IL-4 Suppresses Dendritic Cell Response to Type I Interferons1
Uma Sriram,* Chhanda Biswas,†Edward M. Behrens,* Joudy-Ann Dinnall,*
Debra K. Shivers,* Marc Monestier,‡Yair Argon,†and Stefania Gallucci2*
Cytokines play an important role in modulating the development and function of dendritic cells (DCs). Type I IFNs activate DCs
and drive anti-viral responses, whereas IL-4 is the prototype of a Th2 cytokine. Evidence suggests that type I IFNs and IL-4
influence each other to modulate DC functions. We found that two type I IFNs, IFN-? and IFN-?, stimulated a similar costimu-
latory profile in myeloid resting DCs. IL-4 suppressed the response of myeloid DCs to both type I IFNs in vitro and in vivo by
impairing the up-regulation of MHC and costimulatory molecules and the production of cytokines, such as IL-6 and IL-15, and
anti-viral genes, such as Mx-1, upon type I IFN stimulation. In dissecting the mechanism underlying this inhibition, we charac-
terized the positive feedback loop that is triggered by IFN-? in primary DCs and found that IL-4 inhibited the initial phosphor-
ylation of STAT1 and STAT2 (the transducers of signaling downstream of IFN-? and -? receptors (IFNARs)) and reduced the
up-regulation of genes involved in the amplification of the IFN response such as IRF-7, STAT1, STAT2, IFN-?, and the IFNARs
in vitro and in vivo. Therefore, IL-4 renders myeloid DCs less responsive to paracrine type I IFNs and less potent in sustaining
the autocrine positive loop that normally amplifies the effects of type I IFNs. This inhibition could explain the increased suscep-
tibility to viral infections observed during Th2-inducing parasitoses. The Journal of Immunology, 2007, 179: 6446–6455.
hanced expression of MHC and costimulatory molecules and the
secretion of proinflammatory cytokines allow DCs to activate na-
ive and memory T cells and modulate the immune response. Con-
curring immune responses (i.e., Th1 vs Th2) often show compe-
tition and mutual inhibition that still require investigation (1). For
example, the mechanisms underlying the increased susceptibility
to viral infections observed in animals infected with Th2-inducing
parasites (e.g., Schistosoma) (2) are unclear. DCs may play a piv-
otal role in these processes (3). The high plasticity of DC function
is regulated by the activators and the cytokines that DCs encounter
(1). DCs can be activated by endogenous and exogenous danger
signals (4). Among the former, type I IFNs, specifically IFN-? and
endritic cells (DCs)3are key players in the activation of
adaptive immunity. In the resting/immature state DCs
efficiently take up Ag, whereas after activation the en-
IFN-?, are considered major players in the innate and adaptive
immune responses (5). We and other groups have shown that IFN-?
activates DCs in vitro and acts as adjuvant in vivo (6, 7). IFN-? is
secreted by virally infected cells (8) and by plasmacytoid DCs (9,
10), whereas IFN-? is produced by many types of cells, such as
myeloid DCs, following stimuli not necessarily of a viral nature
(11). Type I IFNs, which share a ubiquitous heterodimeric receptor
(8), mediate the innate response to viral infections and are also re-
quired for full DC response to TLRs (12) and their stimulation of T
and B cells (13).
Activated lymphocytes produce cytokines that strongly influ-
ence DC function. In particular, Th2 lymphocytes produce IL-4, a
key player in driving Th2 differentiation of naive T cells and in B
cell activation (14). Although IL-4 is commonly used to generate
murine bone marrow (BM)-derived DCs, its effects on DC differ-
entiation and activation are only partially known; mouse BM-DCs
generated in the presence of IL-4 appear to be more activated and
stronger stimulators of T cells in response to several danger signals
(15). Human monocytes grown in the presence of either IL-4 or
type I IFNs differentiated into DCs similarly, and IFN-? DCs
showed a more activated phenotype and function (16, 17). Those
studies did not, however, examine the effect of IL-4 on the ability
of DCs to respond to type I IFNs, a situation resembling a viral
infection occurring during Th2-induced parasitosis in vivo. Previ-
ous reports indicate that IL-4 and type I IFNs influence each other.
In peritoneal macrophages, IL-4 blocks IFN-?-mediated antiviral
activity (18), and in human monocytes it attenuates IFN-dependent
transcriptional activity (19). The reciprocal effect has been reported as
signaling, possibly by blocking STAT6 activation (20).
Although this evidence suggests that the signaling pathways
of type I IFNs and IL-4 “cross-talk” and possibly repress each
other, no study has specifically addressed whether IL-4 influ-
ences the response of DCs to type I IFNs. In this work, we
asked whether DCs grown in the presence of IL-4 or briefly
exposed to IL-4 responded differently to type I IFNs in terms of
activation and signal transduction, as compared with DCs that
*Laboratory of Dendritic Cell Biology, Joseph Stokes Jr. Research Institute, Division
of Rheumatology, Department of Pediatrics,†Division of Cell Pathology, Department
of Pathology and Laboratory Medicine, University of Pennsylvania School of Med-
icine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, and‡Depart-
ment of Microbiology and Immunology, Temple University School of Medicine,
Philadelphia, PA 19140
Received for publication September 18, 2006. Accepted for publication August
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health National Institute of Al-
lergy and Infectious Diseases Grant AI049892 (to S.G.), the Lupus Foundation South-
eastern Pennsylvania Chapter, the Arthritis Foundation (Innovative Grant to S.G.),
and by a grant from the Pennsylvania Department of Health. U.S. was supported by
a postdoctoral fellowship from the Arthritis Foundation. E.M.B. was supported by
National Institute of Health Grant T32-HD0043021. The Pennsylvania Department of
Health specifically disclaims responsibility for any analyses, interpretations, or
2Address correspondence and reprint requests to Dr. Stefania Gallucci, 1107C
Abramson Research Center, Children’s Hospital of Philadelphia, 3615 Civic Center
Boulevard, Philadelphia, PA 19104. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; Ct, thresh-
old cycle; IFNAR, IFN-? and -? receptor; IRF, IFN regulatory factor; KO, knockout;
MdFI, median fluorescence intensity; Mx, myxovirus resistance.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
were never exposed to IL-4. Our study clearly indicates that
IL-4 suppresses type I IFN-induced activation of DCs and their
expression of cytokines and antiviral genes, in vitro and in vivo,
by inhibiting STAT1 and STAT2 phosphorylation and reducing
the autocrine loop that normally amplifies the effects of type
Materials and Methods
C57BL/6-RAG-knockout (KO) mice and C57BL/6 mice (The Jackson
Laboratory) were bred and maintained in accordance with the guidelines of
the Institutional Animal Care and Use Committee of the Children’s Hos-
pital of Philadelphia, an American Association for the Accreditation of
Laboratory Animal Care-accredited facility.
