Type I Interferons IL-4 Suppresses Dendritic Cell Response to

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The Journal of Immunology

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 costimulatory 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 characterized the positive feedback loop that is triggered by IFN-␣ in primary DCs and found that IL-4 inhibited the initial phosphorylation 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 susceptibility to viral infections observed during Th2-inducing parasitoses. The Journal of Immunology, 2007, 179: 6446 – 6455.

D

endritic cells (DCs)3 are key players in the activation of adaptive immunity. In the resting/immature state DCs efficiently take up Ag, whereas after activation the enhanced expression of MHC and costimulatory molecules and the secretion of proinflammatory cytokines allow DCs to activate naive and memory T cells and modulate the immune response. Concurring immune responses (i.e., Th1 vs Th2) often show competition 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 pivotal 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

*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 Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, and ‡Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140 Received for publication September 18, 2006. Accepted for publication August 30, 2007. 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. 1 This work was supported by National Institutes of Health National Institute of Allergy and Infectious Diseases Grant AI049892 (to S.G.), the Lupus Foundation Southeastern 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 conclusions. 2 Address 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: [email protected] 3 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; Ct, threshold 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 www.jimmunol.org

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 composed of IFN-␣ and -␤ receptor (IFNAR) 1 and IFNAR2 subunits (8), mediate the innate response to viral infections and are also required for full DC response to TLRs (12) and their stimulation of T and B cells (13). Activated lymphocytes produce cytokines that strongly influence 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 differentiation 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. Previous 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 well; in monocytes/macrophages, type I IFNs negatively regulate IL-4 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 influences 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

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 I IFNs.

Materials and Methods Mice 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 Hospital of Philadelphia, an American Association for the Accreditation of Laboratory Animal Care-accredited facility.

BM-DC BM-DCs were generated as described previously (6). Briefly, BM precursors from C57BL/6-RAG-KO mice were seeded at 1 ⫻ 106/ml in complete IMDM (10% FBS, penicillin/streptomycin, gentamicin, and ␤-mercaptoethanol) 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.

Mice injections 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 FACS analysis. 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.

Flow cytometry BM-DCs. BM-DCs were washed in cold PBS, incubated with rat antimouse 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 cytometer (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 ⫻ 106 cells per tube, and stained with the mAbs as described above. PerCP-Cy5.5-conjugated anti-mouse CD19 mAb was

6447 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).

Quantitative PCR 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⬘ (forward) 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 transcriptase. To analyze gene expression in ex vivo DCs, a different protocol was used that was suitable for very small amounts of RNA. cDNA was synthesized 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 experiment.

ELISA 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 AlphaImager 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 harvested 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 Pharmingen). Cells were then stained with Alexa Fluor 647-conjugated antiphospho-STAT1 (BD Pharmingen) for 1 h at room temperature. Cells were then washed and immediately analyzed on a FACSCanto flow cytometer.

Statistical analysis We performed two-tailed Student’s t tests and considered significant values of p ⬍ 0.05 (marked in the figures as ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001; ⴱⴱⴱ, p ⬍ 0.0001).

Results 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

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Table I. Myeloid signature and costimulatory molecule profile in resting BM-DCs grown with and without IL-4a

Absolute number (⫻106) Percentage CD11c⫹ P ⬙ ⬙ CD11c⫹CD11b⫹ P ⬙ ⬙ CD86 P ⬙ ⬙ CD80 P ⬙ ⬙ CD40 P ⬙ ⬙ MHC Class I P ⬙ ⬙ MHC Class II

No IL-4b

IL-4b

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

a We 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 costimulatory 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. b Mean ⫾ SE.

grew mouse BM precursors in medium supplemented with GMCSF and IL-4 or GM-CSF only (no IL-4) and analyzed their lineage 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 cultures 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 capacity 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 BMDCs 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

FIGURE 1. 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 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 (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001; and ⴱⴱⴱ, p ⬍ 0.0001).

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FIGURE 2. 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-␣ (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.

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 primarily 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 expression (Fig. 2A). To determine whether this profile is specific to IFN-␣ or is shared by other type I IFNs, we studied the costimulatory phenotype 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 (Fig. 2B). It has long been recognized that type I IFNs exert pleiotropic effects on a variety of target cells. The costimulatory profile induced 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-␣ upregulated 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 BMDCs 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 response of myeloid DCs to type I IFNs by suppressing the capability 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 modulates 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-␤. Furthermore, 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). Therefore, we propose that IL-4 suppresses type I IFN-induced activation of DCs and exerts this suppression if given during the generation 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 determine 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

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FIGURE 4. IL-4 suppresses type I IFN-induced production of IL-6. We 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 (ⴱ, p ⬍ 0.05).

