Transcription and Mrna Stabilization Multiple Pathways to Induce IL-4

Elevating Calcium in Th2 Cells Activates Multiple Pathways to Induce IL-4 Transcription and mRNA Stabilization

This information is current as Liying Guo, Joseph F. Urban, Jinfang Zhu and William E. of October 2, 2021. Paul J Immunol 2008; 181:3984-3993; ; doi: 10.4049/jimmunol.181.6.3984 http://www.jimmunol.org/content/181/6/3984 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Elevating Calcium in Th2 Cells Activates Multiple Pathways to Induce IL-4 Transcription and mRNA Stabilization1

Liying Guo,2* Joseph F. Urban,† Jinfang Zhu,* and William E. Paul*

PMA and ionomycin cause T cell cytokine production. We report that ionomycin alone induces IL-4 and IFN-␥, but not IL-2, from in vivo- and in vitro-generated murine Th2 and Th1 cells. Ionomycin-induced cytokine production requires NFAT, p38, and calmodulin-dependent kinase IV (CaMKIV). Ionomycin induces p38 phosphorylation through a calcium-dependent, cyclosporine A-inhibitable pathway. Knocking down ASK1 inhibits ionomycin-induced p38 phosphorylation and IL-4 production. Ionomycin also activates CaMKIV, which, together with p38, induces AP-1. Cooperation between AP-1 and NFAT leads to Il4 gene transcription. p38 also regulates IL-4 production by mRNA stabilization. TCR stimulation also phosphorylates p38, partially through the calcium-dependent pathway; activated p38 is required for optimal IL-4 and IFN-␥. The Journal of Immunology, 2008, 181: 3984–3993. Downloaded from ptimal activation of T cells requires engagement of the radiation, heat shock, and osmotic shock, as well as by proinﬂam- TCR-CD3 complex and of costimulatory molecules matory cytokines, such as TNF-␣ and IL-1. Ligation of the TCR whose ligands are expressed on APCs (1). The binding also activates p38. TCR stimulation triggers small GTP-binding O 2ϩ of Ag/MHC complexes to the TCR elevates intracellular Ca proteins such as Ras, Rac-1, and Cdc42, leading to the activation 2ϩ 3 ␪ ␪ 180 concentration ([Ca ]i) and activates protein kinase C (PKC ). of MAPK cascades (7, 8). p38 is dually phosphorylated at Thr 182 The combination of a calcium ionophore (e.g., ionomycin) and and Tyr in a highly conserved motif in the p38 activation loop. http://www.jimmunol.org/ phorbol ester tumor promoters (e.g., PMA), which bypass TCR Salvador and colleagues reported an alternative p38 activation signals, results in T cell activation (2) and production of a series of pathway in T cells, in which ZAP70 phosphorylates p38␣ on cytokines, including IL-2, IL-4, and IFN-␥. Tyr323, leading to autophosphorylation of the Thr and Tyr in the 2ϩ Elevation of [Ca ]i is an essential event following TCR stim- activation loop and to “self-activation” (9). TCR stimulation thus ulation (3). Imaging studies reveal that TCR stimulation causes results in induction of both the classic MAPK cascade and this 2ϩ sustained elevation in [Ca ]i over a period of 1–2 h. Such sus- alternative p38 inhibitor-sensitive self-activation pathway; the rel- tained signaling allows dephosphorylated NFAT, a key transcrip- ative contributions of the two pathways and their physiological tional regulator of cytokine genes, to be maintained in the nucleus functions remain to be determined. (4). The cooperative action of NFAT and AP-1 is generally The importance of p38 activity to cytokine production is still by guest on October 2, 2021 thought to be necessary for full cytokine transcription (5). controversial. Herein we report that ionomycin alone induces IL-4 PKC␪ is a signal regulator of several T cell activation pathways and IFN-␥ production from in vivo- and in vitro-generated Th2 (6). The MAP kinases ERK, JNK, and p38 are among the targets and Th1 cells, in contrast to IL-2, for which both PMA and iono- of PKC␪. The ERK and JNK pathways are involved in IL-2 pro- mycin are essential for production. The production of IL-4 requires 2ϩ duction, predominantly through activation of AP-1 (7). However, elevation of [Ca ]i, NFAT dephosphorylation, and activation of relatively little is known about the role of p38 MAPK in cytokine p38 and CaMKIV. Phospho-p38 generated through elevation of 2ϩ induction. [Ca ]i plays a crucial role in ionomycin-induced IL-4 production Four p38 MAPK isoforms have been characterized: p38␣, p38␤, by promoting Il4 gene transcription and stabilizing its mRNA. Fur- p38␥, and p38␦. CD4 T cells predominantly express p38␣ and thermore, we show that phospho-p38 is essential for optimal IL-4 p38␦ (8). p38 can be activated by multiple stressors, such as UV production induced through TCR stimulation.