BM-DCs were generated as described previously (6). Briefly, BM precur-
sors from C57BL/6-RAG-KO mice were seeded at 1 ? 106/ml in complete
IMDM (10% FBS, penicillin/streptomycin, gentamicin, and ?-mercapto-
ethanol) enriched with 3.3 ng/ml GM-CSF alone or with 2.5 ng/ml IL-4
(BD Biosciences) in 24-well plates. One milliliter of medium was added on
day 3 and half of the medium was replaced on day 5 and subsequently each
day until the culture was used (day 6 or 7). Generating DCs from RAG-KO
BM does not require depletion of T and B cells, and RAG-KO BM-DCs
behave identically as those from normal mice. Resting DC cultures were
stimulated at day 6 or 7 of culture with 100 ng/ml LPS (Sigma-Aldrich) or
with IFN-? or IFN-?, both at 2500U/ml (HyCult Biotechnology). For both
cytokines, different lots were used obtaining similar results. To guard
against the occasional LPS contamination, DCs were always incubated
with IFN-? in the presence of 50 ?g/ml polymyxin B (Sigma-Aldrich).
BM-DCs were maintained in GM-CSF with or without IL-4 until the end
of the experiment, because GM-CSF sustains DC viability and washing out
those cytokines would require harvesting DCs, a procedure that others and
we have shown to induce murine DC activation (6, 21). BM-DCs were
harvested after 30 min or 8 h for Western blotting, 30 min for intracellular
staining of phospho-STAT1, 6 h for RNA, and 24 h for FACS analysis of
surface activation markers.
Stimulation of DCs in vivo. C57BL/6 mice were injected s.c. in the base
of the thigh region with 0.5 ?g of rIL-4 (BD Bioscience) in complex with
anti-IL-4 Ab (clone 11B11) using a protocol developed by Finkelman et al.
(22). The isotype Ab was injected in the control mice. After 24 h, 20,000
U of IFN-? or PBS was injected s.c. in both groups of mice and 24 h later
the mice were sacrificed and the inguinal lymph nodes were harvested for
Stimulation of splenic DCs ex vivo. C57BL/6 mice were injected in the
tail vein with the IL-4 complexed with anti-IL-4 Ab (IL-4C) or the
isotype Ab and 24 h later the spleens were harvested in medium with
collagenase/DNase as described previously (23). Single cell suspensions
were made using cell strainer; RBCs were lysed, cells were washed, and
total spleen cells were plated in 24-well plate in medium with 3.3 ng/ml
GM-CSF alone or GM-CSF plus 2.5 ng/ml IL-4 and then stimulated with
IFN-? (20,000U/ml) for 3 h at 37°C. Cells were harvested, washed, stained
with anti-CD11c and anti-CD19 mAbs, and sorted by gating on CD11c?
CD19?DCs with a FACSAria flow cytometer (BD Bioscience). The sorted
cells were resuspended in TRIzol reagent for RNA extraction to study type
I IFN-responsive gene expression.
mouse CD16/CD32 (clone 2.4G2) mAb for 10 min to block Fc?R, and then
stained for 30 min on ice with the allophycocyanin-conjugated hamster
anti-mouse CD11c, PE-conjugated rat anti-mouse MHC class II, CD80,
CD86, FITC-conjugated hamster anti-mouse CD40, and mouse anti-mouse
H2Kb mAbs and isotype control Abs (BD Biosciences). Cells were fixed
in 1% formaldehyde and analyzed on a FACSCalibur or FACSCanto cy-
tometer (BD Biosciences).
In vivo stimulated DCs.
Inguinal lymph nodes were collected in IMDM
with collagenase and DNase, cut in half, and incubated at 37°C for 30 min
as described previously (23). The lymph nodes were then smashed in a
strainer and the cells were washed in the presence of 0.5 mM EDTA,
distributed with 2 ? 106cells per tube, and stained with the mAbs as
described above. PerCP-Cy5.5-conjugated anti-mouse CD19 mAb was
BM-DCs were washed in cold PBS, incubated with rat anti-
also included in the staining to gate out activated B cells that may be
CD11c positive. To measure the effectiveness of the IL-4 in vivo, we
stained with anti-CD23 Ab (BD Biosciences) to look for the expression on
B cells (22). Cells were fixed in 1% formaldehyde and analyzed on a
FACSCalibur or FACSCanto cytometer (BD Biosciences).
To analyze gene expression in BM-DCs, RNA was extracted using TRIzol
followed by DNase digestion and repurification with columns (Qiagen).
cDNAs were prepared using random hexamers and avian myeloblastosis
virus reverse transcriptase (Promega). Real-time PCR was performed in
triplicate using an ABI 7900HT machine in 384-well plates and the SYBR
Green system (Applied Biosystems). The following forward and reverse
primers were used: cyclophilin, 5?-GGCCGATGACGAGCCC-3? (for-
ward) and 5?-TGTCTTTGGAACTTTGTCTGCAA-3? (reverse); IFN-?,
5?-ATGAGTGGTGGTTGCAGGC-3? (forward) and 5?-TGACCTTTCAA
ATGCAGTAGATTCA-3? (reverse); IFNAR1 (5?-AGCAGGCATGAAC
CATTCAGT-3? (forward) and 5?-GGACACGGTCTTCTTTCACCAT-3?
