-fold increase in nucleotide incorporation after infection with tachyzoites (0.2/cell) and addition of
IFN-
dramatically reduced tachyzoite proliferation in cultures from both mouse strains. Similar results were obtained
using thioglycollate-elicited or resident macrophage-enriched
populations (Fig. 7 A).
The above data indicate that IFN--dependent effector
functions downstream from IL-12 p40 synthesis are not directly affected by the ICSBP mutation. To evaluate this question in vivo, ICSBP
/
mice were injected with rIL-12 and
control of infection assessed by monitoring mortality (Fig.
7 B). Animals were infected i.p. with 20 cysts of ME49 at
day 0 and treated with 0.5 µg of rIL-12 i.p. on days 0, 1, 2, 3, and 4 after infection. Transient administration of rIL-12
prolonged the mean survival time of ICSBP
/
mice from
10 d in the PBS group to 20 d in the cytokine-treated group (Fig. 7 B). Because we have been unable to demonstrate any IFN-
-independent mechanisms of IL-12-stimulated host resistance against T. gondii (27), it is likely that
the enhanced survival of rIL-12-treated ICSBP
/
mice is
due to induction of the former cytokine. Consistent with this argument, ICSBP
/
spleen cells were able to produce
an IFN-
response when stimulated in vitro with rIL-12,
although the levels of cytokine were markedly less than
that observed in ICSBP+/+ cultures (Table 2).
|
In this study, we show that ICSBP/
mice rapidly succumb to infection with the intracellular pathogen T. gondii.
This enhanced susceptibility appears to be due to the inability of the mutant animals to produce IL-12 p40 and hence to
stimulate IFN-
during parasitic infection. Notably, however, cytokine expression and host defense pathways that
are either unrelated to or downstream from IL-12 do not
appear to be affected by ICSBP since T cells from ko mice
synthesized IFN-
when triggered with Con A (Fig. 3) and
administration of rIL-12 to infected ICSBP
/
mice prolonged their survival (Fig. 7 B). In support of this argument, macrophage populations from ICSBP
/
animals inhibited parasite replication in vitro when exposed to IFN-
(Fig. 7 A) and synthesized significant amounts of nitric oxide (data not shown), a known correlate of macrophage activation (45). Moreover, it has been previously shown that
splenic T cells from ICSBP
/
animals are capable of restoring IFN-
-dependent resistance against the bacterium
Listeria monocytogenes when transferred to lymphocyte-deficient mice, indicating that these cells retain the ability to
respond to antigen after the appropriate stimulatory signals
(16). Indeed, we found that treatment of spleen cells with
IL-12 induced significant quantities of IFN-
(Table 2).
Nonetheless, the response observed in cells from ICSBP
/
mice was less than that of cultured ICSBP+/+ cells. One
interpretation of these data is that the expression of IL-12R
is diminished in ICSBP
/
cells, a finding which may result
from diminished levels of endogenous IL-12 p40 and IFN-
in the uninfected ICSBP-deficient animals (13, 46).
In addition to their inability to produce IL-12 p40 in response to T. gondii infection, ICSBP/
mice displayed a
generalized impairment in macrophage production of the
cytokine when stimulated with IFN-
, LPS, SAC, or T. gondii itself. This defect appears to be selective for IL-12
p40 induction, since ICSBP
/
cells expressed normal levels of IL-1Ra, IL-1
, and IL-1
mRNA as assessed by
RNAse protection (Fig. 5 B) and IL-6, IL-10, and TNF
by RT-PCR (Fig. 6) when exposed to the same stimuli
with the possible exception of a defect in the TNF
response to SAC alone. Moreover, spleen cells from T. gondii-infected ICSBP
/
animals produced IL-10 and TNF
mRNA in quantities comparable to splenocytes from wt control mice (Fig. 4). Since ICSBP
/
macrophages are clearly
capable of responding to IFN-
, signal transduction events
immediately downstream from IFN-
-IFN-
receptor interaction, including STAT1 activation, are likely to be intact. In further support of this argument, we recently observed that ICSBP
/
cells respond to IFN-
to express
IRF-1 a gene controlled by STAT1 (Masumi, A., and
Ozato, K., unpublished observation). Based on these observations and findings that ICSBP
/
macrophages produce
nitric oxide (16), a STAT1-regulated molecule, we suggest
that ICSBP acts downstream in the STAT1 signal transduction pathway and that it affects only a subset of IFN-
-
inducible genes, which includes the IL-12 p40 gene. In this
regard, naive ICSBP-deficient mice are known to have a
number of hematologic abnormalities which are unlikely to
result solely from the IL-12 p40 defect since animals with a
targeted disruption in the latter gene are phenotypically
normal (13, 30).
