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Gadd45 Regulates p38-Dependent Dendritic Cell Cytokine Production and Th1 Differentiation¹

Ludmila Jirmanova^*, Dragana Jankovi, Albert J. Fornace, Jr. and Jonathan D. Ashwell^2,*

^* Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, MD 20892; and Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Gadd45 inhibits the activation of p38 by the T cell alternative pathway involving phosphorylation of p38 Tyr³²³. Given that T cell p38 may play a role in Th1 development, the response to Th-skewing Ags was analyzed in Gadd45^–/– mice. Despite constitutively increased p38 activity in Gadd45^–/– T cells, the Th1 immune response to Toxoplasma gondii Ag (STAg), was diminished. In contrast to T cells, dendritic cells (DC) lacked the alternative p38 activation pathway. Gadd45^–/– DCs responded to STAg with low levels of MAP kinase cascade-dependent p38 activation, IL-12 production, and CD40 expression. Wild-type T cells transferred into Gadd45^–/– recipients had a diminished Th1 response to STAg, whereas Gadd45^–/– T cells transferred into wild-type hosts behaved normally. Therefore, Gadd45 has tissue-specific and opposing functions on p38 activity, and Gadd45-regulated p38 activation in DCs is a critical event in Th1 polarization in vivo.

	Introduction

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Upon activation, naive CD4⁺ T cells expand and differentiate into effector Th cells that regulate immune responses by producing specific cytokines. Th1 cells produce mainly IFN-, whereas Th2 cells typically produce IL-4 and to a lesser extent other cytokines (1, 2). Activation of naive T cells is also dependent on dendritic cells (DC),³ which play a critical role in the development of effector cells by directing Th differentiation through a balanced production of various cytokines: DC-derived IL-12 p70 and IFN- are crucial for Th1 priming; whereas IL-6 and IL-10 are involved in Th2 polarization (3). p38 is selectively induced in TCR-stimulated Th1 but not Th2 cells, and has an essential role in effector T cell function (4, 5, 6). However, the role of p38 in Th1 development is not entirely clear. In vitro polarized Th1 cells expressing constitutively active MAP kinase kinase (MKK) 6, an upstream activator of p38, displayed increased IFN- production, whereas expression of a dominant negative form of p38 or treatment with the p38 inhibitor SB203580 decreased IFN- expression by Th1 effectors (4, 5). Recently, however, p38^–/– T and B cells were found to develop normally (7), and p38-deficient CD4⁺ T cells expressed normal levels of IFN- when differentiated in vitro under Th1-polarizing conditions (8). It was also reported that p38 inhibitors blocked cytokine-induced Th1 IFN- production without disrupting the activation and differentiation of new Th1 effector (5). Hence, there is good evidence of the importance of p38 in Th1 IFN- production, but the role of p38 in Th1 cell differentiation needs to be clarified.

The Gadd45 genes (Gadd45, Gadd45, and Gadd45) (9, 10) have been implicated in regulation of Th1 differentiation: Gadd45- and Gadd45-deficient mice display a diminished Th1 response (11, 12), Gadd45 overexpression increases IFN- production, and Gadd45 deficiency impairs TCR-mediated cytokine expression (13). Given that all Gadd45 family members can bind and activate MEK kinase (MEKK) 4, a MAPK upstream of MKK3, MKK6, and p38 (14), the defect of Gadd45- and Gadd45-deficient effector cells was thought to be due to decreased activation of p38 in these cells (11, 12, 13). In addition to its ability to facilitate MEKK4 activation, Gadd45 has a specific negative role in regulating the activity of T cell p38. T cells stimulated via the TCR use an alternative, ZAP70-dependent, pathway that leads to phosphorylation of p38 Tyr³²³. This enhances catalytic activity and subsequent autophosphorylation on the canonical activating residues Thr¹⁸⁰/Tyr¹⁸² (15). Lack of Gadd45 results in constitutively active T cell p38, hyperproliferation in response to TCR-mediated signals, and development of a lupus-like autoimmune disease (16, 17). Because loss of Gadd45 results in spontaneous ZAP70-mediated T cell p38 activity (17) but decreased MAP kinase cascade-mediated p38 activity in stress or oncogene-activated nonimmune cells (fibroblasts and keratinocytes; Refs. 18 and 19), we reasoned that such animals could be used to investigate the role of p38 activation in T vs non-T cells in Th1 development. Here we find that Th1 development is markedly impaired in Gadd45^–/– animals due to a requirement for Gadd45-dependent p38 activation in DCs, which cannot be compensated for by increased T cell p38 activity.