(reverse); IFNAR2, 5?-CCGCCACTTTTTAACCTGGAT-3? (forward)
and 5?-AGCCGATCGATGGCTTCTG-3? (reverse). We used the standard
curve method for quantitative analysis of gene expression normalized to
the cyclophilin gene product. Genomic DNA contamination was tested in
all samples performing PCRs without avian myeloblastosis virus reverse
To analyze gene expression in ex vivo DCs, a different protocol was
used that was suitable for very small amounts of RNA. cDNA was syn-
thesized using the cDNA archive kit (Applied Biosystems) followed by a
preamplification reaction (Applied Biosystems). Premade TaqMan primers
and probes from Applied Biosystems were used to study the expression of
IL-15, IL-6, STAT1, STAT2, IFN regulatory factor (IRF)-7, IRF-3, and
myxovirus resistance (Mx)-1. Cyclophilin was used as the reference gene.
The comparative threshold cycle (Ct) method or ??Ct method of relative
quantitation of gene expression (24) (Applied Biosystems) was used for
these TaqMan PCRs, and the normalized Ct values (against cyclophilin)
were calibrated against the control sample (GM-CSF only) in each
We used ELISA kits (BD Pharmingen) to measure the levels of IL-6,
IL-10, TNF-?, and IL-12p70 in the supernatants of BM-DC cultures grown
in the presence or absence of IL-4 and stimulated with type I IFNs or LPS
(as positive control) for 24 h.
Western blot analysis
We performed Western blotting as described previously (25) using 30–50
?g of total DC cell protein. We used rabbit polyclonal anti-STAT1 and
STAT2, phospho-STAT1 (Tyr701) and phospho-STAT2 (Tyr689) (Upstate
Biotechnology). Anti-actin, anti-GAPDH, or anti-tubulin Ab (Santa-Cruz
Biotech) was used as a loading control. We detected primary Abs with
anti-rabbit HRP-Ab, chemiluminescence reagents (Pierce), and the Alpha-
Imager documentation system and software (Alpha Innotech).
Flow cytometric analysis of STAT1 phosphorylation
BM-DCs were stimulated for 30 min with 2500 U of IFN-? per milliliter
and harvested into BD Phosflow Fix Buffer I (BD Pharmingen) following
the manufacturer’s recommendation. Control untreated cells were har-
vested in a similar fashion. Cells were fixed for 10 min at 37°C. Following
fixation, cells were permeabilized with Phosflow Perm Buffer III for 30
min on ice. After washing and resuspending in PBS, cells were stained with
biotinylated anti-CD11c Ab followed by PE-Streptavidin (BD Pharmin-
gen). Cells were then stained with Alexa Fluor 647-conjugated anti-
phospho-STAT1 (BD Pharmingen) for 1 h at room temperature. Cells were
then washed and immediately analyzed on a FACSCanto flow cytometer.
We performed two-tailed Student’s t tests and considered significant values
of p ? 0.05 (marked in the figures as ?, p ? 0.05; ??, p ? 0.001; ???, p ?
IL-4 is not required for the generation of resting BM-DCs
IL-4 is included in the standard protocol for growing human DCs
and is also used in the mouse system, where its effects on DC
differentiation and activation are only partially known (15). We
6447 The Journal of Immunology
grew mouse BM precursors in medium supplemented with GM-
CSF and IL-4 or GM-CSF only (no IL-4) and analyzed their lin-
eage and costimulatory phenotype by flow cytometry on days 6 or
7 of culture. There was no difference in the absolute number of
DCs between the two culture conditions (no IL-4 vs IL-4) as
judged by trypan blue exclusion (Table I). However, as reported by
many groups (26, 27), we found a small but statistically significant
increase in the percentage of CD11c-positive BM-DCs in the cul-
tures grown in the presence of IL-4 (analyzed from 11 independent
cultures). In both types of culture ?90% of DCs were
CD11c?CD11b?double positive, resembling the myeloid DCs
(Table I). The analysis of the costimulatory phenotype showed that
both BM-DCs, grown in the presence or absence of IL-4, were in
a resting state expressing low levels of MHC and costimulatory
molecules (Fig. 1, A and C, and Table I). Thus, IL-4 is not an
essential requirement for the generation of mouse resting DCs
from BM precursors. To determine the effects of IL-4 on the ca-
pacity of DCs to activate, we studied the response to LPS and
found that IL-4 had a significant enhancing effect on LPS-induced
activation (Fig. 1, B and C) as previously reported (15), with the
exception of the inhibitory effect on the up-regulation of MHC
class I, which was not considered in the previous publication (15).
Although the constitutive expression of CD86 was higher in BM-
DCs grown in the presence of IL-4, this difference did not affect
the response to LPS that increased CD86 expression by 4-fold in
both IL-4- and no IL-4-BM-DCs (Fig. 1C).
In summary, IL-4 did not activate truly resting DCs per se, and
it enhanced their activation in response to a classic DC stimulus
such as LPS. Therefore, we could use these two sets of DCs to
stimulation with 100 ng/ml LPS (B); in both panels the black lines represent DCs grown in the absence of IL-4 (no IL-4) and area shaded gray represent
DCs grown in the presence of IL-4 (IL-4). Filled black curves represent isotype-control Abs. We gated DCs on CD11c?cells. C, Averages and SE of seven
experiments conducted with seven independent BM-DC cultures (?, p ? 0.05; ??, p ? 0.001; and ???, p ? 0.0001).
IL-4 does not activate resting BM-DCs. A and B, Costimulatory profile of unstimulated BM-DC controls (A) and BM-DCs after 24 h of
Table I. Myeloid signature and costimulatory molecule profile in
resting BM-DCs grown with and without IL-4a
Absolute number (?106)
MHC Class I
MHC Class II
0.5 ? 0.1
68.6 ? 1.9
97 ? 0.3
8.6 ? 1.8
22.7 ? 3.0
9.6 ? 1.7
22.4 ? 6.3
28.1 ? 3.8
0.6 ? 0.05
78.7 ? 1.5
92 ? 2.2
19.3 ? 1.5
27.7 ? 2.3
11.6 ? 1.8
30.5 ? 5.8
33.6 ? 2.5
aWe report absolute numbers of trypan blue-negative cells, percentages of live
DCs, and percentages of CD11c?cells positive for the indicated activation markers.
