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The Journal of Immunology, 2006, 177: 7579-7587.
Copyright © 2006 by The American Association of Immunologists, Inc.

IL-27 Induces Th1 Differentiation via p38 MAPK/T-bet- and Intercellular Adhesion Molecule-1/LFA-1/ERK1/2-Dependent Pathways1

Toshiyuki Owaki*,{dagger}, Masayuki Asakawa*,{ddagger}, Fumio Fukai{dagger}, Junichiro Mizuguchi{ddagger},* and Takayuki Yoshimoto2,*

* Intractable Immune System Disease Research Center, Tokyo Medical University, Tokyo, Japan; {dagger} Department of Patho-Physiology, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan; and {ddagger} Department of Immunology, Tokyo Medical University, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-27, a novel member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in positive and negative regulations of immune responses. We recently demonstrated that IL-27 induces Th1 differentiation through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-independent mechanism. In this study, we further investigated the molecular mechanisms by focusing on p38 MAPK and ERK1/2. IL-27-induced Th1 differentiation was partially inhibited by lack of T-bet expression or by blocking ICAM-1/LFA-1 interaction with anti-ICAM-1 and/or anti-LFA-1, and further inhibited by both. Similarly, the p38 MAPK inhibitor, SB203580, or the inhibitor of ERK1/2 phosphorylation, PD98059, partially suppressed IL-27-induced Th1 differentiation and the combined treatment completely suppressed it. p38 MAPK was then revealed to be located upstream of T-bet, and SB203580, but not PD98059, inhibited T-bet-dependent Th1 differentiation. In contrast, ERK1/2 was shown to be located downstream of ICAM-1/LFA-1, and PD98059, but not SB203580, inhibited ICAM-1/LFA-1-dependent Th1 differentiation. Furthermore, it was demonstrated that STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK, and that IL-27 directly induces mRNA expression of growth arrest and DNA damage-inducible 45{gamma}, which is known to mediate activation of p38 MAPK. Finally, IL-12Rbeta2 expression was shown to be up-regulated by IL-27 in both T-bet- and ICAM-1/LFA-1-dependent mechanisms. Taken together, these results suggest that IL-27 induces Th1 differentiation via two distinct pathways, p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways. This is in contrast to IL-12, which induces it via only p38 MAPK/T-bet-dependent pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-27 is a novel member of the IL-6/IL-12 family that consists of an IL-12 p40-related protein, EBV-induced gene 3, and a newly discovered IL-12 p35-related protein, p28 (1). The orphan cytokine receptor WSX-1/T cell cytokine receptor (TCCR),3 which is homologous to IL-12Rbeta2 subunit, and gp130 constitute a functional signal-transducing receptor for IL-27 (1, 2). IL-27 activates JAK1, JAK2, TYK2, STAT1, STAT2, STAT3, STAT4, and STAT5 in naive CD4+ T cells (3, 4, 5, 6), and enhances proliferation in naive, but not memory CD4+ T cells. IL-27 also induces the expression of T-box expressed in T cells (T-bet), a master transcriptional regulator for Th1 differentiation (7), and subsequent IL-12Rbeta2, and synergizes with IL-12 in primary IFN-{gamma} production (1, 3, 4, 6).

Previous studies using mice lacking one of the IL-27R subunits, TCCR (8)/WSX-1 (9), revealed that IL-27 is required for the early initiation of Th1 responses, and that WSX-1/TCCR-deficient mice have enhanced susceptibility to infection with intracellular pathogens such as Leishmania major (9, 10) and Listeria monocytogenes (8). In contrast, WSX-1 is not essential to develop the protective Th1 responses against Toxoplasma gondii parasites, and rather acts to attenuate the inflammatory responses induced by protozoan infection, including cellular hyperactivation and overproduction of proinflammatory cytokines such as IFN-{gamma}, IL-4, TNF-{alpha}, and IL-6 (5). Thus, IL-27 plays critical roles not only in the early regulation of Th1 differentiation, but also in inhibitory regulation of immune responses, including suppression of cellular activation and proinflammatory cytokine production (11, 12).

We recently demonstrated that IL-27 differentiates naive CD4+ T cells into Th1 cells under Th1-polarizing conditions, but without IL-12, through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-, IFN-{gamma}-, and STAT4-independent mechanism, and that the IL-27-induced Th1 differentiation is overruled by IL-12 dose dependently (13). IL-27 rapidly up-regulates ICAM-1 expression on naive CD4+ T cells stimulated with plate-coated anti-CD3 plus anti-CD28 or with anti-CD3 in the presence of T/NK cell-depleted spleen cells as APCs. The IL-27-induced Th1 differentiation is significantly, but partially inhibited by anti-ICAM-1 and anti-LFA-1. Therefore, to further elucidate the molecular mechanism underlying IL-27 signaling pathways leading to Th1 differentiation, we focused on MAPK family signal cascades, especially ERK1/2 and p38 MAPK, which are well known to be involved in the regulation of various cytokine signalings (14, 15). The present study suggests that IL-27 induces Th1 differentiation via p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways. This is in contrast to IL-12, which induces it via only the p38 MAPK/T-bet-dependent pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and mice

Naive CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS and 50 µM 2-ME. Wild-type BALB/c mice were purchased from Japan SLC. STAT4-deficient mice (16) and T-bet-deficient mice (17) of BALB/c background were purchased from The Jackson Laboratory. Wild-type 129/Sv mice and STAT1-deficient mice (18) of 129/Sv background were purchased from Taconic Farms. All animal experiments were performed in accordance with our institutional guidelines.