*Laboratory of Immunology, National Institute of Allergy and Infectious Dis- Materials and Methods eases, National Institutes of Health, Bethesda, MD 20892; and †Nutrient Require- Mice and cell culture ments & Functions Laboratory, Beltsville Human Nutrition Research Center, Ag- ricultural Research Service, United States Department of Agriculture, Beltsville, Cells from C57BL/6 mice infected with Schistosoma mansoni cercariae for MD 20705 8 wk and splenocytes from C57BL/6 mice infected with Toxoplasma gon- Received for publication April 7, 2008. Accepted for publication July 11, 2008. dii for 10 days were kindly provided by Dr. Dragana Jankovic of the Na- tional Institute of Allergy and Infectious Diseases (10, 11). C57BL/6 mice The costs of publication of this article were defrayed in part by the payment of page were inoculated with third-stage Heligmosomoides polygyrus per os, and charges. This article must therefore be hereby marked advertisement in accordance spleens were isolated 14 days later, as previously described (12). with 18 U.S.C. Section 1734 solely to indicate this fact. CD4 T cells were purified from 5C.C7 transgenic Rag2Ϫ/Ϫ mice by 1 This research was supported by the Intramural Research Program of the National negative selection. Cells were cultured under Th1 and TH2 conditions as Institutes of Health, National Institute of Allergy and Infectious Diseases. described before (13). CD4 T cells were purified from Il4/Gfp heterozy- 2 Address correspondence and reprint requests to Dr. Liying Guo, Laboratory of Im- gous mice by negative selection. The cells were primed with APC, anti- munology, National Institute of Allergy and Infectious Diseases, National Institutes of CD3 (3 ␮g/ml), and anti-CD28 (3 ␮g/ml) under Th2 conditions. Health, Building 10, Room 11N322, 10 Center Drive–MSC 1892, Bethesda, MD 20892-1892. E-mail address: [email protected] Intracellular staining 3 2ϩ 2ϩ Abbreviations used in this paper: [Ca ]i, intracellular Ca concentration; ARE, adenylate/uridylate-rich elements; CsA, cyclosporine A; LN, lymph node; MFI, mean In vitro differentiated Th1 and Th2 cells were rechallenged under different fluorescence intensity; PB, plate-bound; PKC␪, protein kinase C␪; qPCR, quantitative conditions for 4 h, in the presence of monensin, to check cytokine produc- PCR; shRNA, small hairpin RNA; TG, thapsigargin; UTR, untranslated region. tion. The conditions used to rechallenge the cells were: APC ϩ peptide, www.jimmunol.org The Journal of Immunology 3985

FIGURE 1. Ionomycin induces substantial IL-4 and IFN-␥ production, but not IL-2, from in vivo- and in vitro-generated Th1 and Th2 cells. A, Mesenteric LNs were isolated from C57BL/6 mice 8 wk after S. mansoni infection and cells were stimulated for 4 h. IL-4 production by CD44brightCD4

T cells was measured by intracellular staining. B, Splenocytes were isolated from C57BL/6 mice 2 wk after H. polygyrus infection. IL-4 (upper panel) and Downloaded from IL-2 (lower panel) production by CD44brightCD4 T cells. C and D, Two round-primed Th1 or Th2 cells were stimulated under different conditions; percentage of cytokine-producing cells and MFI of the cytokine-producing cells are shown. IL-4 production (C, upper panel) and IL-2 (D, upper panel) in Th2 cells; IFN-␥ production (C, lower panel) and IL-2 (D, lower panel) in Th1 cells. The in vivo experiments were conducted twice and the in vitro experiments multiple times.

cognate cytochrome C peptide (1 ␮M)-loaded APC; anti-CD3/CD28, tion, cells were maintained in medium containing IL-2 and puromycin (5 http://www.jimmunol.org/ plate-bound (PB) anti-CD3 (3 ␮g/ml) and anti-CD28 (3 ␮g/ml); PϩI, PMA ␮g/ml) (Sigma-Aldrich). Another 2 days later, puromycin-resistant cells (10 ng/ml) and ionomycin (1 ␮M), and ionomycin (1 ␮M) alone. were purified by cell sorting. Harvested cells were fixed with 4% paraformadehyde, washed with 0.1% BSA-containing PBS, and stored at 4°C. For staining, cells were Retroviral infection incubated with permeabilization buffer (PBS supplemented with 0.1% GFP-Cre viral supernatant was prepared as described before (14). Splenic BSA/0.1% Triton X-100) and various Abs for 20 min. All Abs were pur- CD4 T cells were purified from mice homozygous for a “floxed” TAK1 chased from BD Pharmingen. gene (15) and cultured under Th2 conditions as described above. Two days To test p38 phosphorylation in PB anti-CD3 and/or anti-CD28 stimu- after a third round of Th2 priming, cells were incubated with a GFP-Cre lated Th2 cells, cells were put in Ab-coated 6-well plates and were spun at retrovirus-containing supernatant. Two days after infection, cells were

