IL-4 is a pleiotropic immunomodulatory cytokine produced by Th2 lymphocytes, mast cells, and eosinophils (7). IL-4 is the key cytokine for generating Th2 effector cells from naive CD4 T cells (8). In addition, IL-4 activates B cells but inhibits macrophage activation, which results in humoral immunity (9, 10). Although IL-4, together with GM-CSF, is commonly used to differentiate DCs from BM precursors, the effect of IL-4 on DC maturation remains largely unknown. Studies have demonstrated that IL-4 enhances the production of IL-12p70 by DCs (11, 12). Because IL-12 is a potent cytokine directing Th1 cell differentiation (13), IL-4 is suggested to have a positive role for Th1 cell differentiation via DCs. Indeed, it was reported that IL-4 can instruct DCs to promote the Th1 response, which depends on IL-12 (14). However, it is unclear how IL-4 induces IL-12 production in DCs that leads to Th1 cell differentiation. A possible mediator for this process is IL-10 because IL-10 inhibits IL-12 production by DCs (15). However, it remains unknown whether IL-10 is responsible for IL-4–mediated induction of the Th1 response.

We investigated the molecular mechanisms by which IL-4 directs Th1 cell differentiation. Our data showed that IL-4 up-regulates IL-12 while inhibiting IL-10 production by DCs, but not by B cells. The inhibition of IL-10 by IL-4 appears necessary for the induction of Th1 cell differentiation because the ability of IL-4 to induce IL-12 was lost in IL-10–deficient DCs. IL-4 inhibits IL-10 promoter activity, which depends on Stat6 and involves histone deacetylation of the IL-10 promoter. As a result, IL-4 plays a key role for Th1 cell differentiation by inhibiting IL-10 gene expression to up-regulate IL-12 production by DCs.

	Results and discussion

Top Abstract Results and discussion MATERIALS AND METHODS References

 
IL-4 inhibits IL-10 while enhancing IL-12p70 production by BM-derived DCs (BM-DCs)
To examine the effects of IL-4 on DC maturation, we cultured BM cells with GM-CSF for 5 d, followed by stimulation with LPS in the presence or absence of IL-4 for 2 d. IL-4–treated DCs had similar populations of CD11c⁺CD11b⁺CD8^–B220^– DCs and expressed comparable levels of MHC II and the costimulatory molecules CD40, CD80, and CD86 as controls (unpublished data). When cytokine production was examined, DCs stimulated in the presence of IL-4 produced less IL-10 but more IL-12p70 (Fig. 1 A). IL-6 and TNF- levels were not altered by IL-4 (Fig. 1 A). IL-4 treatment showed a similar effect on cytokine production from DCs stimulated by CpG (unpublished data), indicating that culture with IL-4 affects stimulation by both TLR4 and TLR9 ligands (16). Thus, IL-4–treated BM-DCs produce less IL-10 and more IL-12p70.

View larger version (25K): [in this window] [in a new window]   Figure 1. IL-4 inhibits IL-10 while enhancing IL-12 expression in BM-DCs. (A) BM-DCs from C57BL/6 mice were stimulated by LPS with or without IL-4 for 2 d. ELISA was performed to measure IL-6, IL-10, IL-12p70, and TNF- production. (B) BM-DCs were stimulated for 24 h as indicated. qRT-PCR was performed to assess the amount of mRNA for IL-10, IL-6, IL-12p35, and IL-12p40. The relative mRNA levels were normalized to the GAPDH gene. Data are means ± SE of at least three independent experiments.

Induction of Th1 cell differentiation by IL-4–treated DCs is IL-10 dependent
IL-10 is known to inhibit IL-12 expression (15). Therefore, it is possible that the induction of IL-12 by IL-4 is caused by the reduction of IL-10 production. To test this, we examined cytokine production by IL-10^–/– DCs. IL-10^–/– BM-DCs secreted more IL-12 and expressed higher IL-12p35 mRNA than control DCs on LPS stimulation (Fig. 2, A and B). More importantly, the effect of IL-4 on IL-12 production and IL-12p35 mRNA expression was abolished in IL-10^–/– DCs (Fig. 2, A and B). These data indicate that the up-regulation of IL-12 by IL-4 correlates with decreased IL-10 levels.