Reagents

SB203580 and PD98059 were purchased from Calbiochem. Anti-CD3 (145-2C11), anti-IL-4 (11B11), and anti-LFA-1 (FD441.8) were from American Type Culture Collection. Anti-T-bet (4B10), anti-STAT1, anti-STAT4, anti-ERK1, anti-ERK2, and anti-p38 MAPK were from Santa Cruz Biotechnology. Anti-actin was from Sigma-Aldrich. Anti-phosphotyrosine STAT1 (Tyr701), anti-phospho-ERK1/2 (Thr202/Tyr204) (197G2), and anti-phospho-p38 MAPK (Thr180/Tyr182) (3D7) were from Cell Signaling Technology. Anti-CD28 (37.51) and anti-IL-12Rbeta2 (HAM10B9) were from BD Biosciences. Anti-ICAM-1 (YN1/1.7.4) was from eBioscience. Mouse rIL-12 and mouse ICAM-1/Fc chimera were from R&D Systems. Human rIL-2 and mouse rIFN-{gamma} were provided by Shionogi.

Preparation of purified rIL-27 protein

rIL-27 was prepared as a FLAG-tagged single chain protein by flexibly linking EBV-induced gene 3 to p28 using HEK293-F cells (Invitrogen Life Technologies), as described before (19).

Preparation of naive CD4+ T cells

Primary T cells were purified by passing spleen cells depleted of erythrocytes through nylon wool. The flow-through fraction was incubated with biotin-conjugated anti-CD8{alpha}, anti-B220, anti-Mac-1, anti-Ter-119, and anti-DX5, followed by incubation with anti-biotin magnetic beads (Miltenyi Biotec), and passed through a magnetic cell-sorting column (Miltenyi Biotec), and the negative fraction was collected (CD4+ T cells >95%). These purified T cells were then incubated with anti-CD62 ligand magnetic beads (Miltenyi Biotec), and the positive fraction was collected as purified naive CD4+ T cells (CD62 ligand+ cells >99%).

Th1 differentiation assay

Naive CD4+ T cells (5 x 105 cells/ml) were primed with plate-coated anti-CD3 (2 µg/ml) and anti-CD28 (0.5 µg/ml) in the presence or absence of IL-27 (10 ng/ml) under the Th1-polarizing conditions or those without IL-12 (10 ng/ml), including anti-IL-4 (10 µg/ml). On day 3, cells were split 1:4 and expanded in IL-2 (50 U/ml)-containing complete medium. On day 6, cells were collected, washed, and restimulated at 5 x 105 cell/ml with plate-coated anti-CD3 (2 µg/ml). After 24 h, culture supernatants were harvested and assayed for IFN-{gamma} production by ELISA.

Western blotting

Cells were lysed in a lysis buffer containing protease inhibitors, and resultant cell lysates were separated on an SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membrane (Millipore), as described previously (20). The membrane was then blocked, probed with primary Ab and then with appropriate secondary Ab conjugated to HRP, and visualized with the ECL detection system (Amersham Biosciences), according to the manufacturer’s instructions.

RT-PCR

Total RNA was extracted using a guanidine thiocyanate procedure, cDNA was prepared using oligo(dT) primer and SuperScript RT (Invitrogen Life Technologies), and RT-PCR was performed using TaqDNA polymerase, as described previously (21). Cycle conditions were 95°C for 20 s, 57°C for 30 s, and 72°C for 45 s. The following primers were also used: growth arrest and DNA damage-inducible (GADD)45beta sense primer, 5'-GGGGGATTTTGCAATCTTCT-3'; GADD45beta antisense primer, 5'-ACGGTGAGGCGATCCTGA-3'; GADD45{gamma} sense primer, 5'-CATTTCACGTTGATTCAGGCG-3'; and GADD45{gamma} antisense primer, 5'-AACAAACT GAGCTTCTCCAAGGC-3'. Cycle conditions and primers used for hypoxanthine phosphoribosyltransferase were as previously described (22).

Statistical analysis

Statistical analysis was performed by Student’s t test. A p value of <0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-27 induces Th1 differentiation in T-bet- and ICAM-1/LFA-1-dependent mechanisms

We recently demonstrated that IL-27 differentiates naive CD4+ T cells into Th1 cells under Th1-polarizing conditions, but without IL-12 through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-, IFN-{gamma}-, and STAT4-independent mechanism (13). To further elucidate the molecular mechanisms underlying IL-27- induced Th1 differentiation, we first explored the role of T-bet and compared it with that of ICAM-1. Naive CD4+ T cells from wild-type BALB/c and T-bet-deficient mice were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or absence of IL-27 for 16 h, and analyzed for ICAM-1 expression by FACS (Fig. 1, A and B). As reported previously (13), IL-27 rapidly up-regulated ICAM-1 expression on not only wild-type naive CD4+ T cells, but also T-bet-deficient cohorts. Wild-type naive CD4+ T cells were also stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 in the presence or absence of blocking Abs against ICAM-1 and LFA-1 for 48 h, and analyzed for T-bet expression by Western blotting (Fig. 1C). Blocking ICAM-1/LFA-1 interaction by their Abs did not affect the augmentation of T-bet expression by IL-27. We then examined the effect of lack of T-bet expression and/or blocking ICAM-1/LFA-1 interaction on IL-27-induced Th1 differentiation. Wild-type and T-bet-deficient naive CD4+ T cells were primed with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12 in the presence or absence of anti-ICAM-1/anti-LFA-1, expanded with IL-2 on day 3, restimulated with plate-coated anti-CD3 for 24 h on day 6, and analyzed for IFN-{gamma} production in culture supernatants by ELISA (Fig. 1D). Lack of T-bet expression partially (~50%) inhibited the ability of IL-27 to induce Th1 differentiation. Blocking ICAM-1/LFA-1 interaction also partially (~50%) inhibited the ability, as reported previously (13). Both lack of T-bet expression and blocking ICAM-1/LFA-1 interaction further inhibited the ability of IL-27 to induce Th1 differentiation. These results suggest that IL-27 induces Th1 differentiation via T-bet- and ICAM-1/LFA-1-dependent pathways, which are presumably regulated by distinct mechanisms.