	Materials and Methods

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Mice

Gadd45^–/– mice made in the 129/Ola background (20) were backcrossed onto the C57BL/6 (B6) background for eight generations, and mice heterozygous for Gadd45 were intercrossed to obtain the wild-type (WT) and Gadd45^–/– littermates used in these studies. B6 mice that carry the Thy-1a (Thy1.1) allele were obtained from The Jackson Laboratory (strain CBy.PL(B6)-Thy1a/ScrJ). Experiments were performed on 6- to 8-wk-old mice. Mice were maintained in a National Institutes of Health animal facility, and experiments followed approved National Institutes of Health animal study protocols.

Reagents

Soluble extract from Toxoplasma gondii tachyzoites (STAg) and extract from Schistosoma mansoni eggs (SEA) were prepared as described (21, 22). Recombinant proteins and Abs were purchased from Sigma-Aldrich, Cell Signaling, and BD Pharmingen except anti-IL-4 (11B.11), which was obtained from the National Institutes of Health Biological Resources Branch repository, and anti-ZAP70 Ab, which was kindly provided by Dr. Larry Samelson (National Cancer Institute, National Institutes of Health, Bethesda, MD) (23). Anti-rabbit and anti-mouse Abs conjugated to AlexaFluor680 or IRDye800 were obtained from Molecular Probes or Rockland Chemicals, respectively.

Cell culture and Th polarization

Cells were cultured in RPMI 1640 supplemented with 10% FCS, 250 µg/ml gentamicin, 100 U/ml penicillin, 4 mM glutamine, and 50 µM 2-ME (complete medium). DCs were purified from collagenase D-treated spleen by positive selection using CD11c (N418) MicroBeads and LS separation columns (Miltenyi Biotec). CD4⁺ T cells were purified with Mouse CD4 Immunocolumns (Cedarlane Laboratories). CD4⁺ T cells were cultured for 4 days in the presence of WT irradiated (30 Gy) splenocytes (5 x 10⁵ per well) in the presence of Con A (1 µg/ml) and IL-2 (1 ng/ml). The medium was supplemented with IL-12 (2 ng/ml) and anti-IL-4 (10 µg/ml) for Th1 polarization and with anti-IL-12 (10 µg/ml) and IL-4 (10 ng/ml) for Th2 polarization.

Purification of splenic DCs and generation of bone marrow DCs

DCs were purified from collagenase D-treated spleen by positive selection using CD11c (N418) MicroBeads and LS separation columns (Miltenyi Biotec). To generate DCs from bone marrow, cells from femurs were cultured at 5 x 10⁵ cells/ml in complete medium supplemented with GM-CSF (30 ng/ml) and IL-4 (1 ng/ml). On day 3, the cultures were supplied with fresh medium, and bone marrow DCs were harvested on day 6. The purity of splenic CD11c⁺ DCs was 90–95%, and that of bone marrow-derived DCs was 70–75%.