View larger version (27K): [in this window] [in a new window]   Figure 2. IL-4 up-regulates IL-12 and promotes Th1 cell differentiation by inhibiting IL-10. (A) BM-DCs were prepared from BALB/c WT and IL-10–/– mice and stimulated with LPS alone or LPS with IL-4 for 1 d. ELISA was performed to detect IL-10 and IL-12p70 production. (B) BM-DCs from BALB/c WT and IL-10–/– mice were activated for 6 h with the indicated stimuli. IL-12p35 mRNA expression was measured by qRT-PCR. (C) BM-DCs were generated from BALB/c WT and IL-10–/– mice and treated with LPS alone or LPS with IL-4 for 6 h. 2 x 104 BM-DCs were then washed and added to 2 x 105 CD4+ T cells enriched from splenocytes of DO11.10 TCR-transgenic mice together with 0.01µM OVA peptide and 50 U/ml rIL-2. 5 d later, CD4 T cells were restimulated overnight with plate-bound anti-CD3 antibody. Supernatants were collected to measure IL-4 and IFN- production by ELISA. Data are means ± SE of three independent experiments.

Two interesting observations emerged. First, CD4 T cells primed by IL-4–treated BM-DCs from WT mice produced more IFN- and less IL-4 than cells primed by DCs treated without IL-4 (Fig. 2 C). In contrast, this effect of IL-4 was not observed when DCs from IL-10^–/– mice were used as APCs to prime CD4 T cells. Second, IL-10^–/– DCs were more potent to direct Th1 cell differentiation than WT DCs. This finding may be caused by the elevated IL-12 production by IL-10^–/– DCs, as shown in Fig. 2 A.

IL-4 inhibits IL-10 production by DCs but not by B cells
IL-10 is produced not only by DCs but also by other APCs, including B cells (17). In addition, IL-4 activates B cells and induces Ig isotype switching (7). Therefore, we asked whether IL-10 production by B cells is also suppressed by IL-4. Splenic B cells were purified and cultured in the presence of LPS alone or LPS with IL-4 for 2 d. As a control, we also stimulated purified splenic DCs with LPS or CpG in the presence or absence of IL-4. Consistent with BM-DCs, splenic DCs produced less IL-10 if IL-4 was added to the culture (Fig. 3 A). On the contrary, IL-4 did not inhibit IL-10 production by B cells. Rather, the IL-10 level was increased in the presence of IL-4 (Fig. 3 B). Thus, the inhibitory effect of IL-4 on IL-10 production is specific for DCs and the molecular mechanisms governing IL-10 expression must be distinct between DCs and B cells.

View larger version (14K): [in this window] [in a new window]   Figure 3. IL-4 inhibits IL-10 production by splenic DCs, but not by splenic B cells. CD11c+ DCs (A) and B220+ B cells (B) were enriched from the spleen of C57BL/6 mice using magnetic selection and stimulated with LPS or CpG in the presence or absence of IL-4, as described in Materials and methods. ELISA was performed to detect IL-10 production. Data are means ± SE of three independent experiments.

View larger version (14K): [in this window] [in a new window]   Figure 4. IL-4 inhibits IL-10 gene expression in a Stat6-dependent manner. BM-DCs from C57BL/6 WT and Stat6–/– mice were stimulated by LPS with or without IL-4 for 24 h. ELISA and qRT-PCR were performed to detect IL-10 protein (A) and mRNA (B), respectively. Data are means ± SE of three independent experiments.

View larger version (29K): [in this window] [in a new window]   Figure 5. IL-4 inhibits LPS-inducible IL-10 gene expression. (A) BM-DCs from C57BL/6 mice were stimulated 6 h with LPS alone or LPS with IL-4, followed by a 5-µg/ml actinomycin D treatment to stop RNA synthesis. The remaining IL-10 mRNA levels were measured by qRT-PCR at the indicated time points. (B) DC2.4 cells were transfected with the IL-10 promoter–driven luciferase reporter, stimulated by 2.5 µg/ml LPS with or without 10 ng/ml IL-4 overnight, and harvested to assess luciferase activity. Relative luciferase activity was normalized by protein concentrations. (C) BM-DCs from C57BL/6 and Stat6–/– mice were stimulated by LPS with or without IL-4 for 90 min. ChIP was performed using Abs specific for the acetylated histone H4 or normal rabbit serum. Purified DNA fragments were amplified using primers specific for the IL-10 or the HPRT promoter, as described in Materials and methods. Data are means ± SE (A and B) or representative (C) of at least three independent experiments.