We found the percentage of CD11c?DCs to be significantly higher in the presence
of IL-4 (p ? 0.0002), whereas we did not find significant differences in the costimu-
latory phenotype of unstimulated DCs grown in presence or absence of IL-4 with the
exception of CD86, which was significantly higher (p ? 0.0005) in DCs grown in
presence of IL-4. Averages and SE of eleven experiments conducted with eleven
independent BM-DC cultures are shown.
bMean ? SE.
6448 IL-4 SUPPRESSES DC RESPONSE TO TYPE I IFNs
determine the effects of IL-4 on the activation induced by type I
IFNs in resting DCs.
IFN-? and IFN-? induce similar patterns of costimulatory
molecules in BM-DCs
We previously demonstrated that IFN-? at high doses (10,000
U/ml) is a potent stimulator of DCs (6). In recent investigations,
we used lower doses of IFN-? (2500U/ml) to study its effects on
the activation of resting DCs. We first tested BM-DCs grown in
medium supplemented with GM-CSF alone. IFN-? induced pri-
marily the up-regulation of MHC class I and the costimulatory
molecule CD86 at levels similar to those induced by LPS, used
here as a positive control (Fig. 2A). In contrast to LPS, IFN-? had
only a modest effect on CD40 and almost no effect on CD80 ex-
pression (Fig. 2A).
To determine whether this profile is specific to IFN-? or is
shared by other type I IFNs, we studied the costimulatory pheno-
type induced by IFN-?. IFN-? stimulated a profile very similar to
that induced by IFN-?, namely the up-regulation of MHC class I
and CD86, although at lower levels of expression. Similar to
IFN-?, IFN-? had minimal effects on CD40 and CD80 expression
It has long been recognized that type I IFNs exert pleiotropic
effects on a variety of target cells. The costimulatory profile in-
duced by IFN-? and IFN-? partially overlaps with that elicited by
TLR ligands and may contribute to the specific immune responses
promoted by these endogenous danger signals.
IL-4 inhibits the up-regulation of costimulatory molecules
induced by type I IFNs
We then determined whether IL-4 could affect the response of
resting DCs to type I IFNs. On day 6 or 7 of culture we stimulated
the two sets of DCs with IFN-? and analyzed the costimulatory
phenotype after 24 h of stimulation. We found that IFN-? up-
regulated MHC class I and CD86 in DCs from both cultures (Fig.
3, A–D). We focused our attention on MHC class I and CD86,
because we found that they are the most consistent indicators of
DC response to type I IFNs in vitro (Fig. 2, A and B). The analysis
of seven experiments revealed that DCs grown in the absence of
IL-4 responded to IFN-? with a significantly higher percentage of
cells expressing CD86 and high levels of MHC class I than DCs
grown in the presence of IL-4, suggesting that IL-4 suppresses the
DC response to IFN-? (Fig. 3, A–D).
To determine whether IL-4 affected the DC response to IFN-?
specifically or to type I IFNs more generally, we stimulated BM-
DCs with IFN-? and found that IL-4 suppressed the up-regulation
of MHC class I induced by IFN-? (Fig. 3E), whereas it had lesser
effects on CD86 expression (data not shown but comparable to
those in Fig. 3G). The presence of IL-4 did not affect the modest
modulation of CD80 and CD40 induced by IFN-? and IFN-? (data
not shown). Therefore, we concluded that IL-4 inhibits the re-
sponse of myeloid DCs to type I IFNs by suppressing the capa-
bility of DCs to up-regulate MHC and costimulatory molecules in
response to IFN-? and MHC class I in response to IFN-?.
To determine whether IL-4 suppresses DC activation or modu-
lates DC differentiation, we analyzed the effects of a short-term
pretreatment with IL-4 on BM-DCs grown in GM-CSF alone. We
added 2.5 ng/ml IL-4 to DCs at day 5 of culture, 24 h before the
stimulation with type I IFN, and found that IL-4 suppressed the
ability of DCs to respond to type I IFNs (Fig. 3, F and G). Indeed,
BM-DCs pretreated with IL-4 showed a reduced up-regulation of
CD86 and MHC class I upon incubation with IFN-?, and a reduced
up-regulation of MHC class I upon incubation with IFN-?. Fur-
thermore, the up-regulation of MHC class I upon LPS stimulation,
which is considered dependent on autocrine type I IFNs, was also
inhibited by the short-term treatment with IL-4 (Fig. 3F) as it was
in DCs grown in the presence of IL-4 (see Fig. 1, B and C). There-
fore, we propose that IL-4 suppresses type I IFN-induced activa-
tion of DCs and exerts this suppression if given during the gener-
ation of DCs and also to fully differentiated DCs.
Because the responses of BM-DCs to IFN-? and IFN-? were
affected by IL-4 to different extents, we performed a dose titration
of these type I IFNs in the presence or absence of IL-4 to deter-
mine whether these differences are due to qualitative differences in
the ability of IL-4 to inhibit DC response to IFN-? vs IFN-? or
whether they are simply due to differences in specific activities of
(IFN-a) or 2500 U/ml IFN-? (IFN-b) (with 50 ?g/ml polymyxin B) and 24 h later we analyzed DC activation markers by flow cytometry. We gated DCs
on CD11c?cells. A, Costimulatory molecules expressed by DCs stimulated with LPS (thin lines), IFN-? (thick lines), or control with medium alone (area
shaded gray). B, We stimulated DCs with LPS (thin lines), IFN-? (thick lines), or control (with polymyxin B) (area shaded gray). In both panels the filled
black lines represent isotype control Abs. Plots are representative of seven experiments.
Both IFN-? and IFN-? induce up-regulation of MHC class I and CD86. We stimulated BM-DCs with 100 ng/ml LPS or 2500 U/ml IFN-?
6449 The Journal of Immunology
the two IFN preparations. We found that: 1) IL-4 inhibited the
up-regulation of MHC class I induced by all of the doses of IFN-?
tested and by medium to low doses of IFN-?; 2) IL-4 inhibited the
up-regulation of CD86 induced by medium to low doses of IFN-?;
and 3) IL-4 had no effect on the expression of CD86 induced by
IFN-? at all of the doses tested (Fig. 3, H–K). These data exclude
the explanation that a difference in the specific activity of the two
IFN preparations plays a role in the different kind of suppression
by IL-4 on IFN-? and IFN-?. Instead, they indicate that there are
qualitative differences in the response of BM-DCs to IFN-? and
IFN-? that influence the inhibitory effect of IL-4.