Immunization and adoptive transfer

Mice were immunized with STAg (50 or 100 µg/footpad) or with SEA (80 µg/footpad), and cells from popliteal lymph nodes were analyzed (24). For adoptive transfer, CD4⁺ T cells were purified from lymph nodes and spleens of 6- to 8-wk-old mice. Ten million WT (Thy1.1⁺) or Gadd45^–/– (Thy1.2⁺) CD4⁺ T cells in 0.3 ml of PBS were transferred i.v. into recipient WT (Thy1.2⁺ or Thy1.1⁺) or Gadd45^–/– (Thy1.2⁺) B6 mice. Recipient mice were immunized with 20 µg of STAg in 0.5 ml of PBS i.p. on the same day (day 0).

Flow cytometry and intracellular staining

Cells were stimulated for 4.5 h with PMA (10 ng/ml) and ionomycin (1 µg/ml), the last 2 h in the presence of monensin (2 µM; Calbiochem); stained with CD4, CD8, Thy1.1, or Thy1.2 Abs; and fixed using BD Cytofix/Cytoperm (BD Biosciences). Fixed cells were stained with anti-IL-4 and anti-IFN- in the presence of 2.4G2 Ab to block FcR binding. DCs were stimulated or not with STAg (10 µg/ml) for 16 h and stained with CD11c and CD40 Abs. Flow cytometry was conducted with a FACSCalibur using CellQuest software (BD Biosciences). Analysis was performed with FlowJo software (Tree Star).

ELISA

DCs were purified from spleen and stimulated with the indicated amounts of STAg or LPS for 16 h. Medium was collected and analyzed by ELISA for the indicated cytokines.

Immunoblotting

Protein extracts were prepared from equivalent numbers of cells, separated by SDS-PAGE, transferred to nitrocellulose membranes (Invitrogen Life Technologies), and immunoblotted with the indicated Abs. Signals were detected with anti-rat or anti-mouse secondary Abs and visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences).

RT-PCR

Total RNA was extracted from bone marrow-derived DCs using Uneasy Mini Kit (Qiagen), and an equal amount of RNA was reverse transcribed with ThermoScript RT-PCR System (Invitrogen Life Technologies). Gadd45 was amplified as described (20), and Gadd45, Gadd45 and -actin were amplified using the following primers: Gadd45, 5'-GCAAGCGATCTGTCTTGCTCA-3' and 5'-TGTGGAGCCAGGAGCAGCA-3; Gadd45, 5'-GCTGTGCTTTCCGGAACTGTA-3', CCTGCCGCCTCATTTGCA-3' and 5'-GTGGGCTCTATGGCTTCTGA-3'; -actin, 5'-CCAGCCTTCCTTCCTGGGTA-3' and 5'-CTAGAAGCATTTGC GGTGCA-3'.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Lack of Gadd45 increases p38 phosphorylation in Th cells

Increased T cell p38 activity is associated with robust Th1 immune responses and IFN- production (4), and Gadd45- and Gadd45-deficient mice have low T cell p38 activation and diminished Th1 immune responses (11, 12). Observations such as these have led to the notion that intrinsic T cell p38 activity is a key determinant of Th1 development. Because the loss of Gadd45 results in ZAP70-dependent constitutive p38 activation in resting T cells (17), it therefore seemed possible that Th1 development would be favored in these animals. Initial studies were performed to determine whether p38 phosphorylation is increased in Th1 effector cells lacking Gadd45. CD4⁺ T cells from WT or Gadd45-deficient (knockout; KO) mice were cultured for 4 days under Th1-polarizing conditions and stimulated with plate-bound anti-CD3 and anti-CD28 Abs. Although undetectable in unstimulated WT Th1 cells, there was an increase of dual p38 phosphorylation following stimulation (Fig. 1A). In contrast, Gadd45^–/– Th1 cells had a high level of phosphorylated p38 with or without stimulation, demonstrating that the alternative p38 activation pathway is active in Th1 as well as resting T cells (17). Th2 polarized WT T cells did not up-regulate p38 phosphorylation after TCR stimulation (Fig. 1B). Gadd45^–/– Th2 cells had a very low amount of phosphorylated p38 in the absence of TCR signaling, but this increased after TCR stimulation, demonstrating that in Th2 cells Gadd45 dampens p38 responses to TCR signaling.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Lack of Gadd45 increases the phosphorylation of p38 in Th cells. In vitro generated Th1 (A) or Th2 (B) cells were stimulated with plate-bound anti ()-CD3 and anti-CD28 for the indicated times. Lysates from 3 x 106 cells were immunoblotted with dual phospho-specific anti-p38 Ab (P-p38), and the membrane was reblotted with p38 Ab.