IL-4 inhibits the LPS-inducible, but not the basal, level of IL-10 expression (Fig. 1 B and Fig. 5 B), suggesting cross talk between IL-4–induced and LPS-mediated signaling pathways. Mitogen-activated protein kinase and NF-B signals have been demonstrated to be involved in DC activation and cytokine production (16). However, IL-4 did not affect LPS-mediated mitogen-activated protein kinase or NF-B activation in BM-DCs (unpublished data). This finding is not surprising because IL-4 did not have a global inhibitory effect on cytokine production by DCs. It is not clear how IL-4 down-regulates IL-10 gene expression via Stat6. We suspect that IL-4–activated Stat6 may compete with other transcription factors and/or cofactors that are essential for LPS-inducible expression of the IL-10 gene in DCs. Whatever the mechanisms might be, IL-4–mediated regulation of IL-10 in DCs could play an important role in mounting a proper Th response. However, we cannot rule out the possibility that IL-4 may affect additional DC functions that have not been investigated.

A recent study has demonstrated that IL-4 instructs Th1 responses, which in turn protects mice from Leishmania major infections (14). Interestingly, the authors elegantly showed that the effect of IL-4 in eliciting the Th1 response is limited to the initial stage of infection, although the mechanisms for this observation were not provided. Similarly, other studies also have shown initial Th1 responses during the early stage of infection with L. amazonensis or Schistosomiasis mansoni in IL-10^–/– mice (20, 21). Despite this early response, parasites persisted in chronic lesions with normal Th2 responses in the absence of IL-10 (20, 21). We showed that IL-4 inhibits IL-10 production by both splenic and BM-DCs, but not by B cells, on LPS stimulation (Fig. 3). This suggests that all DCs share common regulatory mechanisms to produce IL-10. Given the potency of DCs as APC-activating naive CD4 T cells, the presence of IL-4 during the initial phase of the immune response would favor the generation of Th1 cells. B cells primarily reside in the secondary lymphoid organs, which prevents them from encountering antigens early in the response. If an infection progresses, B cells would be activated and be able to present antigens to naive CD4 T cells. In B cells, however, IL-4 does not inhibit IL-10 production. Instead, IL-4 would help B cells to produce more IL-10 and skew the immune response toward Th2 cells. Therefore, a shift from Th1 to Th2 responses by IL-4 is at least partly caused by a change in the cell type presenting antigens to T cells.

With this scenario, one would expect to see a dominant Th1 response in the absence of B cells. Indeed, DCs produced more IL-12 in the absence of B cells, resulting in Th1 cell deviation (22). This study also showed that B cells regulate the capacity of DCs to promote IL-4 secretion, further enhancing the Th2 response. Therefore, there is a negative feedback loop of cytokine production controlled by different APCs. In addition, B cell–deficient mice did not show recovery from disease in a Th1 cell–dependent model of experimental autoimmune encephalomyelitis (23). Experimental autoimmune encephalomyelitis recovery was dependent on IL-10 production by B cells. In the absence of B cells, DCs would be the primary APC-activating CD4 T cells, generating pathogenic Th1 cells and enhancing the severity of the disease. It is likely that the presence of both DCs and B cells is important to balance Th1 and Th2 cell generation and, moreover, proper temporal regulation of cytokine production for an effective immune response.

In conclusion, we showed that IL-4 directs DCs to produce less IL-10, resulting in more IL-12 production to promote the Th1 response. Therefore, IL-4 has a dual role for Th cell differentiation, and the type of APCs present during the initial activation of CD4 T cells appears critical for the CD4 T cell effector function.

	MATERIALS AND METHODS

Top Abstract Results and discussion MATERIALS AND METHODS References

 
Mice and cells
C57BL/6, BALB/c, and IL-10–deficient (IL-10^–/–) mice were purchased from the Jackson Laboratory. Stat6^–/– mice were as previously described (18). All mice were maintained under specific pathogen-free conditions at the Indiana University School of Medicine animal facility.

BM-DCs were prepared as previously described (24). In brief, total BM cells depleted of T and B cells were cultured for 5 d in RPMI 1640 with 5% FBS and 10 ng/ml murine rGM-CSF (BD Biosciences). These cells were considered immature DCs. Immature DCs were replated at 10⁶ cells/ml and matured in the presence of 1 µg/ml LPS (Escherichia coli O55:B5 serotype; Sigma-Aldrich) or 2 µg/ml CpG (TCCATGACGTTCCTGATGCT) with or without 10 ng/ml murine rIL-4 (BD Biosciences) for up to 2 d.