Figure 1
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  		FIGURE 1. IL-27 induces Th1 differentiation in T-bet- and ICAM-1/LFA-1-dependent mechanisms. A, T-bet is not required for rapid up-regulation of ICAM-1 expression by IL-27. Wild-type BALB/c and T-bet-deficient naive CD4+ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or absence of IL-27 under Th1-polarizing conditions without IL-12 for 16 h, and analyzed for cell surface expression of ICAM-1 by FACS using anti-ICAM-1 (solid line) and control rat IgG (plain line with shading). Mean fluorescence intensity (MFI) of each histogram is shown as the mean ± SD of three independent experiments (B). C, ICAM-1/LFA-1 is not required for augmentation of T-bet expression by IL-27. Wild-type naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 in the presence or absence of blocking Abs against ICAM-1 and LFA-1 (10 µg/ml each) for 48 h. Total cell lysates were then prepared and subjected to Western blotting using anti-T-bet and anti-actin. D, IL-27-induced Th1 differentiation is reduced in T-bet-deficient naive CD4+ T cells and further inhibited by blocking ICAM-1/LFA-1 interaction. Wild-type and T-bet-deficient naive CD4+ T cells were primed with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12 in the presence or absence of anti-ICAM-1/anti-LFA-1 (10 µg/ml each). Primed CD4+ T cells were expanded with IL-2 on day 3 and restimulated with plate-coated anti-CD3 for 24 h on day 6, and culture supernatants were analyzed for IFN-{gamma} production in triplicate by ELISA. Data are shown as means ± SD. *, Indicates p < 0.05. Similar results were obtained in three independent experiments.

 
IL-27-induced Th1 differentiation is mediated by p38 MAPK and ERK1/2 independently

p38 MAPK and ERK1/2 are well known to be involved in the regulation of various cytokine signalings (14, 15). It was demonstrated previously that IL-12 activates p38 MAPK, but not ERK1/2, and that activation of p38 MAPK is required for Th1 differentiation in a STAT4-independent manner (23). Therefore, we next explored the role of these MAPKs in the signaling of IL-27. Naive CD4+ T cells were primed with plate-coated anti-CD3 and either IL-27 or IL-12 for 48 h, then restimulated with IL-27 or IL-12, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting (Fig. 2A). IL-27 induced phosphorylation of both p38 MAPK and ERK1/2, whereas IL-12 induced that of p38 MAPK, but not ERK1/2, as reported previously (23). Then the effect of their inhibitors on IL-27-induced Th1 differentiation was examined. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 for 1 h, then primed with plate-coated anti-CD3/anti-CD28 and IL-27, expanded with IL-2 on day 3, restimulated with plate-coated anti-CD3 for 24 h on day 6, and analyzed for IFN-{gamma} production in culture supernatants by ELISA (Fig. 2B). Pretreatment with either SB203580 or PD98059 partially, but dose dependently inhibited IL-27-induced Th1 differentiation to the level that corresponds to ~50% of that without inhibitors. In contrast, pretreatment with SB203580, but not PD98059, inhibited IL-12-induced Th1 differentiation, as reported previously (23). Moreover, the combined pretreatment with SB203580 and PD98059 completely inhibited IL-27-induced Th1 differentiation (Fig. 2C). These results suggest that IL-27 activates p38 MAPK and ERK1/2, and that IL-27-induced Th1 differentiation is mediated by both independently.


Figure 2
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  		FIGURE 2. IL-27-induced Th1 differentiation is mediated by p38 MAPK and ERK1/2 independently. A, IL-27 induces phosphorylation of both p38 MAPK and ERK1/2, whereas IL-12 induces that of p38 MAPK, but not ERK1/2. Naive CD4+ T cells were primed with plate-coated anti-CD3 and either IL-27 or IL-12 for 48 h, and restimulated with IL-27 or IL-12 for 20, 60, and 180 min. Total cell lysates were then prepared and subjected to Western blotting using anti-phospho (p)-p38 MAPK and anti-p-ERK1/2. Each blot was stripped and reprobed with anti-total ERK1/2 and anti-total p38 MAPK to control for loading. B, IL-27-induced Th1 differentiation is inhibited by SB203580 or PD98059, whereas IL-12-induced Th1 differentiation is inhibited by SB203580, but not PD98059. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 for 1 h, and then primed with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12. Primed CD4+ T cells were expanded with IL-2 on day 3 and restimulated with plate-coated anti-CD3 for 24 h on day 6, and culture supernatants were analyzed for IFN-{gamma} production in triplicate by ELISA. The results are shown as ratio of Th1 differentiation, which was calculated by the following formula: amount of IFN-{gamma} produced from CD4+ T cells differentiated by IL-27 or IL-12 in the presence of inhibitors/amount of IFN-{gamma} produced from CD4+ T cells differentiated by IL-27 or IL-12 without inhibitors. Data are shown as means ± SD. C, IL-27-induced Th1 differentiation is partially inhibited by either B203580 or PD98059 and completely inhibited by both. Wild-type naive CD4+ T cells were pretreated with either SB203580 or PD98059 (10 µM each) or both for 1 h, primed, restimulated, and analyzed for IFN-{gamma} production, as described above. Data are shown as means ± SD. **, Indicates p < 0.01. Similar results were obtained in three independent experiments.