The Th1 but not Th2 immune response is attenuated in Gadd45^–/– mice

To test whether spontaneously high p38 activity in Gadd45^–/– T cells enhances Th1 skewing, WT or Gadd45^–/– animals were immunized with STAg, which induces an IL-12-dependent Th1 response in B6 mice through TLR11 (25, 26). The animals were immunized with 50 µg/footpad (low dose) or 100 µg/footpad (high dose) of STAg. Popliteal lymph node cells taken 8 days later were stimulated with STAg for 3 days and analyzed. Surprisingly, immunization with low dose STAg resulted in easily detectable IFN- production by WT CD4⁺ T cells but very little from Gadd45^–/–CD4⁺ T cells (Fig. 2A). High dose STAg generated more IFN-⁺ Gadd45^–/– CD4⁺ T cells, but still much less than that found in WT mice. The results obtained from multiple mice are shown in Fig. 2B. On average, we detected 9 ± 1.5 and 14 ± 1.7% IFN-⁺CD4⁺ WT T cells after immunization with 50 and 100 µg of STAg, respectively, as opposed to 3.2 ± 1.4 and 5.6 ± 1% in the knockout CD4⁺ T cells. The Th2 immune response can be tested by immunization with soluble extracts isolated from S. mansoni eggs (27). WT or Gadd45^–/– animals were immunized with SEA, and after 8 days popliteal lymph node cells were stimulated with SEA for 3 days and analyzed. CD4⁺ T cells of both genotypes preferentially produced IL-4. An analysis of four independent immunizations with SEA found similar numbers of IL-4⁺ CD4⁺ T cells from WT and Gadd45^–/– mice (n = 4, 10 ± 2 and 12 ± 1.6%, respectively; Fig. 2, C and D). Therefore, the presence or absence of Gadd45 does not seem to affect the Th2 response to SEA, whereas the Th1 response was markedly diminished. This result suggested the existence of a Gadd45-dependent mechanism that is independent of its effects on T cell p38 for full Th1 differentiation.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. The Th1 immune response is diminished in Gadd45-deficient mice. A, WT and KO mice were immunized with either 50 µg (left) or 100 µg (right) of STAg. Cells from popliteal lymph nodes were stimulated with PMA and ionomycin and analyzed by intracellular staining for IL-4 and IFN-. The plots are gated on CD4+ T cells, and the numbers indicate the frequency of positive cells in each quadrant. Data are representative for one of two independent experiments. B, Percent of CD4+ IFN-+ cells from individual animals immunized with STAg. A summary of two independent experiments is shown (bars indicate, means). C, WT and KO mice were immunized with 80 µg of SEA. Cells from popliteal lymph nodes were stimulated with PMA and ionomycin and analyzed by intracellular staining as above. Data are representative for one of two independent experiments. D, Percent of CD4+ IL-4+ cells obtained from individual animals immunized with SEA. A summary of two independent experiments is shown.