1600 rpm for 20 s to allow cells to attach to the bottom. Paraformadehyde placed in IL-2-containing medium. Twenty-four to 48 h later, cells were by guest on October 2, 2021 (4% final concentration) was added to cell culture to fix the cells imme- stimulated to check cytokine production or p38 phosphorylation in diately after the cells had been cultured in the plates for the indicated response to ionomycin. periods. For phospho-p38 staining, fixed cells were incubated with a rabbit RNA purification and quantitative PCR (qPCR) monoclonal phospho-p38 Ab (1/25) (336 ng/ml) (Cell Signaling Technol- ogy) or a nonspecific rabbit isotype control Ab (336 ng/ml) (Imgenex) for Total RNA was isolated using RNeasy Mini kits (Qiagen) (treated with 30 min at room temperature. After extensive washing, cells were stained RNase-free DNase I); first-strand cDNA was prepared using SuperScript with a FITC-conjugated donkey anti-rabbit Ab (1/200) (Jackson Immuno- III (Invitrogen). All PCR was performed on a 7900HT sequence detection Research Laboratories). systems (Applied Biosystems). The TaqMan universal PCR SuperMix and all the primer and probe sets were purchased from Applied Biosystems. To Small hairpin (sh)RNA lentiviral infection test Gfp mRNA level, SYBR Green technology was utilized with the prim- ers as reported before (13). To exclude the possible false signal introduced p38␣, ASK1, CaMKII, and CaMKIV shRNA and nonsilencing-control by binding of SYBR Green to likely primer dimer, the PCR conditions shRNA constructs were purchased from Sigma-Aldrich. All the plasmids were set as 95°C for 15 s, 60°C for 1 min, and 78°C for 30 s for 40 cycles, were purified with EndoFree plasmid purification kit (Qiagen). 293FT and the increase in fluorescence was detected only at the 78°C “packaging” cells were transfected with shRNA plasmids or nonsilencing incubation step. control shRNA plasmids, together with the packaging plasmid pCMV⌬8.9 and the envelope plasmid pHCMV-G using the Fugene 6 transfection re- Immunoblotting agent (Roche Diagnostics) according to the manufacturer’s protocol. Pack- ϫ aging plasmid pCMV⌬8.9 and envelope plasmid pHCMV-G were kindly Stimulated cells were lysed in 1 SDS sample buffer (0.05 M Tris-HCl provided by Dr. N. Hacohen at Harvard Medical School. Lentivirus-con- (pH 6.8), 1% SDS (w/v), 10% glycerol (v/v), bromophenol blue 1.009% (w/v)) freshly supplemented with the following inhibitors: 1 mM PMSF, 10 taining supernatant was collected twice, at 48 and 72 h after transfection, ␮ ϫ respectively. g/ml aprotinin, leupeptin, pepstatin, and 1 phosphatase inhibitor cock- To “knockdown” p38, Th2 cells were cultured for a second round under tail 1 and cocktail 2 (Sigma-Aldrich). Cell lysates were sonicated for 10 s Th2 conditions. Two days later, the lentivirus, collected as described to shear DNA. above, and polybrene (8 ␮g/ml) (Sigma-Aldrich) were added and the cells Proteins were separated on 12% pre-made tricine-glycine gels (Invitro- were centrifuged at 2500 rpm for 90 min at room temperature. The super- gen) and then transferred onto nitrocellulose membranes (Millipore) by the natant was removed immediately and fresh medium was added to the cul- Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad). Membranes ture, which continued under Th2 conditions. Forty-eight hours after infec- were probed with phospho-p38 MAPK Ab (Cell Signaling Technology) tion, cells were placed in medium containing IL-2 and puromycin (5 ␮g/ (1/1000), followed by a HRP-labeled anti-rabbit Ab (Pierce) (1/1000). The ml) (Sigma-Aldrich). One week later, puromycin-resistant cells were signal was visualized with ECL. Membranes was stripped by Restore purified by cell sorting and cultured with cytochrome C peptide (1 ␮M)- Western blot stripping buffer (Pierce) and then reprobed with p38 MAPK loaded APC in the presence of anti-IL-4 (10 ␮g/ml), anti-IFN-␥ (10 ␮g/ Ab (Cell Signaling Technology) (1/1000). ml), and anti-IL-12 (10 ␮g/ml) (null Th cell conditions) for 4 days. Nuclear extraction and EMSAs To knockdown ASK1, CaMKII, and CaMKIV, two round-primed Th2 cells that had just finished 4-day priming under Th2 condition were used. Cells were stimulated with ionomycin for 50 min. Nuclear protein extracts Infection was performed as described above. Forty-eight hours after infec- were prepared as previously described (16), and protein concentration was 3986 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION

A (10µM) Inhibitors: BC two p38 inhibitors: mock non-silencing mock SB203580 SB202190 MEK JNK p38 control p38shRNA-1 p38shRNA-2 IL-4 IL-4 80% 54% 59% 80% 52% 88% MFI: IL-4 66% MFI: 52% 34% 23% 433 349 250 P+I 343 232 240 P+I P+I

46% 7% 4% 54% 49% 66% 11% MFI: MFI: 28% 12% 5% 173 108 103 219 178 139 I I I 24% 2% 2% CD4 MFI: P+I I CD4 147 104 103 noitcudorp 4-L noitcudorp 1.4 1.4 1.2 1.2 RNA: 1 : 0.38 : 0.13 TG 1 1 MEK inhibitor 0.8 0.8 0.6 0.6 JNK inhibitor Protein: 0.4 0.4 CD4 0.2 0.2 ni g 1 P38 inhibitor c A- 2-AN 0 0 ne N isl Rhs R

I hs83 1µM 3µM 10µM 1µM 3µM 10µM -n lo 83p no rt n p inhibitors concentration co FIGURE 2. Ionomycin and TG induce IL-4 production through a p38-dependent pathway. A, Ionomycin-induced IL-4 production by Th2 cells is highly sensitive to p38 inhibition. Th2 cells were preincubated with DMSO (mock), 10 ␮M of inhibitors (upper panel), or various concentrations of inhibitors, as indicated (lower panel), of the three distinct MAPK pathways for 30 min and then stimulated. In the lower panel, the degree of IL-4 production by DMSO-treated cells was set as “1.” B, Knocking down p38 inhibits ionomycin-induced IL-4 production. Th2 cells were lentivirally infected with nonsilencing or p38 shRNAs; puromycin-resistant cells were puriﬁed and further stimulated under ThN conditions. These cells were divided into three portions: one portion to check IL-4 production in response to PϩI or ionomycin (upper panel), one portion to analyze p38 mRNA by qPCR, and one portion to check p38 protein by immunoblotting (lower panel). C, TG similarly induces IL-4 production through a p38-dependent pathway. Th2 cells were pretreated then ␮ ␮

stimulated as described. SB203580, 5 M; SB202190, 5 M; TG, 10 nM. The experiments were conducted three times. Downloaded from

determined with the BCA kit (Pierce). EMSAs were conducted with the EMSA kit (Promega). The oligonucleotides corresponding to sense and antisense sequences within the Il4 promoter (17) were 5Ј-biotin labeled

(Bio-Synthesis), HPLC puriﬁed, and annealed for use as probe. Samples http://www.jimmunol.org/ were resolved on a 6% DNA retardation gel (Invitrogen) in 0.5ϫ Tris- borate-EDTA buffer. The protein-DNA binding complex was detected by the LightShift chemiluminescent EMSA kit (Pierce). The sequences of the oligonucleotides were: sense, 5Ј-TAATGTAAACTCATTTTCCCTTG-3Ј and antisense, 5Ј-CAAGGGAAAATGAGTTTACATTA-3Ј. AP-1 ELISA An ELISA-based TransAM AP-1 kit, in which oligonucleotide containing AP-1 binding motif has been immobilized to 96-well plate, was used to