 
T-bet-dependent Th1 differentiation by IL-27 is mediated by p38 MAPK, but not ERK1/2

Next, we investigated the role of these two MAPKs in T-bet-dependent Th1 differentiation induced by IL-27. We first explored the effect of MAPK inhibitors on augmentation of T-bet expression by IL-27. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 for 1 h, stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 for 48 h, and analyzed for T-bet expression by Western blotting (Fig. 3A). IL-27- or IL-12-induced augmentation of T-bet expression was greatly suppressed by SB203580, but not PD98059. We then examined the effect of lack of T-bet expression on activation of MAPKs by IL-27. Wild-type and T-bet-deficient naive CD4+ T cells were primed with plate-coated anti-CD3 and either IL-27 or IL-12 for 48 h, then restimulated with IL-27 or IL-12 for 20 min, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting (Fig. 3B). Lack of T-bet expression did not affect activation of p38 MAPK and ERK1/2 induced by IL-27 and also activation of p38 MAPK by IL-12. Furthermore, the effect of MAPK inhibitors on IL-27-induced Th1 differentiation from wild-type and T-bet-deficient naive CD4+ T cells was examined (Fig. 3C). IL-27-induced Th1 differentiation was reduced in T-bet-deficient naive CD4+ T cells, as shown in Fig. 1C. Notably, PD98059, but not SB203580, further suppressed the Th1 differentiation. These results suggest that p38 MAPK is located upstream of T-bet, and that T-bet-dependent Th1 differentiation by IL-27 is mediated by p38 MAPK, but not ERK1/2.


Figure 3
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  		FIGURE 3. T-bet-dependent Th1 differentiation by IL-27 is mediated by p38 MAPK, but not ERK1/2. A, IL-27- or IL-12-induced augmentation of T-bet expression is inhibited by SB203580, but not PD98059. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 (10 µM each) for 1 h, and stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12 for 48 h. Total cell lysates were then prepared and subjected to Western blotting using anti-T-bet and anti-actin. B, T-bet is not required for IL-27-induced activation of p38 MAPK and ERK1/2 and for IL-12-induced activation of p38 MAPK. Wild-type and T-bet-deficient naive CD4+ T cells were primed, restimulated, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting, as described in Fig. 2A. C, IL-27-induced Th1 differentiation in T-bet-deficient naive CD4+ T cells is further inhibited by PD98059, but not SB203580. Wild-type and T-bet-deficient naive CD4+ T cells were pretreated with SB203580 or PD98059 (10 µM each) for 1 h, primed, expanded, restimulated, and analyzed for IFN-{gamma} production by ELISA, as described in Fig. 2C. Data are shown as means ± SD. *, Indicates p < 0.05. Similar results were obtained in three independent experiments.

 
ICAM-1/LFA-1-dependent Th1 differentiation by IL-27 is mediated by ERK1/2, but not p38 MAPK

Next, we investigated the role of these two MAPKs in ICAM-1/LFA-1-dependent Th1 differentiation induced by IL-27. We first explored the effect of MAPK inhibitors on ICAM-1 expression up-regulated by IL-27. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 for 1 h, stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 for 16 h, and analyzed for ICAM-1 expression by FACS (Fig. 4, A and B). IL-27-induced up-regulation of ICAM-1 expression was not suppressed by SB203580 or PD98059. We then examined the effect of blocking by anti-ICAM-1/anti-LFA-1 on activation of these MAPKs by IL-27. Wild-type naive CD4+ T cells were primed with plate-coated anti-CD3 and either IL-27 or IL-12 for 48 h, then restimulated with IL-27 or IL-12 for 20 min, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting (Fig. 4C). Blocking by anti-ICAM-1/anti-LFA-1 greatly suppressed IL-27-induced activation of ERK1/2, but not p38 MAPK. Consistent with these results, we also confirmed that ICAM-1/Fc chimera induced phosphorylation of ERK1/2, but not p38 MAPK in naive CD4+ T cells stimulated with plate-coated anti-CD3 (data not shown). Furthermore, the effect of SB203580 or PD98059 on IL-27-induced Th1 differentiation from naive wild-type CD4+ T cells in the presence or absence of anti-ICAM-1/anti-LFA-1 was examined (Fig. 4D). Blocking by anti-ICAM-1/anti-LFA-1 partially (~50%) suppressed IL-27-induced Th1 differentiation, as shown in Fig. 1D. Notably, SB203580, but not PD98059, further inhibited the Th1 differentiation to a level similar to that without IL-27. These results suggest that ERK1/2 is located downstream of ICAM-1/LFA-1, and that ICAM-1/LFA-1-dependent Th1 differentiation by IL-27 is mediated by ERK1/2, but not p38 MAPK.