IL-4 suppresses the production of IL-6 in BM-DCs upon type I
Both IFN-? and IFN-? induce IL-6 production in DCs (17, 28).
Therefore, we measured by ELISA the levels of IL-6 in the su-
pernatants of BM-DC cultures grown in the presence or absence of
IL-4. Among the type I IFNs tested, IFN-? induced much higher
IL-6 secretion in DCs than IFN-? did (Fig. 4). We did not find any
difference in the levels of IL-6 between unstimulated DCs left in
medium alone and those with polymyxin B alone added that we
used as control for IFN-? (data not shown). IL-4, which induced
the production of small amounts of IL-6 in unstimulated DCs,
significantly suppressed the up-regulation of IL-6 upon IFN-? and
IFN-? stimulation (Fig. 4). Type I IFNs did not induce IL-10,
TNF-?, or IL-12 in either IL-4 or no IL-4-BM-DCs (data not
shown). Therefore, IL-4 suppresses the production of IL-6 induced
by type I IFNs in BM-DCs, indicating that IL-4 not only inhibits
the expression of costimulatory molecules but also the secretion of
IL-4 suppresses the expression of IFN-? and IFNARs
in BM-DCs upon type I IFN stimulation
Type I IFNs amplify their own function via an autocrine loop (29,
30) by inducing production of IFN-? and/or IFN-?, depending on
the cell type analyzed. Under normal circumstances, myeloid DCs
preferentially produce IFN-? (8). In other cell types, the expres-
sion of the two subunits of the receptors for type I IFNs, IFNAR1
and IFNAR2, can also be modulated by their ligands (31). To
determine whether IL-4 inhibits this positive feedback loop in
induced by type I IFNs in vitro. A, MHC class I and CD86 expression
induced by 24 h of stimulation with IFN-? in the presence or absence of
IL-4. Black lines represent DCs grown in the absence of IL-4 (no IL-4) and
the shaded gray areas represent DCs grown in the presence of IL-4. Filled
black curves represent isotype-control Abs. B–E, Histogram bars represent
averages and standard errors of the results from seven experiments con-
ducted with seven independent BM-DC cultures. B and C, Percentages of
DCs expressing high levels of MHC class I and CD86 after IFN-? stim-
ulation, respectively. The percentages of positive cells were determined by
setting the threshold of positivity, indicated in A by the vertical line, on the
unstained control background at ?1%. D and E, MdFI of MHC class I in
BM-DCs after 24 h of stimulation with IFN-? (IFN-a) or IFN-? (IFN-b)
(with polymyxin B). F and G, MHC class I and CD86 MdFI of DCs
IL-4 inhibits the up-regulation of costimulatory molecules
pretreated or not pretreated for 24 h with IL-4 and then stimulated with
IFN-?, IFN-? (with polymyxin B), and LPS for 24h. H–K, Dilution curves
of IFN-? (H and J) and IFN-? (with polymyxin B) (I and K) on BM-DCs
exposed or not exposed to 2.5 ng/ml IL-4. Values are averages and SE of
MHC class I and CD86 MdFI of at least two sets of experiments (?, p ?
0.05; ??, p ? 0.001; ???, p ? 0.0001).
measured by ELISA the levels of IL-6 in the supernatants of DC cultures
grown in the presence or absence of IL-4 and stimulated with 2500 U/ml
IFN-? (IFN-a) or IFN-? (IFN-b) (with polymyxin B) for 24 h. Averages
and SE of four independent cultures are shown (?, p ? 0.05).
IL-4 suppresses type I IFN-induced production of IL-6. We
6450 IL-4 SUPPRESSES DC RESPONSE TO TYPE I IFNs
DCs, we analyzed by real-time RT-PCR the expression of IFN-?
and IFNAR1 and IFNAR2 in BM-DCs grown in the presence or
absence of IL-4. Resting BM-DCs expressed very small amounts
of IFN-? RNA, and this production was not significantly influ-
enced by IL-4. IFN-? stimulation induced IFN-? transcript in BM-
DCs and IL-4 clearly inhibited this activation (Fig. 5A). The anal-
ysis of IFNAR1 and IFNAR2 transcripts gave similar results: IL-4
did not affect IFNAR constitutive expression and suppressed its
up-regulation by IFN-? (Fig. 5, B and C). Together with the results
of the suppression of the up-regulation of costimulatory molecules
and IL-6 upon stimulation with type I IFNs, these data indicate that
IL-4 renders myeloid DCs less responsive to paracrine type I IFNs
and suggest that it diminishes the autocrine positive feedback loop
that normally amplifies the effects of this family of cytokines.
IL-4 suppresses the expression of IFN-responsive genes
Type I IFNs stimulate murine DCs to produce cytokines such as
IL-6 (17, 28) and IL-15 (32, 33), although they do not induce
classic proinflammatory cytokines like IL-12 (34). In Fig. 4, we
show that IL-4 inhibits IFN-?-induced secretion of IL-6. To de-
termine whether IL-4 also affects IFN-induced IL-15, we measured
by quantitative real-time RT-PCR IL-15 mRNA in IL-4-BM-DCs
and no IL-4-BM-DCs stimulated with 2500 U/ml IFN-? for 6 h.
We found that BM-DCs expressed little IL-15 constitutively and that
IL-4 did not affect this constitutive expression. IFN-? stimulation in-
duced IL-15 expression that was inhibited by IL-4 (Fig. 5D).
Type I IFNs were first discovered for their property of interfer-
ing with viral replication (35, 36). They cause interference by in-
ducing the expression of genes that can directly inhibit viral rep-
lication in infected cells (36). Mx-1 is one of these anti-viral genes
up-regulated by type I IFNs (37). To determine whether the inhib-
itory effect of IL-4 could also influence the induction of anti-viral
genes, we measured the RNA expression of Mx-1 by quantitative
real-time RT-PCR in IL-4-BM-DCs and no IL-4-BM-DCs stimu-
lated with 2500 U/ml IFN-? for 6 h. We found that both IL-4-
BM-DCs and no IL-4-BM-DCs expressed low levels of Mx-1
mRNA constitutively; they up-regulated Mx-1 upon IFN-? stim-
ulation, and IL-4-BM-DCs showed a reduced up-regulation of
Mx-1 (Fig. 5D), indicating that IL-4 not only inhibits MHC and
costimulatory molecules and cytokines but is also able to inhibit
the expression of anti-viral genes induced by type I IFNs.