quantify activated AP-1 components (Active Motif). Five-microgram nu- by guest on October 2, 2021 clear extracts prepared as above were used and c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD Abs were added at 1/2000 dilution. The AP-1 binding assay was performed according to the instructions of the manufacturer. Results Ionomycin induces IL-4 and IFN-␥, but not IL-2, production from in vivo- and in vitro-differentiated Th cells Mesenteric lymph nodes (LNs) were isolated from mice 8 wk after S. mansoni infection, which strikingly induces a Th2 response. LN cells were either unstimulated, immediately stimulated with PB anti-CD3/CD28, with a combination of PMA and ionomycin (PϩI), or with ionomycin alone. The percentage of CD44brightCD4 T cells from infected mice that produced IL-4 in response to ionomycin was similar to that in response to PϩIor to PB anti-CD3/CD28; mean ﬂuorescence intensities (MFI) of the IL-4-expressing cells induced by PϩI or by ionomycin alone were also similar (Fig. 1A). Splenocytes isolated from mice 2 wk after H. polygyrus infection, another typical Th2 response inducer, showed only slight dif- ferences in the percentage of IL-4-producing cells and in their MFI in response to PϩI or to ionomycin. No IL-2 production was observed when cells were stimulated with ionomycin, whereas IL-2 FIGURE 3. p38 is required for optimal IL-4 and IFN-␥ production, but expression was induced in 36% of the cells stimulated with PϩI is not necessary for IL-2 production. A, Splenocytes isolated from (Fig. 1B). C57BL/6 mice 2 wk after H. polygyrus infection were pretreated for 30 min and then stimulated. The cells shown were gated on CD44brightCD4 T cells. Ionomycin-induced IL-4 production was also observed in Th2 B, Spleens were isolated from C57BL/6 mice 10 days after T. gondii in- cells primed in vitro. A similar percentage of cells produced IL-4 fection, and CD4 T cells were puriﬁed with CD4 beads by positive selec- ϩ when stimulated with peptide/APC, anti-CD3/CD28, P I, or iono- tion. The cells shown were gated on CD44brightCD4 T cells. C, Two round- mycin alone (Fig. 1C). The MFI of IL-4-producing cells induced primed Th1 cells were pretreated for 30 min and then stimulated to check by ionomycin alone was about half that of the IL-4-producing cells IL-2 production. The in vivo experiments were conducted twice and the in induced by PB anti-CD3/CD28 or by PϩI. vitro experiments multiple times. The Journal of Immunology 3987

B FIGURE 4. Calcium is important in p38 phosphorylation mediated by ionomycin and by PB anti-CD3. A, Iono- mycin induces p38 phosphorylation. Th2 (left panel) and Th1 (right panel) cells were stimulated for 15 min and then p38 phosphorylation was examined by intracellular staining. C and E, PB anti-CD3 but not anti-CD28 induces p38 C E phosphorylation. Th2 cells were stimulated with PB Abs for indicated periods and analyzed with intracellular staining (C) and immunoblotting (E). EGTA, CsA, and FK520 abolish p38 phosphorylation induced by ionomycin (B and F) and by anti-CD3 (D and F). Th2 cells were preincubated with indicated inhibitors for 30 min to 1 h and stim- F Downloaded from ulated with ionomycin for 20 min (B and F) or with PB anti-CD3 for 40 min (D–F) to measure phospho-p38. EGTA, 0.8 mM; CsA, 50 ng/ml; FK520, 1 ␮M; SB203580, 5 ␮M; BAPTA, 10 ␮M; BAPTA/AM, 10 ␮M. The experiments were conducted at least three times. http://www.jimmunol.org/ D by guest on October 2, 2021 Most in vitro-primed Th1 cells produced IFN-␥ (Fig. 1C). Based inhibition of PϩI-induced IL-4 production suggests that PϩI ac- on the percentage of IFN-␥-producing cells and MFI of these cells, tivates multiple pathways. By contrast, ionomycin appears to se- the amount of IFN-␥ induced by ionomycin was ϳ21.5% of that lectively activate the p38 pathway. induced by PϩI. Ionomycin-induced IFN-␥ production by in vivo- generated Th1 cells was also observed, which will be discussed in Knocking down p38 inhibits ionomycin-induced IL-4 production detail subsequently. p38 was knocked down by introducing p38 shRNAs into Th2 cells. Neither in vitro-generated Th1 or Th2 cells produced IL-2 when The shRNA lentiviruses contain a puromycin resistance gene, al- stimulated with ionomycin (Fig. 1D). lowing for selection of cells that express the viral-encoded sequences. Puromycin-resistant cells were purified and challenged Inhibiting p38 blocks ionomycin-induced IL-4 production with PϩI or ionomycin. Cells infected with either of the two dif- Ionomycin has been shown to activate calcineurin, which in turn ferent p38 shRNA lentiviruses expressed less p38 mRNA and pro- dephosphorylates NFAT, causing the latter to enter the nucleus and tein and produced less IL-4 than did cells infected with the non- promote gene transcription. Since AP-1, a target of MAPKs, is silencing control shRNA (Fig. 2B). p38 shRNA construct-2, which generally thought to cooperate with NFAT to induce gene tran- caused more efficient diminution of p38 mRNA and protein, re- scription, we asked whether ionomycin activated a MAPK path- sulted in greater inhibition of IL-4 production. p38 shRNA con- way. We found that 1 ␮M of the p38 inhibitor SB203580 almost struct-2 reduced the response to PϩI by 75%, suggesting that the completely abolished IL-4 production, whereas little inhibition importance of p38 in IL-4 production in response to PϩI was was observed in cells pretreated with 1–10 ␮M of a JNK inhibitor underestimated by experiments relying on the p38 inhibitor. or 1–3 ␮M of a MEK inhibitor (Fig. 2A). MEK is upstream of the ERK; thus, the inhibitor blocks ERK activation. Also, 10 ␮Mof Thapsigargin (TG) induces IL-4 production through a the MEK inhibitor partially inhibited ionomycin-induced IL-4 pro- p38-dependent pathway duction. However, it is possible that chemical toxicity accounted We also tested TG for its capacity to stimulate IL-4 production. TG for this decrease since we could detect no phosphorylation of is an endoplasmic reticulum calcium-ATPase inhibitor that in- ERK1/2 in the cells stimulated by ionomycin alone (data not duces calcium depletion from endoplasmic reticulum stores and, in shown). turn, causes extracellular calcium influx through Ca2ϩ release-ac- The p38 inhibitor diminished IL-4 production induced by PϩI tivated Ca2ϩ channels. TG caused 24% of Th2 cells to produce by 50% (Fig. 2A). MEK inhibitor pretreatment also decreased IL-4, which was almost completely abolished by pretreatment with PϩI-induced IL-4 production, presumably due to diminished ac- p38 inhibitors (Fig. 2C). This strongly supports the concept that 2ϩ tivation of AP-1. The striking inhibition of ionomycin-induced elevating [Ca ]i activates p38, which, together with NFAT, plays IL-4 production by the p38 inhibitor compared with the partial a critical role in IL-4 production. 3988 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION p38 is required for optimal TCR-stimulated effector cytokine production A p38 inhibitor caused a 50% reduction in PB anti-CD3/CD28- or PϩI-stimulated IL-4 production by CD44brightCD4 T cells from H. polygyrus infected-mice; the response to ionomycin alone was reduced by 80% (Fig. 3A). The p38 inhibitor similarly blocked the IL-4 response to in vitro-primed Th2 cells stimulated with peptide- loaded APC or PB anti-CD3/CD28 (data not shown). p38 inhibition resulted in 50% diminution of PB anti- CD3/CD28-stimulated IFN-␥ production by CD44brightCD4 T cells from T. gondii-infected mice (Fig. 3B). Similarly, p38 inhibition reduced IFN-␥ production by in vitro-primed Th1 cells in response to peptide-loaded APC or PB anti-CD3/CD28 (data not shown). p38 is dispensable for IL-2 production The p38 inhibitor failed to diminish TCR- or PϩI-mediated IL-2 production by in vitro-primed Th1 cells (Fig. 3C). A similar failure of inhibition of IL-2 production by in vivo-generated Th1 cells and Downloaded from in vitro-primed Th2 cells in response to TCR-mediated stimuli was observed (data not shown). Thus, p38 is required for optimal IL-4 and IFN-␥ production, but it is not necessary for IL-2 production.