Figure 4
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  		FIGURE 4. ICAM-1/LFA-1-dependent Th1 differentiation by IL-27 is mediated by ERK1/2, but not p38 MAPK. A, Activation of p38 MAPK and ERK1/2 is not required for IL-27-induced up-regulation of ICAM-1 expression. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 (10 µM each) for 1 h, stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12 for 16 h, and analyzed for cell surface expression of ICAM-1 by FACS using anti-ICAM-1 (solid line) and control rat IgG (plain line with shading). MFI of each histogram is shown as the mean ± SD of three independent experiments (B). C, ICAM-1/LFA-1 is required for IL-27-induced activation of ERK1/2, but not p38 MAPK. Naive CD4+ T cells were primed with plate-coated anti-CD3 and either IL-27 or IL-12 for 48 h in the presence or absence of anti-ICAM-1/anti-LFA-1 (10 µg/ml each) or after pretreatment with SB203580 or PD98059 (10 µM each), restimulated, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting, as described in Fig. 2A. D, IL-27-induced Th1 differentiation in the presence of anti-ICAM-1/anti-LFA-1 is further inhibited by SB203580, but not PD98059. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 (10 µM each) for 1 h, primed in the presence or absence of anti-ICAM-1/anti-LFA-1 (10 µg/ml each), expanded, restimulated, and analyzed for IFN-{gamma} production by ELISA, as described in Fig. 2C. Data are shown as means ± SD. **, Indicates p < 0.01. Similar results were obtained in three independent experiments.

 
STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK

We previously demonstrated that STAT1 is important for IL-27 signaling to induce T-bet and ICAM-1 expression and subsequent Th1 differentiation (6, 13). Therefore, we next investigated the role of STAT1 in the activation of p38 MAPK and ERK1/2 by IL-27. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 for 1 h, stimulated with plate-coated anti-CD3 for 16 h, restimulated with IL-27 for 20 min, and analyzed for tyrosine phosphorylation of STAT1 (Fig. 5A). Pretreatment with SB203580 or PD98059 did not affect tyrosine phosphorylation of STAT1. Then wild-type and STAT1-deficient naive CD4+ T cells were primed with plate-coated anti-CD3 and either IL-27 or IL-12 for 48 h, then restimulated with IL-27 or IL-12 for 20 min, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting (Fig. 5B). In STAT1-deficient naive CD4+ T cells, activation of ERK1/2 by IL-27 was abolished, whereas that of p38 MAPK was still observed. These results suggest that STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK.


Figure 5
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  		FIGURE 5. STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK. A, STAT1 is not located downstream of p38 MAPK and ERK1/2. Wild-type naive CD4+ T cells were pretreated with SB203580 or PD98059 (10 µM each) for 1 h and primed with plate-coated anti-CD3/anti-CD28 for 16 h. After washing, these cells were stimulated with IL-27 for 20 min. Total cell lysates were then prepared and subjected to Western blotting using anti-phosphotyrosine STAT1. Each blot was stripped and reprobed with anti-total STAT1 to control for loading. B, IL-27-induced activation of ERK1/2, but not p38 MAPK is abolished in STAT1-deficient naive CD4+ T cells. Wild-type and STAT1-deficient naive CD4+ T cells were primed, restimulated, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting, as described in Fig. 2A. Similar results were obtained in three independent experiments.

 
STAT4 is not required for IL-27-induced activation of p38 MAPK and ERK1/2 and up-regulation of ICAM-1 expression

STAT4 is required for IL-12-induced Th1 differentiation (16, 24, 25). It was reported previously that IL-27 also induces tyrosine phosphorylation of STAT4 (26). However, we have demonstrated recently that STAT4 is not necessary for IL-27-induced up-regulation of T-bet and also for IL-27-induced Th1 differentiation (13). Therefore, we further investigated the role of STAT4 in IL-27-induced activation of p38 MAPK and ERK1/2 and up-regulation of ICAM-1 expression. Wild-type and STAT4-deficient naive CD4+ T cells were primed with plate-coated anti-CD3 and either IL-27 or IL-12 for 48 h, then restimulated with IL-27 or IL-12 for 20 min, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting (Fig. 6A). In STAT4-deficient naive CD4+ T cells, activation of p38 MAPK and ERK1/2 by IL-27 was intact, and that of p38 MAPK by IL-12 was also observed, as reported previously (16, 24, 25). Furthermore, wild-type and STAT4-deficient naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 for 16 h, and analyzed for ICAM-1 expression by FACS (Fig. 6, B and C). IL-27-induced rapid up-regulation of ICAM-1 expression was still observed in STAT4-deficient naive CD4+ T cells. These results suggest that STAT4 is not required for IL-27-induced activation of p38 MAPK and ERK1/2, and up-regulation of ICAM-1 expression.


Figure 6
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  		FIGURE 6. STAT4 is not required for IL-27-induced activation of p38 MAPK and ERK1/2 and up-regulation of ICAM-1 expression. A, STAT4 is not necessary for IL-27-induced activation of p38 MAPK and ERK1/2. Wild-type and STAT4-deficient naive CD4+ T cells were primed, restimulated, and analyzed for phosphorylation of p38 MAPK and ERK1/2 by Western blotting, as described in Fig. 2A. Similar results were obtained in three independent experiments. B, STAT4 is not necessary for IL-27-induced rapid up-regulation of ICAM-1 expression. Wild-type and STAT4-deficient naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 in the presence or absence of IL-27 under Th1-polarizing conditions without IL-12 for 16 h, and analyzed for cell surface expression of ICAM-1 by FACS using anti-ICAM-1 (solid line) and control rat IgG (plain line with shading). MFI of each histogram is shown as the mean ± SD of three independent experiments (C).