Another important IFN responsive gene is IRF-7, which has
been shown to be the master regulator of type I IFN-dependent
immune responses (38) and, in cell types other than DCs, has been
shown to increase upon IFN stimulation and be part of the IFN
positive feedback loop (38). We measured the expression of IRF-7
mRNA by quantitative real-time RT-PCR in IL-4-BM-DCs and no
IL-4-BM-DCs stimulated with IFN-? and found that both IL-4-
BM-DCs and no IL-4-BM-DCs expressed low levels of IRF-7
mRNA constitutively and up-regulated IRF-7 mRNA upon IFN-?
stimulation, and IL-4-BM-DCs showed a reduced up-regulation of
IRF-7 mRNA (Fig. 5D). As negative control, we also measured the
expression of IRF-3, a member of the same family of IFN regu-
latory factors that is involved in the signaling pathway downstream
of TLRs and is not induced by type I IFNs (39). We confirmed that
IRF-3 was not up-regulated by IFN-? and found that IL-4 did not
affect its expression (Fig. 5D), indicating the specificity of the
effect of IL-4 on the response of IRF-7 to type I IFN.
sive genes upon IFN-? stimulation. We analyzed the gene expression of
IFN-? (IFN-b) (A), IFNAR1 (B), and IFNAR2 (C) by real-time RT-PCR
using the SYBR Green system in BM-DCs stimulated with IFN-? (IFN-a)
(2500 U/ml) for 6 h. We used cyclophilin for gene normalization by using
the standard curve method. Error bars represent the averages and SE of
three experiments conducted with three independent BM-DC cultures. D,
We analyzed the gene expression of IFN inducible genes in BM-DCs cul-
tured with and without IL-4 and stimulated with IFN-? (2500 U/ml) for
3 h. PCRs were performed using the TaqMan gene expression system and
IL-4 suppresses the expression of IFN-? and IFN respon-
analyzed by the ??Ct method. All of the conditions were calibrated against
the control (GM-CSF medium only) in each experiment. Graphs are rep-
resentative of two experiments (?, p ? 0.05).
6451 The Journal of Immunology
In summary, these results indicate that IL-4 inhibits the expres-
sion of several kinds of IFN-? responsive genes, from MHC and
costimulatory molecules and cytokines to the antiviral gene Mx-1
and the signaling factor IRF-7 and suggest that it may well be a
general inhibitor of responses to IFN-?.
IL-4 suppresses STAT1 and STAT2 phosphorylation and
The transduction of the IFNAR signaling requires the phosphory-
lation of the signal transducers STAT1 and STAT2 (8). Therefore,
we analyzed STAT1 and STAT2 phosphorylation to determine
whether IL-4 exerts its effects on the signaling pathway directly
downstream of IFNAR. Thus, we stimulated BM-DCs with IFN-?
and processed them for protein detection by Western blotting after
30 min or 8 h of incubation. Resting BM-DCs showed minimal
amounts of phosphorylated STAT1 and STAT2 irrespective of the
presence of IL-4 (see controls in Fig. 6A), indirectly confirming
that these cells do not produce much IFN-? constitutively (see Fig.
5A). Soon after IFN-? stimulation (30 min), we observed increased
phosphorylation of both STATs (pSTAT1 and pSTAT2) in DCs
grown in the absence of IL-4; this increase was maintained, albeit
at lower levels, after 8 h of stimulation (Fig. 6A). In contrast, DCs
differentiated in the presence of IL-4 were suppressed in their
phosphorylation of STAT1 and STAT2 molecules at both time
points (Fig. 6A), indicating that IL-4 inhibits DC phosphorylation
of STAT1 and STAT2.
In our BM-DC culture, 20–30% of the cells are not CD11c-
positive DCs but rather CD11b-single positive macrophages or
CD11c-CD11b-double negative fibroblast-like cells. To deter-
mine whether the IL-4-induced inhibition of the type I IFN
response was specific to the CD11c-positive population, we an-
alyzed STAT-1 phosphorylation by intracellular staining in
cells stained for the surface marker CD11c. We found that IL-4
suppressed the phosphorylation of STAT1 upon IFN-? stimu-
lation both in the CD11c and non-CD11c cells (Fig. 6B) (the
median fluorescence intensity (MdFI) values of non-CD11c
cells were 75 for no IL-4-control, 322 for no IL-4-IFN-?, 95 for
IL-4-control, and 267 for IL-4-IFN-?). These results indicate
that IL-4 inhibits IFN-induced STAT-1 phosphorylation in all
of the cell types present in the culture, suggesting that it may be
a general phenomenon that affects the response to the type I
IFNs of several cellular types.
In other cell types, the protein levels of STAT1 and STAT2 are
positively regulated by type I IFNs (40). Therefore, we analyzed
STAT1 and STAT2 expression in BM-DCs after type I IFN stim-
ulation to shed light on the regulation of STATs in DCs and to
further address the IL-4 inhibition of the type I IFN autocrine loop
in DCs (Fig. 6C). We found that resting DCs expressed detectable
levels of STAT1 and STAT2 proteins and that IL-4 treatment led
to constitutively lower levels of STAT2 and, to a lesser extent,
STAT1 (Fig. 6C). In BM-DCs grown in absence of IL-4, STAT1
and STAT2 expression were up-regulated 8 h after stimulation
with IFN-? (Fig. 6C), demonstrating that STAT1 and STAT2 are
IFN-induced genes in DCs and part of the type I IFN autocrine
loop. IL-4 strongly inhibited the up-regulation of STAT1 and
STAT2 by IFN-? (Fig. 6C).