Calcium-dependent p38 phosphorylation p38 is generally phosphorylated at Thr180 and Tyr182 in the highly http://www.jimmunol.org/ conserved Thr-Gly-Tyr motif of the activation loop. To check the ability of ionomycin to induce p38 phosphorylation, we stained Th2 cells with an Ab that recognizes p38 only when it is dually phosphorylated. Cells stimulated with ionomycin or PϩI for 15 min showed an equal increase in staining intensity, involving the entire population of stimulated cells (Fig. 4A). PMA alone had little or no effect on p38 phosphorylation. Ionomycin-induced p38 phosphorylation in Th1 cells was similarly observed (Fig. 4A). FIGURE 5. ASK1 is the major kinase upstream of p38. A, Ionomycin by guest on October 2, 2021 Ionomycin-induced p38 phosphorylation was dependent on cal- induces phosphorylation of p38, MKK3/6, and TAK1 in a CsA-sensitive manner. After pretreatment for 30 min, cells were stimulated with iono- cium. Pretreatment of the cells with extracellular calcium chelator, mycin for 20 min and lysed in 1ϫ SDS sample buffer to perform immu- EGTA, completely abolished p38 phosphorylation (Fig. 4B). Iono- noblotting. B, Ionomycin-induced IL-4 production and p38 phosphoryla- mycin-induced p38 phosphorylation was also inhibited by cyclo- tion show modest decrease in TAK1-deficient Th2 cells. Th2 cells derived sporine A (CsA) or ascomycin (FK520), a chemical analog of ta- from TAK1-floxed splenocytes were retrovirally infected with GFP-Cre crolimus (FK506), suggesting a linkage between calcineurin and viral supernatant. They were stimulated to check cytokine production and the p38 pathway. Ionomycin-induced p38 activation was not af- ionomycin-induced p38 phosphorylation. C, Knocking down ASK1 strik- fected by p38 inhibition, consistent with the concept that p38 au- ingly diminishes ionomycin-induced IL-4 production and p38 phosphory- tophosphorylation is mediated through ZAP70, which is bypassed lation. Th2 cells were lentivirally infected with ASK1 shRNA or nonsi- by ionomycin. lencing control shRNA viral supernatant. Puromycin-resistant cells were In contrast to the rapid induction of phospho-p38 in cells stim- sorted and then divided into three groups: to check cytokine production, to check ionomycin-induced p38 phosphorylation, and to check Ask1 mRNA ulated with ionomycin, only a portion of cells showed p38 phos- level. D, Ionomycin-induced p38 phosphorylation is CaMK independent. phorylation in the first 15 min after stimulation with anti-CD3 or Th2 cells were pretreated with KN-93 (5 ␮M) or KN-92 (5 ␮M) for 30 min anti-CD3/CD28 (Fig. 4C). The proportion of phospho-p38-ex- and then challenged to detect ionomycin-induced p38 phosphorylation. The pressing cells increased with time, reaching essentially 100% by experiments were conducted at least twice. 90 min. Cells stimulated with PB anti-CD28 alone showed no p38 activation, and anti-CD3/CD28-stimulated cells showed no greater staining with anti-phospho-p38 at any time point than did cells treated with anti-CD3 alone, suggesting that CD28 does not play a anti-CD3-dependent p38 phosphorylation, which may represent role in mediating p38 activation. the previously reported p38 autophosphorylation pathway. To determine whether calcium plays an important role in anti- p38 phosphorylation was also examined by immunoblotting. It CD3-stimulated p38 activation, cells were pretreated BAPTA, an was minimal at 15 min, but easily detectable at 30 min after anti- extracellular calcium chelator, and BAPTA/AM, an intracellular CD3 stimulation, peaked at 45–60 min, and decreased at 90 min calcium chelator. Each dramatically decreased p38 phosphoryla- (Fig. 4E). p38 phosphorylation was not observed in cells stimu- tion in response to anti-CD3 stimulation (Fig. 4D), implying that lated by anti-CD28 at any time. calcium also plays a critical role in TCR-stimulated p38 activation. Immunoblotting was also performed to confirm calcium-medi- Anti-CD3-induced p38 phosphorylation was also inhibited by CsA ated p38 phosphorylation (Fig. 4F). Phosphorylation was observed and FK520. By contrast, anti-CD3-induced ERK phosphorylation in cells stimulated by ionomycin for 20 min and abolished in stim- was not inhibited by calcium chelators or by CsA (data not shown). ulated cells pretreated with EGTA, CsA, or FK520. More p38 Pretreatment with the p38 inhibitor resulted in partial decrease in phosphorylation was observed in cells stimulated by anti-CD3 for The Journal of Immunology 3989