 
IL-27 directly induces GADD45{gamma} expression, which presumably mediates activation of p38 MAPK

It was demonstrated previously that GADD45beta expression in naive CD4+ T cells activates p38 MAPK and selectively increases cytokine-induced IFN-{gamma} production (27). It was also reported that GADD45{gamma} mediates activation of the p38 MAPK pathway to increase IFN-{gamma} production in effector Th1 cells (28). Because IL-27 activated p38 MAPK even in STAT1-deficient naive CD4+ T cells (Fig. 5B), we next investigated whether IL-27 can induce expression of GADD45beta and/or GADD45{gamma}. Wild-type naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 for 48 h, and analyzed for mRNA expression of GADD45beta and GADD45{gamma} by RT-PCR (Fig. 7A). IL-27 enhanced mRNA expression of GADD45{gamma}, but not GADD45beta, the expression of which appears to be regulated by signaling through TCR, as reported (28, 29). IL-27-induced enhancement of GADD45{gamma} expression was not inhibited by SB203580, PD98059, or anti-ICAM-1/anti-LFA-1. Then the role of STAT1, STAT4, and T-bet in IL-27-induced enhancement of GADD45{gamma} expression was examined using respective deficient mice. Enhancement of GADD45{gamma} expression by IL-27 was still observed in naive CD4+ T cells from all these deficient mice (Fig. 7, B–D). These results suggest that IL-27 directly induces GADD45{gamma} expression, independently of p38 MAPK, ERK1/2, ICAM-1/LFA-1, STAT1, STAT4, and T-bet.


Figure 7
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  		FIGURE 7. IL-27 directly induces GADD45{gamma} expression, which presumably mediates activation of p38 MAPK. A, IL-27 induces GADD45{gamma} expression independently of p38 MAPK, ERK1/2, and ICAM-1/LFA-1. Naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12 in the presence or absence of anti-ICAM-1/anti-LFA-1 (10 µg/ml each) or after pretreatment with SB203580 or PD98059 (10 µM each) for 1 h. Forty-eight hours later, total RNA was prepared and subjected to RT-PCR analysis for GADD45beta, GADD45{gamma}, and hypoxanthine phosphoribosyl transferase mRNA expression. B–D, STAT1, STAT4, and T-bet are not required for IL-27-induced enhancement of GADD45{gamma} expression. Wild-type, and STAT1-, STAT4-, and T-bet-deficient naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12 for 48 h, and total RNA was prepared and subjected to RT-PCR analysis, as described above. Similar results were obtained in three independent experiments.

 
IL-27-induced up-regulation of IL-12Rbeta2 expression is mediated by both T-bet- and ICAM-1/LFA-1-dependent mechanisms

IL-27 induces expression of T-bet and subsequent IL-12beta2 on naive CD4+ T cells, resulting in synergistic IFN-{gamma} production with IL-12 (3, 6). In addition, IL-27 rapidly up-regulates ICAM-1 expression on naive CD4+ T cells (13). Therefore, we finally investigated the interaction among ICAM-1, T-bet, and IL-12Rbeta2. Wild-type and T-bet-deficient naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 in the presence or absence of anti-LFA-1 for 16 and 48 h, and analyzed for expression of ICAM-1 and IL-12Rbeta2 by FACS (Fig. 8, A and B). Sixteen hours after the stimulation with IL-27, ICAM-1 expression was rapidly up-regulated on naive CD4+ T cells regardless of the presence or absence of T-bet expression, whereas IL-12Rbeta2 expression was not increased. Forty-eight hours later, IL-12Rbeta2 expression was also increased on wild-type naive CD4+ T cells. In contrast, lack of T-bet expression or blocking by ant-LFA-1 reduced the IL-12Rbeta2 expression to a level corresponding to ~50% of that on wild-type naive CD4+ T cells without treatment by anti-LFA-1. Notably, both lack of T-bet expression and blocking by anti-LFA-1 almost completely inhibited the up-regulation of IL-12Rbeta2 expression. Moreover, pretreatment with SB203580 or PD98059 did not affect IL-27-induced up-regulation of IL-12Rbeta2 expression on wild-type and T-bet-deficient naive CD4+ T cells (Fig. 8, C and D). These results suggest that IL-27-induced up-regulation of IL-12Rbeta2 expression is mediated by both T-bet- and ICAM-1/LFA-1-dependent mechanisms, and that p38 MAPK and ERK1/2 are not essential for it.