Regulation of IL-12 p40 synthesis is known to occur
primarily at the level of transcription (26, 47) and to involve both IFN--dependent and -independent mechanisms of induction in vivo (48). In the case of T. gondii-
stimulated responses, IFN-
, while dramatically enhancing
production of IL-12 p40 by macrophages in vitro, is clearly
not necessary for induction of the monokine in infected
animals (27). The data presented here indicate that ICSBP
regulates both the IFN-
-dependent and -independent pathways of IL-12 p40 synthesis (Table 1; Fig. 5). The effects of
ICSBP deficiency were most pronounced in the case of
IFN-
-primed macrophages where reductions of >95% in
IL-12 p40 were observed in the responses to SAC, LPS,
live RH tachyzoites, or STAg. The same defect was apparent when IL-12 p40 responses of macrophages to STAg
were measured in the cultures initiated in the absence of
IFN-
priming (Fig. 5). Nevertheless, cultures of unprimed
ICSBP
/
cells did produce detectable, although reduced
levels of cytokine in response to SAC, LPS, or RH.
Since STAg-stimulated adherent thioglycollate exudate
populations produce detectable levels of IFN- (unpublished observations), it was possible that the unresponsiveness of ICSBP
/
macrophages to that agent reflects an indirect effect on endogenous IFN-
priming rather than a
direct effect on p40 gene expression. This explanation was
ruled out by showing that spleen cells from uninfected
ICSBP
/
mice are totally defective in IL-12 p40 production when exposed to STAg. As confirmed here (Table 1),
the IL-12 response of splenocytes from ICSBP+/+ mice to
STAg is independent of endogenous priming by IFN-
and, therefore, the defective response of the mutant cells
cannot be explained by a priming deficiency. Taken together these findings argue that ICSBP plays a major role in
regulating both IFN-
-dependent and -independent IL-12
p40 synthesis, a conclusion consistent with the failure of
ICSBP
/
mice to develop an IL-12 response to T. gondii
infection in vivo. Preliminary results suggest that Toxoplasma itself can induce ICSBP expression in macrophages
in the absence of IFN-
priming, a finding that reinforces
the concept that IFN-
signaling may not be essential for
ICSBP function (Scharton-Kersten, T., unpublished results).
The mechanism(s) by which ICSBP regulates IL-12 p40
gene transcription appear to be complex. Thus, although a
consensus ISRE sequence is present in the p40 promoter
we have so far been unable to demonstrate a function for
this element in activation of the gene. It is thus possible
that ICSBP binds to a previously unidentified cis regulatory
element in the IL-12 promoter. Alternatively, ICSBP may
act indirectly by interacting with other transcriptional regulators of the IL-12 p40 gene. Murphy et al. (47) have reported that NFB plays a major role in the induction of the
mouse IL-12 p40 promoter by IFN-
/LPS. Since cytokine
genes known to be induced by NF
B, such as IL-1
, IL-1
,
and TNF-
are induced normally in ICSBP
/
cells, the
defect is not likely to be dependent on NF
B. In addition, Ma et al. (26) have identified an upstream ets sequence, distinct from the NF
B site which they propose is as an essential regulator of the human IL-12 p40 promoter. One hypothesis bridging this and our observations is that ICSBP
acts by forming a complex with ets binding transcription
factors thereby modifying their activity. Finally Screpanti et al.
(49) have suggested that the C/EBP
(NF-IL-6) (50) influences IL-12 expression in macrophages, perhaps through its
regulation of IL-6 synthesis. As IL-6, a direct target of C/
EBP
is induced normally in ICSBP
/
macrophages (Fig.