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 IFN stimulation Both IFN-␣ and IFN-␤ induce IL-6 production in DCs (17, 28). Therefore, we measured by ELISA the levels of IL-6 in the supernatants 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 proinflammatory cytokines. IL-4 suppresses the expression of IFN-␤ and IFNARs in BM-DCs upon type I IFN stimulation

FIGURE 3. IL-4 inhibits the up-regulation of costimulatory molecules 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 conducted with seven independent BM-DC cultures. B and C, Percentages of DCs expressing high levels of MHC class I and CD86 after IFN-␣ stimulation, 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

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 expression 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

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 (ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001; ⴱⴱⴱ, p ⬍ 0.0001).

The Journal of Immunology

6451 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 influenced by IL-4. IFN-␣ stimulation induced IFN-␤ transcript in BMDCs and IL-4 clearly inhibited this activation (Fig. 5A). The analysis 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 in BM-DCs

FIGURE 5. IL-4 suppresses the expression of IFN-␤ and IFN responsive 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 cultured 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

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 determine 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 induced IL-15 expression that was inhibited by IL-4 (Fig. 5D). Type I IFNs were first discovered for their property of interfering with viral replication (35, 36). They cause interference by inducing the expression of genes that can directly inhibit viral replication in infected cells (36). Mx-1 is one of these anti-viral genes up-regulated by type I IFNs (37). To determine whether the inhibitory 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 stimulated with 2500 U/ml IFN-␣ for 6 h. We found that both IL-4BM-DCs and no IL-4-BM-DCs expressed low levels of Mx-1 mRNA constitutively; they up-regulated Mx-1 upon IFN-␣ stimulation, 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-4BM-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 regulatory 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.

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 (ⴱ, p ⬍ 0.05).

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IL-4 SUPPRESSES DC RESPONSE TO TYPE I IFNs

In summary, these results indicate that IL-4 inhibits the expression 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 expression The transduction of the IFNAR signaling requires the phosphorylation 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 CD11cpositive DCs but rather CD11b-single positive macrophages or CD11c-CD11b-double negative fibroblast-like cells. To determine whether the IL-4-induced inhibition of the type I IFN response was specific to the CD11c-positive population, we analyzed 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-␣ stimulation 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 stimulation 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 response of BM-DCs to type I IFNs by inhibiting the initial phosphorylation of STAT1 and STAT2 and by reducing the autocrine positive feedback loop that normally amplifies the effects of type I IFNs.

FIGURE 6. IL-4 suppresses STAT1 and STAT2 phosphorylation and expression. A and C, Left panels, Western Blot analyses of the phosphorylation (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 stimulation 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 (ⴱ, p ⬍ 0.05).

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

The Journal of Immunology

6453

FIGURE 7. 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) 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.

vitro was reproducible in vivo. First, we injected 2 ⫻ 104 U 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 complexing 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-

FIGURE 8. IL-4 suppresses gene expression induced by IFN-␣ in 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). Real-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 (ⴱ, p ⬍ 0.05).

jecting s.c. 2 ⫻ 104 U 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 ⫻ 104 U 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-␣ (Fig. 8B). 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.

Discussion 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

6454 I IFNs by rendering DCs less capable of phosphorylating STAT1 and STAT2 and thus limiting the autocrine positive loop that normally 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 cytokines 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 proinflammatory 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 activation 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 transferring 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 molecules 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 specific 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 respond 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 amplification 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 macrophages/monocytes, IL-4-activated STAT6 negatively regulates IFN-stimulated STAT1-dependent transcription (19), possibly through the interaction of the transactivation domain of STAT6,

IL-4 SUPPRESSES DC RESPONSE TO TYPE I IFNs suggesting the importance of transcriptional coactivators and corepressors (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. Because STAT2 facilitates STAT1 recruitment to the IFNAR complex (45), the decreased STAT2 expression that is caused by IL-4 may further reduce recruitment of STATs and strengthen the suppressive 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 finding 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-4dominated environment in which they are less capable of responding 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 parasites (e.g., Schistosoma) (2). Our results also suggest the use of IL-4 as a novel therapeutic strategy for diseases in which an excessive 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 erythematosus.

Acknowledgments 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 assistance.

Disclosures The authors have no financial conflict of interest.

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