In conclusion, our data indicate that IL-4 suppresses the re-
sponse of BM-DCs to type I IFNs by inhibiting the initial phos-
phorylation of STAT1 and STAT2 and by reducing the autocrine
positive feedback loop that normally amplifies the effects of type
IL-4 inhibits the up-regulation of costimulatory molecules
induced by type I IFNs in DCs in vivo
Although the BM-DCs are a well accepted model of myeloid DCs,
we sought to determine whether the suppressive effect observed in
expression. A and C, Left panels, Western Blot analyses of the phosphor-
ylation (p) and expression of STAT1 and STAT2 in BM-DCs grown in the
presence or absence of IL-4 and harvested 30 min or 8 hours after stimu-
lation with 2500 U/ml IFN-?. Actin, tubulin, or GAPDH were used as
loading controls. Right panels, Bars represent the intensities of each STAT
band, normalized to the respective loading controls, from one experiment
representative of three experiments conducted with three independent
BM-DC cultures. B, Flow cytometric phospho-STAT (pSTAT) analysis of
BM-DCs grown in the presence or absence of IL-4 and stimulated for 30
minutes with 2500 U/ml IFN-?. We then performed staining for the surface
marker CD11c and intracellular staining for STAT1. Results are means of
triplicates from one culture (?, p ? 0.05).
IL-4 suppresses STAT1 and STAT2 phosphorylation and
6452 IL-4 SUPPRESSES DC RESPONSE TO TYPE I IFNs
vitro was reproducible in vivo. First, we injected 2 ? 104U of
IFN-? s.c. into C57BL/6 (B6) mice and, 24 h later, analyzed the
draining inguinal lymph nodes. We stained the total population of
the lymph nodes and gated for CD11c-positive DCs and found an
increase in MHC class I and CD86 expression in DCs (Fig. 7), as
IFN-? does in vitro.
To test the hypothesis that IL-4 suppresses the response of DCs
to IFN-? in vivo, we treated B6 mice with IL-4 in vivo. Because
rIL-4 has a very short life span, we used the protocol developed by
Finkelman et al. (22) to increase the effectiveness of IL-4 by com-
plexing it with a neutralizing anti-IL-4 mAb (from clone 11B11).
IL-4 complexed with anti-IL-4 mAb (IL-4C) slowly releases the
cytokine, extending its effects to 2 or 3 days. We pretreated mice
with IL-4C or an isotype control Ab injected s.c. 24 h before in-
jecting s.c. 2 ? 104U of IFN-? or an equivalent volume of PBS.
Twenty-four hours later, we stained the total population of the
inguinal lymph nodes and gated for CD11c-positive DCs. We
found a clear suppression of the up-regulation of MHC class I and
CD86 induced by IFN-? in the IL-4 injected group as compared
with the controls (injected with an isotype Ab) (Fig. 7), indicating
that IL-4 can suppress the response of DCs to IFN-? in vivo. To
measure the effectiveness of IL-4 in vivo, we assayed in mice
injected with IL-4C alone the up-regulation of CD23 on CD19?B
cells by FACS analysis using anti-mouse CD23 Ab that has been
described as a sign of exposure to IL-4 (data not shown) (22).
IL-4 suppresses IFN-responsive genes in splenic DCs upon ex
vivo IFN-? stimulation
To establish the biological relevance of the suppression of the IFN
response by IL-4 that we observed in vitro, we determined whether
IL-4 has the same effects on DCs that differentiated in vivo. We
injected i.v. IL-4C or isotype control mAbs in normal B6 mice and,
24 h later, stimulated the total population of splenocytes ex vivo
with 2 ? 104U of IFN-? in the presence of 3.3 ng/ml GM-CSF
alone or GM-CSF plus 2.5 ng/ml IL-4 for 3 h at 37°C. We then
sorted the splenic DCs gating on CD11c?CD19?DCs and studied
their RNA expression. We found that the treatment with IFN-?
induced the expression of the mRNA for STAT1, STAT2, and
IRF-7 and that this up-regulation was reduced in DCs isolated
from mice treated with IL-4C, whereas no effects were seen on the
expression of IRF-3, either by IFN-? or IL-4 (Fig. 8, A and B), as
we have found in BM-DCs (Fig. 5D). Similarly, IFN-? induced
the expression of the mRNA for the cytokines IL-6 and IL-15,
and this activation was almost completely inhibited in DCs
from mice treated with IL-4C (Fig. 8, A and B). IL-4 also
reduced the expression of Mx-1 mRNA induced by IFN-?
These results confirm in vivo the data obtained in vitro with
BM-DCs and indicate that IL-4 can suppress the expression of
MHC and costimulatory molecules, proinflammatory cytokines,
anti-viral genes, and genes of the signaling pathway downstream
of the IFNAR that is induced in DCs in vivo by stimulation with
type I IFNs.
The versatility of DCs to grow and differentiate in different kinds
of conditions indicates the importance of the microenvironment in
which the DCs differentiate and activate for the overall immune
response. We report in this article that IL-4, a cytokine widely used
in DC culture, suppresses the capacity of DCs to respond to type
and CD86 (B) in 6- to 8-wk-old C57BL/6 mice injected s.c. with IL-4C (IL-4, filled bars) or isotype control Ab (no IL-4, open bars) and 24 h later with
20,000 U of recombinant IFN-? (IFN-a) or PBS. The draining inguinal lymph nodes were harvested after 24 h and stained for CD11c, CD19, MHC class
I, and CD86. Cells were gated and analyzed on the CD11c?CD19?DCs. Numbers in the contour plots indicate percentage (top right) and MdFI values
(bottom right) of the indicated markers. Error bars are averages and SE of six mice in each group.
IL-4 suppresses the up-regulation of MHC class I and CD86 induced by IFN-? in vivo. We analyzed the expression of MHC class I (A)
splenic DCs. We analyzed the gene expression of the IFN-inducible genes
STAT1, STAT2, and IL-6 (A) and IL-15, Mx1, IRF-7, and IRF-3 (B) after the
injection of IL-4C (IL-4) or isotype control Ab (no IL-4) and ex vivo
treatment of splenocytes with recombinant IFN-? (IFN-a) (20,000 U). Re-
al-time RT-PCR was performed using the TaqMan gene expression system
and analyzed by the ??Ct method. All of the conditions were calibrated
against the control (GM-CSF medium only) in each experiment. Graphs are
representative of two experiments (?, p ? 0.05).