6) we consider it highly unlikely that ICSBP acts through
this transcription factor. An additional implication of our
findings is that ICSBP can act as a positive transcriptional regulator in addition to its known negative activities on
promoters carrying the ISRE sequence (51). Indeed, preliminary data from transfection experiments support the concept that ICSBP enhances, rather than suppresses, IL-12 p40
promoter activity (Wang, I.-M., and K. Ozato, unpublished observation). This dichotomy in ICSBP function is
not completely unexpected since other transcription factors
have been shown to act positively or negatively depending on the promoter context (52).
The impaired IL-12 expression in ICSBP/
mice predicts that these animals might display enhanced susceptibility to other intracellular pathogens, particularly those agents
against which IFN-
is required for protection. In accordance with this view, ICSBP
/
mice have recently been
demonstrated to display enhanced susceptibility to infection
with another protozoan parasite, Leishmania major (Morse,
H.C., personal communication) and were previously reported to show impaired resistance to the intracellular bacterium Listeria monocytogenes (16). Although the latter authors did not evaluate the molecular basis for the increased
susceptibility of the ICSBP
/
mice to listeriosis, resistance
to this pathogen is highly IL-12 dependent (53) and thus,
the sensitivity of the animals to Listeria infection is likely to
be related to defective IL-12 p40 induction. Of interest,
these authors noted that listeriosis is much less severe in
IRF-1
/
or IRF-2
/
mice than in ICSBP
/
mice.
Based on the finding that IRF-2 expression is reduced in ICSB
/
spleen (13), Fehr et al. (16) suggested that the
mutant mice manifest a "double ko phenotype." One implication of this suggestion might be that both IRF-2 and
ICSBP are required for IL-12 gene activation; however,
recent experiments (data not shown) indicate that the expression of IRF-2 protein is unimpaired in macrophage populations or macrophage-like cell lines from ICSBP
/
mice. Thus, we favor the concept that ICSBP plays a central role in triggering IL-12 p40 expression that cannot be
substituted for by other members of the IRF family.
Although the mechanism of action of ICSBP remains to
be elucidated, the discovery of a transcription factor with
highly specific effects on IL-12 p40 gene induction and, in
turn, on IFN--dependent immunity has important implications for our understanding of the regulation of host defense. In the future, it will be important to define the precise microbial signals leading to the induction of ICSBP as
well as the properties of different pathogens that determine
their selective triggering of the transcription factor. Such
information could be useful in defining new approaches for regulating IL-12 expression to both enhance host resistance
and, in the case of IL-12-dependent immunopathology, to
prevent disease.
Address correspondence to Keiko Ozato, LMGR, NICHD, 9000 Rockville Pike, Building 6, Room 2A01, NIH, Bethesda, MD 20892-2753. Phone: (301) 496-9184. Fax: (301) 480-9354. E-mail: ozatok{at}dir6.nichd.nih.gov
Received for publication 30 June 1997 and in revised form 15 August 1997.
1 Abbreviations used in this paper: ICSBP, interferon consensus sequence binding protein; IRF, interferon regulatory factor; ISGF, interferon-stimulated gene factor; ISRE, interferon-stimulated response element; ko, knockout; LMc, large mononuclear cell; PEC, peritoneal cells; SAC, Staphylococcus aureus Cowan strain 1; SMc, small mononuclear cell; STAg, soluble tachyzoite antigen; STAT1, signal transducer and activator of transcription; wt, wild-type.We are grateful to Dr. J. Magram (Hoffmann-LaRoche) for providing breeding pairs of IL-12 p40/
animals; Dr. S. Wolf (Genetics Institute) for provision of rIL-12; Dr. J. Lou for his guidance on RT-PCR; Mr.
S. Khan for genotyping the ICSBP
/
mice; and Ms. S. Hieny and Ms. P. Caspar for technical assistance. We
also thank Drs. W. Leonard, D. Singer, and G. Yap for critical reading of the manuscript and Ms. Brenda
Marshall for editorial assistance.
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