p38 activation is defective in Gadd45^–/– DCs

Innate immune cells such as DCs are important regulators of Th1 development (3). To determine whether Gadd45-deficient DCs, like T cells, use the alternative p38 activation pathway rather than the MAPK cascade, we tested them for expression of ZAP70 (Fig. 3A). Neither WT nor Gadd45^–/– DCs purified from spleen expressed detectable levels of this tyrosine kinase. Direct assessment of p38 Tyr³²³ phosphorylation was performed in DCs stimulated or not with STAg (Fig. 3B). Whereas Tyr³²³ phosphorylation was detected in Gadd45^–/– CD4⁺ T cells, it was undetectable in WT or KO DCs. We conclude, therefore, that DCs do not possess the alternative p38 activation pathway. The MAPK cascade was then characterized in WT or KO DCs stimulated with STAg. Immunoblotting revealed that MKK3 and MKK6 were activated in WT DCs stimulated with as little as 2.5 µg/ml STAg, but Gadd45^–/– DCs failed to up-regulate MKK3/6 even at 5 µg/ml (Fig. 3C). Consistent with this, p38 activity was up-regulated in WT but not in Gadd45^–/– DCs stimulated with STAg (Fig. 3C). Therefore, DCs lacking Gadd45 have suboptimal MAPK signaling and p38 activation in response to STAg.

View larger version (67K): [in this window] [in a new window]   FIGURE 3. p38 phosphorylation is regulated via the alternative ZAP70-dependent pathway in KO Th1 cells but not in KO DCs. A, One million WT lymph node CD4+ T cells, splenic WT, or splenic Gadd45–/– DC were lysed and immunoblotted with anti-ZAP70 Ab (top). The membrane was reblotted with anti--actin. B, One million splenic WT or Gadd45–/– DCs were stimulated with STAg (10 µg/ml) for the indicated times, and lysates immunoblotted with Ab against Tyr323-phosphorylated p38 (P-Y323 p38) or p38. CD4+ KO T cell lysates were used as a positive control for Tyr323 phosphorylation. C, One million splenic WT or Gadd45–/– DCs were stimulated with the indicated concentrations of STAg for 30 min, lysed, and analyzed for MAPKK3/MAPKK6 and p38 phosphorylation using phospho-specific Abs. D, Gadd45, Gadd45, and Gadd45 mRNA expression in bone marrow-derived WT and KO DCs were analyzed by RT-PCR. For amplification, 30, 35, 40, and 45 cycles were used. -Actin was used as a standard.

Gadd45 positively regulates DC IL-12 production

Resistance to T. gondii relies primarily on the production of IFN- (28). IFN- production by Th1-differentiated CD4⁺ cells is largely dependent on IL-12 produced by DCs (25, 29). Splenic DCs are CD11c⁺, and among these the CD8⁺ subset is the main source of IL-12 (3). Given the failure of Gadd45^–/– mice to generate a normal Th1 response and Gadd45^–/– DCs to up-regulate MAP kinase signaling in response to STAg, DCs from WT and KO mice were further characterized.