Splenic DCs and B cells were isolated by positive selection with anti-CD11c and anti-B220 magnetic beads (Miltenyi Biotec), respectively. Splenic DCs were activated for 24 h using the same conditions as BM-DCs. Splenic B cells were cultured at a density of 10⁶ cells/ml in RPMI 1640 with 10% FBS and stimulated for 48 h by 4 µg/ml LPS with or without 5 ng/ml rIL-4.

FACS analysis
Abs used for flow cytometry, CD11c (clone HL3), CD11b (clone M1/70), CD8 (clone 53–6.7), CD45R (B220; clone RA3-6B2), CD40 (clone 3/23), CD80 (B7-1; clone 16-10A1), CD86 (B7-2; clone GL-1), and MHC class II (I-A^b; clone AF6-120.1), were obtained from BD Biosciences. Flow cytometric analysis was performed using FACSCalibur and analyzed using CellQuest software (BD Biosciences).

ELISA
Cytokine concentrations in the culture supernatants were detected by ELISA as previously described (25). Purified anti–mouse capture and biotinylated detection Abs were IL-6 (P5-20F3, MP5-32C11), IL-10, (JES5-2A5, SXC-1), IL-12p70 (9A5, C17.8), TNF- (G281-2626, MP6-XT3), IFN- (R4-6A2, XMG1.2), and IL-4 (11B11, BVD6-24G2). All Abs were obtained from BD Biosciences.

qRT-PCR
Total RNA was prepared using Trizol (Invitrogen), and cDNA was prepared as previously described (26). qRT-PCR was performed by the comparative threshold cycle (C_T) method and normalized to GAPDH. The primers used for IL-10, IL-6, and GAPDH were as described previously (24). The primers used for IL-12p35 were 5'-CCTCAGTTTGGCCAGGGTC-3' and 5'-CAGGTTTCGGGACTGGCTAAG-3' and for IL-12p40 were 5'-GGAAGCACGGCAGCAGAATA-3' and 5'-AACTTGAGGGAGAAGTAGGAATGG-3'.

Transient transfections and luciferase assays
DC2.4 cells were maintained in RPMI 1640 with 10% FBS, 50 µM 2-ME, 2 mM L-glutamine, and 100 µg/ml penicillin and streptomycin. 10⁶ cells were transiently transfected with the 802- bp IL-10 promoter–driven luciferase reporter plasmid (provided by M. Tone, University of Pennsylvania, Philadelphia, PA, and H. Waldmann, University of Oxford, Oxford, UK; reference 27) using the Lipofectamine Plus reagents (Invitrogen). 4 h after transfection, cells were divided into four wells and rested 3 h before receiving different treatments overnight. Cell lysates were prepared and used for luciferase assays as previously described (25). Relative luciferase activity was normalized by protein concentration.

ChIP assay
ChIP assays were performed essentially according to Upstate Biotechnology's protocol. In brief, 10⁷ cells were treated for 10 min with 1% formaldehyde to cross-link DNA binding proteins to the DNA and were lysed in SDS-containing buffer. Cell extracts were sonicated to sheer DNA to 500 bp and immunoprecipitated overnight with Ab specific for acetylated histone H4 (Upstate Biotechnology). The recovered protein–nucleic acid complexes were incubated for 4 h with 0.4 M sodium chloride at 65°C to reverse cross-links. Purified DNA fragments were amplified 30 cycles using PCR and analyzed on 1.5% agarose gels. Immunoprecipitations with normal rabbit serum served as a negative control and PCR for the proximal promoter of the mouse hypoxanthine guanine phosphoribosyl transferase (HPRT) gene was used as an internal control. The primers used for the IL-10 promoter were 5'-GGCACCAGAACTCTCCTCTG-3' and 5'-TGGGTTGAACGTCCGATATT-3' and for the HPRT promoter were 5'-CTGCCTCTGCCTCCTAAATG-3' and 5'-CTCCCAGAGGATTCCCAGAT-3'.

In vitro antigen-specific CD4⁺ T cell priming
CD4⁺ T cells were enriched from total splenocytes of DO11.10 TCR transgenic mice using anti-CD4 magnetic beads (Miltenyi Biotec). BM-DCs from BALB/c WT and IL-10^–/– mice were matured by LPS with or without IL-4 for 6 h and extensively washed before adding to enriched CD4⁺ T cells with 0.01 µM OVA_323-339 peptide (Peptides International) and 50 U/ml human rIL-2. After 5 d, cells were restimulated with plate-bound anti-CD3 (145-2C11) antibody overnight. ELISA was performed to detect IFN- and IL-4 production.

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