IL-4 suppresses gene expression induced by IFN-? in
6453 The Journal of Immunology
I IFNs by rendering DCs less capable of phosphorylating STAT1
and STAT2 and thus limiting the autocrine positive loop that nor-
mally amplifies type I IFN functions. Administration of IL-4 in
vivo inhibits the activation of DCs induced by IFN-? treatment
and suppresses the expression of IFN-responsive genes by DCs
differentiated in vivo.
Different activators induce unique responses in DCs that are
distinguished by the costimulatory molecules involved and cyto-
kines produced, thereby dictating distinct immune responses. Both
IFN-? and IFN-? up-regulate most consistently surface expression
of MHC class I and CD86 without inducing other costimulatory
molecules, such as CD40 and CD80, or the classic proinflamma-
tory cytokines, such as TNF-? or IL-12 (34). However, if DCs are
activated by type I IFNs in the presence of IL-4, this type of ac-
tivation is suppressed.
Previously, Labeur et al. (15) have compared murine BM-DCs
grown in medium supplemented with GM-CSF or GM-CSF plus
IL-4. They used a protocol that yields activated DCs by transfer-
ring cells at days 5 and 7 of culture. We have previously shown
that the transfer procedure activates DCs even in the absence of
activators such as LPS or CD40 ligand (6). Therefore, the data
from Labeur et al. (15) indicate that IL-4 does not affect DC yield
while it enhances the DC activation induced by transfer, LPS, and
CD40 ligand. Our results (Fig. 1 and Table I) confirm that IL-4 is
not required for the generation of BM-DCs. Furthermore, we show
that IL-4 does not activate truly resting DCs per se, because DCs
grown in IL-4 keep a resting costimulatory phenotype, although
IL-4 does enhance LPS-induced activation with the exception of
the inhibitory effect on the up-regulation of MHC class I.
It has been reported that the up-regulation of costimulatory mol-
ecules induced by TLR ligands, such as LPS and polyinosinic/
polycytidylic acid, is mediated by autocrine type I IFNs (12, 41).
Our findings that IL-4 inhibits the LPS-induced up-regulation of
MHC class I while it enhances the other costimulatory molecules
suggest that IL-4 exerts opposite effects on the responses of DCs
to IFN-? and LPS. Furthermore, the finding that IL-4 suppresses
MHC class I up-regulation in response to IFN-? but does not affect
the up-regulation of CD86 supports the notion that the mechanism
of MHC class I regulation is similar upon different stimuli and is
mainly mediated by autocrine IFN-? (42), whereas the regulation
of the costimulatory molecules in DCs is more complex and spe-
cific to distinct activators even if they share the same receptors,
such as IFN-? and IFN-?. The mechanism by which type I IFN
signaling leads to distinct outcomes is still matter of investigation.
From many studies conducted especially in cell lines, we know
that the effects of type I IFNs are amplified by a positive feedback
loop that sustains itself by inducing the expression of type I IFNs
and the genes involved in their response, such as the two subunits
of IFNAR and the intracellular signaling molecules. In this study
we characterized the positive feedback loop that is functional in
primary dendritic cells. Indeed, we found that myeloid DCs re-
spond to type I IFNs by increasing the expression of IFN-?, the
two subunits of IFNAR, and the signal transducers STAT1,
STAT2, and IRF-7. Therefore, components of type I IFN signaling
known from other cells are also part of the autocrine positive am-
plification loop in DCs.
The IL-4-mediated suppression of the response to type I IFNs in
DCs involves decreased phosphorylation of STAT1 and STAT2
that occurs 30 min after IFN-? stimulation. This indicates that the
cross-talk between the two signaling pathways occurs at the level
of STATs or upstream of them. Previous reports show that in mac-
rophages/monocytes, IL-4-activated STAT6 negatively regulates
IFN-stimulated STAT1-dependent transcription (19), possibly
through the interaction of the transactivation domain of STAT6,
suggesting the importance of transcriptional coactivators and core-
pressors (43). Although some argue that IL-4 is necessary for
STAT1 induction in DCs (44), our results are in agreement with
Ohmori and Hamilton (43), who demonstrated that IL-4 suppresses
STAT1 transcription via STAT6 in transfected fibroblasts. Be-
cause STAT2 facilitates STAT1 recruitment to the IFNAR com-
plex (45), the decreased STAT2 expression that is caused by IL-4
may further reduce recruitment of STATs and strengthen the sup-
pressive effect of IL-4.
Our results suggest that IL-4, a prototypic Th2 cytokine, inhibits
the expression of the proinflammatory cytokines IL-6 and IL-15
upon IFN-? stimulation and may be important in the context of
immune responses in which IL-6 and IL-15 play a pivotal role.
Similarly, IL-4 inhibits the up-regulation of Mx-1, suggesting
that IL-4 could suppress the anti-viral response of DCs. The find-
ing by intracellular staining (Fig. 6) that the inhibition of STAT-1
phosphorylation is not limited to DCs but affects all of the cell
types present in the culture leads us to suggest that IL-4 may
negatively affect the general IFN-induced anti-viral response of
immune and nonimmune cells. We envision that, during a Th2
response, myeloid DCs differentiate and/or activate in an IL-4-
dominated environment in which they are less capable of re-
sponding to type I IFNs. These DCs would help maintain the Th2
response but would be impaired in their ability to respond to and
produce type I IFNs upon a possible concurring viral infection.
This mechanism may explain the increased susceptibility to viral
infections observed in animals infected with Th2-inducing para-
sites (e.g., Schistosoma) (2). Our results also suggest the use of
IL-4 as a novel therapeutic strategy for diseases in which an ex-
cessive exposure to type I IFNs could be pathogenic (5). A direct
or indirect strengthening of the effects of IL-4 on DCs may, for
example, be therapeutic in the autoimmune disease systemic lupus
We thank Roberto Caricchio, Randy Cron, Terri Finkel, Terri Laufer, and
Edward Pearce for critically reading the manuscript, Polly Matzinger for
reading the manuscript and helpful suggestions, and Lisa Bain for editorial
The authors have no financial conflict of interest.
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6455The Journal of Immunology