Regardless of Gadd45 expression, we detected that 2% of splenocytes were CD11c⁺ DCs in spleen, and of those 20% were CD8⁺, the main producers of IL-12 (Ref. 3) and Fig. 4A). In addition to the MHC-encoded molecules involved in Ag presentation, DCs express CD40, B7-1, and B7-2, all of which are important in the generation of effector T cells. CD40 binding to its ligand (CD40L) on T cells in particular is essential for T cell activation and expansion (30, 31). Even in the absence of overt stimulation, a distinct subset of WT splenic DCs expressed CD40, and expression of CD40 expression in unstimulated KO DCs was lower (Fig. 4B, left). Whereas the number of DCs expressing high levels of CD40 was increased after stimulation with STAg in both groups, however, WT cells responded much better than Gadd45^–/– KO cells (Fig. 4B, right). There was no reproducible difference between the genotypes in the resting or STAg-stimulated levels of B7-1 and B7-2 (data not shown). Gadd45^–/– DCs also failed to up-regulate CD40 to normal levels in response to LPS (data not shown). The functional capability of isolated splenic CD11c⁺ DCs was determined by incubation with STAg (signals via TLR11), CpG DNA (TLR9), and LPS (TLR2 and TLR4) and measuring the production of IL-12. Even in the absence of in vitro stimulation, a low level of IL-12 was produced by WT but not Gadd45-deficient DCs (Fig. 4C, left). Activation of WT DCs with STAg (10 µg/ml) or LPS (1 µg/ml) increased IL-12 production. Stimulation of Gadd45^–/– DCs, however, resulted in a much reduced (STAg) or no detectable (LPS) enhancement of IL-12 production. We did not detect up-regulation of IL-12 in DCs incubated with CpG DNA in cells of either genotype. In the absence of stimulation or at subthreshold amounts (1.2 and 2.5 µg/ml), we reproducibly detected a slight elevation of IL-12 produced by WT compared with Gadd45^–/– DCs (Fig. 4C, right). At a STAg concentration of 5 µg/ml, WT DCs produced easily detectable IL-12 whereas knockout DCs produced little above background.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. DC IL-12 p70 production is decreased in the absence of Gadd45. A, Splenocytes from WT or Gadd45–/– mice were stained for CD11c and CD8 and analyzed by flow cytometry. Numbers indicate the frequency of CD11c+ cells (left) and CD11c+CD8+ DCs (right). Representative data from one of three independent experiments are shown. B, WT (—) and KO (- - -) DCs were purified from spleen and cultured in complete medium alone (left) or medium containing STAg (5 µg/ml; right) for 16 h. Cells were stained for CD11c and CD40 or with IgG-FITC Ab as isotype control (gray fill). Histograms are gated on CD11c+ cells and show representative data from one of two independent experiments. C, WT and KO DCs were purified from spleen. Left panel, the DCs were stimulated with STAg (10 µg/ml), CpG DNA (20 µg/ml), or LPS (1 µg/ml) for 16 h. Right, DCs were stimulated with the indicated concentrations of STAg for 16 h. IL-12 in the supernatants was measured by ELISA. Values are the mean ± SD from two independent experiments. FSC, Forward scatter.

The failure of Gadd45^–/– T cells to polarize into Th1 cells is not T cell intrinsic

These results suggest that Gadd45^–/– DCs have a severely reduced capacity to respond to STAg and support Th1 differentiation and are consistent with a central role for Gadd45-regulated DC p38 activity in Th1 skewing. To test this, purified CD4⁺ T cells from WT (Thy1.1) mice were transferred into WT (Thy1.2) or Gadd45^–/– (Thy1.2) B6 mice, and Gadd45^–/– CD4⁺ T cells (Thy1.2) were transferred into WT (Thy1.1) mice. The animals were immunized with STAg i.p., and 7 days later splenocytes were harvested, restimulated with STAg in vitro, and analyzed for IFN- production (Fig. 5). The transferred CD4⁺ T cells constituted 1% of splenocytes in all combinations (data not shown). The majority of WT and Gadd45^–/– CD4⁺ T cells that were exposed to STAg in WT hosts differentiated into Th1 cells, as assessed by the production of IFN- (65 ± 9 and 55 ± 3.5% IFN-⁺ cells, respectively; Fig. 5). In contrast, only a minority (28 ± 3%) of WT CD4⁺ T cells that were recovered from Gadd45-deficient hosts produced IFN-. To address the possibility that WT host T cells might somehow affect Th1 differentiation of donor T cells, CD4⁺ T cells from WT or Gadd45-deficient animals were transferred into TCR^–/– mice, which lack T cells (32, 33), and challenged with STAg as above. T cells of both genotypes differentiated to a similar extent along the Th1 pathway as determined by inducible IFN- expression (data not shown). Together, these results demonstrate that the quality of the Th1 response is determined by the host, and Gadd45^–/– CD4⁺ T cells are able to differentiate into Th1 cells when the DCs (and perhaps other APCs) can provide IL-12- and CD40-costimulatory signals.