Figure 8
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  		FIGURE 8. IL-27-induced up-regulation of IL-12Rbeta2 expression is mediated by both T-bet and ICAM-1/LFA-1. A, Both T-bet and ICAM-1/LFA-1 are involved in IL-27-induced up-regulation of IL-12Rbeta2 expression. Wild-type and T-bet-deficient naive CD4+ T cells were stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 under Th1-polarizing conditions without IL-12 in the presence or absence of anti-LFA-1 (10 µg/ml) for 16 and 48 h, and analyzed for expression of ICAM-1 and IL-12Rbeta2 by FACS. Numbers in the panels indicate percentages of cells in each quadrant. Percentage of ICAM-1+IL-12Rbeta2+ cells after the stimulation for 48 h of each dot plot is shown as the mean ± SD of three independent experiments (B). * and **, Indicate p < 0.05 and p < 0.01, respectively. C, p38 MAPK and ERK1/2 are not essential for IL-27-induced up-regulation of IL-12Rbeta2 expression. Wild-type and T-bet-deficient naive CD4+ T cells were pretreated with SB203580 or PD98059 (10 µM each) for 1 h, stimulated with plate-coated anti-CD3/anti-CD28 and IL-27 for 48 h, and analyzed for cell surface expression of IL-12Rbeta2 in triplicate by FACS using anti-IL-12Rbeta2 (solid line) and control hamster IgG (plain line with shading). Numbers represent the MFI of each histogram. MFI of each histogram is shown as the mean ± SD (D). Similar results were obtained in three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrated that IL-27 induces Th1 differentiation via two distinct pathways, p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways, as schematically shown in Fig. 9. Ligation of IL-27R by IL-27 immediately activates STAT1, which mediates rapid up-regulation of ICAM-1 and thereafter augmentation of T-bet expression on naive CD4+ T cells (6, 13). Independently of STAT1, IL-27 also induces GADD45{gamma} expression, which presumably mediates p38 MAPK activation (28), followed by augmentation of T-bet expression. ICAM-1 then ligates LFA-1 on CD4+ T cells, resulting in activation of ERK1/2 and consequent Th1 differentiation. Notably, ICAM-1/LFA-1 interaction, as well as T-bet, cooperatively enhances IL-12beta2 expression, leading to Th1 differentiation. Thus, IL-27 activates two MAPKs, p38 MAPK and ERK1/2, which play critical roles in the T-bet- and ICAM-1/LFA-1-dependent pathways, respectively. Although IL-27 activates STAT1–5 (3, 4, 5, 6), STAT1 is critically important for IL-27 signaling, in particular, leading to up-regulation of ICAM-1 and T-bet expression on naive CD4+ T cells and consequent Th1 differentiation (6, 13). However, augmentation of T-bet expression by IL-27 appears to be also regulated by the GADD45{gamma}/p38 MAPK pathway, independently of STAT1. IL-27 induced GADD45{gamma} expression and activation of p38 MAPK even in STAT1-deficient naive CD4+ T cells. Moreover, p38 MAPK inhibitor, SB203580, greatly inhibited IL-27-induced augmentation of T-bet expression. These results imply that both signals mediated by STAT1 and p38 MAPK may be required for IL-27-induced augmentation of T-bet expression. In contrast, in T-bet-deficient naive CD4+ T cells or in the presence of blocking Ab against LFA-1, the up-regulation of IL-12Rbeta2 expression by IL-27 was reduced, but was still significantly present. These results suggest that either T-bet or ICAM-1/LFA-1 may be necessary for up-regulation of IL-12Rbeta2 expression by IL-27. In contrast, IL-12 induces Th1 differentiation via only the p38 MAPK/T-bet-dependent pathway, as shown in this study. IL-12 also induced GADD45{gamma} expression through STAT4 and activated p38 MAPK in STAT4/GADD45{gamma}-dependent and -independent mechanisms, leading to IFN-{gamma} production and Th1 differentiation. Thus, IL-27 activates two MAPKs for Th1 differentiation by multiple pathways as compared with IL-12, and the p38 MAPK/T-bet pathway appears to be shared by them.


Figure 9
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  		FIGURE 9. IL-27 signaling pathways leading to Th1 differentiation. When naive CD4+ T cells are stimulated with IL-27, the following two molecular events are initiated: one is activation of STAT1, and the other is induction of GADD45{gamma} expression, which is also induced by IL-12/STAT4 signaling. Activation of STAT1 leads to not only augmentation of T-bet expression, but also up-regulation of ICAM-1 expression in T-bet-independent manner. ICAM-1 interacts with LFA-1 on the adjoining CD4+ T cells, resulting in ERK1/2 activation and differentiation into effector Th1 cells. In contrast, GADD45{gamma} activates p38 MAPK and subsequently T-bet, which directly enhances IFN-{gamma} production. T-bet also up-regulates IL-12Rbeta2 expression, which contributes to Th1 differentiation through IL-12/STAT4 signaling. Furthermore, we found that IL-27 also augments IL-12Rbeta2 expression through ICAM-1/LFA-1 signaling in T-bet-independent manner as an alternative pathway. Thus, IL-27 activates two MAPKs, p38 MAPK and ERK1/2, for Th1 differentiation by multiple pathways as compared with IL-12, and the p38 MAPK/T-bet pathway appears to be shared by IL-27 and IL-12.

 
T-bet is a master transcriptional factor that directly regulates Th1 lineage commitment by activating the hallmark Th1 cytokine, IFN-{gamma} (7). In addition, T-bet up-regulates IL-12Rbeta2 expression on CD4+ T cells to induce IL-12 responsiveness (30). Several previous studies revealed the role of T-bet and its effects on IFN-{gamma} production in CD4+ T cells in response to various infections in vivo. Szabo et al. (17) reported that Ag-specific CD4+ T cells from T-bet-deficient mice show little IFN-{gamma} production and increased amounts of Th2 cytokines IL-4 and IL-5 in response to L. major infection. Likewise, CD4+ T cells from T-bet-deficient mice also failed to produce detectable levels of IFN-{gamma} in response to infection with Salmonella (31) or HSV type 2 (32). These data suggest that T-bet is essential for IFN-{gamma} production in CD4+ T cells for pathogen clearance. In contrast to these reports, Way and Wilson (33) revealed that the proportion of IFN-{gamma}-producing Ag-specific CD4+ T cells in T-bet-deficient mice following L. monocytogenes infection is limited to only 50% decrease compared with wild-type mice, and that the IFN-{gamma} secretion by CD4+ T cells is still detectable. Sullivan et al. (34) also showed that the proportion of IFN-{gamma}-producing CD4+ T cells in the lungs of T-bet-deficient mice during Mycobacterium tuberculosis infection is not completely impaired. These results imply that T-bet is not necessarily an absolute factor for IFN-{gamma} production by CD4+ T cells. Considering that IL-27 can induce Th1 differentiation even in the absence of T-bet expression, as shown in this study, it could be conceivable that IL-27 may play a role in generating IFN-{gamma}-producing Ag-specific CD4+ T cells for the resistance to L. monocytogenes and M. tuberculosis infection in T-bet-deficient mice.