View larger version (10K): [in this window] [in a new window]   FIGURE 5. The role of Gadd45 in Th1 skewing is not T cell intrinsic. Purified CD4+ T cells from WT (Thy1.1+) mice were transferred into WT (Thy1.2+) or KO (Thy1.2+) mice, and purified CD4+ T cells from KO (Thy1.2+) mice were transferred into WT (Thy1.1+) recipients. The recipient mice were immunized with 20 µg of STAg i.p.; 7 days later, splenocytes were harvested, stimulated, and analyzed. Values are the percent of donor IFN-+ CD4+ T cells in each combination; each symbol represents an individual mouse.

	Discussion

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IL-12 is a central cytokine in the response to T. gondii because it triggers T cell IFN- synthesis, and IL-12-deficient and IFN--deficient animals succumb to acute toxoplasmosis after infection (36). Although IL-12 can be produced by many different inflammatory cells, including macrophages and neutrophils, in vivo the DCs are the first to synthesize IL-12 in T. gondii-infected mice (37). This early production of IL-12 is induced rapidly and independently of IFN- (38). Pathogen priming of DCs also increases CD40 expression, which makes DCs more responsive to activated T cells via the interaction with CD40L (31). Our finding that STAg- and LPS-mediated activation of the p38 MAPK cascade, with the resultant defect in IL-12 and CD40 expression, is dependent on Gadd45 raises the possibility that the Th1-skewing defect in mice lacking the Gadd45 and Gadd45 isoforms (11, 12) may be largely dependent on p38 defects in DCs as well, rather than in the T cells themselves.

Before the present study, the role of Gadd45 in MEKK4/p38 pathway has been explored in KO mice in vivo in keratinocytes and fibroblasts, and indirect evidence has suggested that Gadd45 may have a physiological role in MAPK signaling in apoptosis and cell cycle control: Gadd45^–/– keratinocytes up-regulate but do not sustain p38 activity after UV irradiation and DNA damage (19), and mouse embryonic fibroblasts require Gadd45 for p38 activation via the H-ras signaling pathway (18). In immune cells, however, and in contrast to the other members of the Gadd45 family, Gadd45 has an inhibitory effect on TCR-induced p38 activation and negatively regulates TCR-ZAP70-mediated induction of p38 (17). Hence, although physiological levels of Gadd45 do not prevent overt TCR-mediated signals from activating p38, they are sufficient to prevent activation by tonic TCR-mediated signals.

The use of Gadd45-deficient mice, which display the unique characteristic of elevated T cell but diminished DC p38 activity, has allowed us to characterize the tissue-specific role of this essential MAPK in Th1 effector cell development. Increased T cell p38 activity positively affects Th1 development, but only when the T cells are polarized in a suitable environment provided by p38-competent DCs. In the absence of Gadd45, there is compromised p38 signaling in DCs and perhaps other APCs, and a defective Th1 immune response even in the presence of hyperactive T cell p38. T cell-intrinsic p38 activity has been regarded as crucial for Th1 immune responses, a notion supported by studies with Gadd45^–/– and Gadd45^–/– mice (11, 12). However, this idea has been challenged by recent observations from chimeric mice expressing p38-deficient T cells, in which Th1 polarization is normal (8). Those findings, together with the present observations, suggest that the role of p38 in Th1 polarization is primarily at the level of the DC rather than the T cell.

	Acknowledgment

 
We are grateful to Remy Bosselut for critical review of the manuscript.

	Disclosures

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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 work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.

² Address correspondence and reprint requests to Dr. Jonathan D. Ashwell, Laboratory of Immune Biology, Building 37, Room 3002, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. E-mail address: jda{at}pop.nci.nih.gov

³ Abbreviations used in this paper: DC, dendritic cells; SEA, Schistosoma mansoni egg Ags; STAg, Toxoplasma gondii Ags; MEKK, MEK kinase; WT, wild type; KO, knockout; MKK, MAP kinase kinase.

Received for publication November 30, 2006. Accepted for publication January 17, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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