In vitro study also showed that T-bet is not always necessary for IFN-{gamma} production in CD4+ T cells differentiated into Th1 cells. Usui et al. (35) reported that CD4+ T cells from T-bet-deficient mice still produce IFN-{gamma} if both IL-12Rbeta2 and STAT4 are stably expressed in the CD4+ T cells. The present study showed that IL-27 up-regulates IL-12Rbeta2 expression via ICAM-1/LFA-1 signaling even in the absence of T-bet (Fig. 8, A and B), suggesting that T-bet is not essential for IL-12-mediated Th1 differentiation in the presence of IL-27. Moreover, we demonstrated that IL-27 itself induces Th1 differentiation of CD4+ T cells from T-bet-deficient mice through STAT1/ICAM-1/LFA-1 signaling (Fig. 3C), followed by ERK1/2 activation (Figs. 4 and 5). These results suggest that IL-27/STAT1 is an alternative signaling pathway leading to Th1 differentiation in addition to IL-12/STAT4.

Previous studies using mice lacking one of the IL-27R subunits, TCCR (8)/WSX-1 (9), revealed that IL-27 is required for the early initiation of Th1 responses, and that WSX-1/TCCR-deficient mice have enhanced susceptibility to infection with intracellular pathogens such as L. major (9, 10) and L. monocytogenes (8). In contrast, WSX-1 is not essential to develop the protective Th1 responses against T. gondii parasites, and rather acts to attenuate the inflammatory responses induced by protozoan infection, including cellular hyperactivation and overproduction of proinflammatory cytokines (5). One of the key differences between infection with L. major or L. monocytogenes and that with T. gondii might be considered to be the induction level of IL-12, a principal mediator of strong type I immunity (36). T. gondii promotes strong innate immune responses that lead to high IL-12 levels early during infection, whereas acute L. major induces much less IL-12 production (37). Consistent with these observations, we previously demonstrated that the ability of IL-27 to induce Th1 differentiation is most prominent under Th1-polarizing conditions, but without IL-12 and overruled by IL-12 dose dependently (13). The IL-27-induced Th1 differentiation is partially mediated by rapid and marked up-regulation of ICAM-1 expression on naive CD4+ T cells through ICAM-1/LFA-1 interaction in STAT1-dependent, but T-bet-, IFN-{gamma}-, and STAT4-independent mechanism. In the present study, we have further clarified the signaling molecules leading to Th1 differentiation induced by IL-27, and demonstrated that IL-27 uses more complicated mechanisms than IL-12. Namely, IL-27 activates both p38 MAPK/T-bet and ICAM-1/LFA-1/ERK1/2 pathways, whereas IL-12 activates only p38 MAPK/T-bet pathway. Although these results may imply that IL-27 has a greater effect on Th1 differentiation than IL-12, comparison of phenotypical analyses of IL-12-deficient mice and WSX-1/TCCR-deficient mice clearly reveals that IL-12 is the Th1-driving cytokine (9, 38). This difference could be explained by the growing evidences that IL-27 possesses not only proinflammatory property, but also anti-inflammatory property, although the molecular mechanisms remain unknown (12, 39). We recently demonstrated that IL-27 plays an important role to suppress excessive progression of CD28-mediated IL-2 production and IL-2 responses by inducing suppressor of cytokine signaling (SOCS)3 expression in a negative feedback mechanism (40). Because SOCS3 was reported to inhibit IL-12-mediated Th1 differentiation through STAT4 activation as well (41), IL-27 may play a role in inhibition of Th1 differentiation induced by IL-12 through SOCS3, and therefore, synergistic induction of Th1 differentiation by IL-12 and IL-27 cannot be seen (13). These possibilities are currently under investigation.

Taken together, it is highly conceivable that IL-12 dominantly regulates Th1 differentiation through p38 MAPK/T-bet pathway under the circumstances that IL-12 is abundantly produced and the IL-12 signaling pathway is intact. However, when IL-12 production is limited or the IL-12 signaling pathway is impaired by infection with certain pathogens, the IL-27-mediated alternative signaling pathway leading to Th1 differentiation may emerge and compensate for it.


   Acknowledgments
 
We thank Drs. L. H. Glimcher and R. D. Schreiber for permission to use T-bet-deficient mice and STAT1-deficient mice, respectively. We also thank the Animal Research Center of Tokyo Medical University for animal care. We are indebted to Dr. J. P. Barron (Tokyo Medical University) for his review of this manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 study was supported by a Grant-in-Aid for Scientific Research; High-Tech Research Center Project; University-Industry Joint Research Project from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and a grant from Astellas Foundation for Research on Metabolic Disorders. Back

2 Address correspondence and reprint requests to Dr. Takayuki Yoshimoto, Intractable Immune System Disease Research Center, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. E-mail address: yoshimot{at}tokyo-med.ac.jp Back

3 Abbreviations used in this paper: TCCR, T cell cytokine receptor; GADD, growth arrest and DNA damage inducible; MFI, mean fluorescence intensity; SOCS, suppressor of cytokine signaling; T-bet, T-box expressed in T cells. Back

Received for publication May 2, 2006. Accepted for publication September 19, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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