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Two round-primed Il4/Gfp Th2 cells were either unstimulated (U) or stimulated with anti-CD3/28 for 1.5 h (A) or with ionomycin for2h(B). Actinomycin http://www.jimmunol.org/ D(2␮g/ml) was added with or without the p38 inhibitor SB203580 (5 ␮M) for indicated periods. Il4 and Gfp mRNAs were measured by qPCR. Half-life was calculated by linear regression analysis of a plot of natural log Il4 (Gfp) mRNA (in arbitrary units) against time. C and D, p38 activation promotes Il4 mRNA transcription. Il4/Gfp Th2 cells were pretreated for 30 min and then stimulated with ionomycin for 1.5 h (C) or with PB anti-CD3/28 (D) for indicated periods. Il4 mRNA and Gfp mRNA were measured by qPCR. C, The mRNA amount in the cells without p38 inhibitor treatment was set as “1.” D, The Gfp mRNA amount in unstimulated cells was set as “1.” E, p38 did not interfere NFAT binding to Il4 promoter. Th2 cells were unstimulated (U), pretreated with DMSO or p38 inhibitor SB203580 (5 ␮M) for 30 min and then stimulated with ionomycin for 50 min. Nuclear extract was collected and EMSA was performed. The experiments were conducted three times. by guest on October 2, 2021 40 min. Such phosphorylation was partially decreased in cells pre- eters, and construct 3 and the nonsilencing control were ineffective treated with SB203580, almost completely abolished in the cells (Fig. 5C). pretreated with CsA or FK520, and partially diminished by pre- The CaMK inhibitor KN-93 and its inactive analog KN-92 treatment with calcium chelators BAPTA or BAPTA/AM. Thus, failed to inhibit ionomycin-induced p38 phosphorylation (Fig. intracellular staining and immunoblotting correlated well, and both 5D), suggesting that calcium activation of ASK1 is mediated showed that calcium plays an important role for both ionomycin through calcineurin rather than through CaMK. We conclude that and anti-CD3-induced p38 phosphorylation. ASK1 is a major upstream kinase of p38 phosphorylation in the ionomycin-activated pathway. ASK1 is the upstream kinase of p38 To understand the upstream signaling pathway that leads to [Ca2ϩ]-dependent p38 activation, we tested the phosphorylation of p38 stabilizes Il4 mRNA upstream MAP kinases. Immunoblotting showed that ionomycin Many cytokine mRNAs are under tight stability regulation through caused phosphorylation of MKK3/6 (map2k3/6) and TGF-␤-acti- proteins that bind to adenylate/uridylate (AU)-rich elements vated kinase 1 (TAK1, map3k7), which was diminished in the (AREs) in their 3Ј untranslated regions (UTR) (18). Ionomycin presence of CsA (Fig. 5A). However, retrovirally introducing GFP- could induce IL-4 through message stabilization, new transcrip- Cre into Th2 cells derived from TAK1-floxed mice (15) resulted in tion, or both. Th2 cells heterozygous for an Il4 allele in which Gfp only a subtle decrease in IL-4 production in response to PϩIor has replaced the first exon and a portion of the first intron of Il4 ionomycin alone (Fig. 5B). TAK1-deficient (GFPϩ) cells demon- were analyzed to take advantage of the absence of AREs in the Gfp strated the same degree of ionomycin-induced p38 phosphoryla- 3ЈUTR (19) and of their presence in the Il4 3ЈUTR. The half-life tion as did controls (GFPϪ, TAK1fl/fl) cells (Fig. 5B). Thus, despite of Il4 mRNA induced by TCR was 27 min in the presence of the its ionomycin-induced phosphorylation, TAK1 is not responsible p38 inhibitor and 145 min in its absence (Fig. 6A). The half-life of for p38 phosphorylation in ionomycin-treated cells. Il4 mRNA induced by ionomycin was 4 min in the presence of the ASK1 (map3k5), another member of the MKKK family, acti- p38 inhibitor and 55 min in its absence (Fig. 6B). TCR- and iono- vates both the JNK and p38 pathways through Map2k3/4/6. Of mycin-induced Gfp mRNA were stable in actinomycin D-treated three ASK1 shRNAs introduced into Th2 cells, two caused dim- cells regardless of whether p38 inhibitor was added. Thus, one inution in both ASK1 and in ionomycin-induced p38 phosphory- mechanism through which p38 controls IL-4 production is by lation and IL-4 production. Construct 2 diminished Ask1 mRNA mRNA stabilization. Il4 mRNA half-life is longer in cells stimu- by 70%, almost completely inhibited ionomycin-induced p38 lated with TCR than that in cells stimulated with ionomycin alone. phosphorylation, and diminished ionomycin-induced IL-4 produc- This could reflect two possibilities: PB anti-CD3/CD28 activates tion by ϳ60%. Construct 1 was less effective for all three param- other pathway(s) that contribute to Il4 mRNA stabilization, or PB 3990 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION

A CaMK inhibitor D mock KN-92 KN-93 1.2 IL-4 37 39.6 8.3 1 0.8 I 0.6 0.4 0.2 0 AP-1 ELISA U I 39NK+I 2 83p NK 9 CD4 I+ I+ B nonsilencing FIGURE 7. CaMKIV and p38 induce AP-1 family control CaMKII shRNA constructs gene transcription. A, A CaMK inhibitor markedly di- IL-4 38.1 32.5 39.2 37.8 36.3 35.3 minishes ionomycin-induced IL-4 production. Two round-primed Th2 cells were pretreated for 30 min and then stimulated to check ionomycin-induced IL-4 production. B, CaMKIV but not CaMKII is involved in I ionomycin-induced IL-4 production. Th2 cells were len- CaMKIV shRNA constructs tivirally infected with the indicated shRNA viral super- 10.8 31.5 29.6 21.7 20.5 natant. Puromycin-resistant cells were sorted and then stimulated with ionomycin for4htocheck IL-4 pro- Downloaded from duction, or collected to check Camk2 or Camk4 mRNA level. The mRNA amount in nonsilencing control CD4 shRNA infected cells was set as “1.” C, fosB and fosl2,

but not fosl1 or junB, mRNA are induced by ionomycinevitaler ANRm CaMKII mRNA CaMKIV mRNA and signiﬁcantly decreased in the presence of p38 in- 1.2 tnuoma tnuoma hibitor and CaMK inhibitor. Th2 cells were pretreated 0.8

with indicated inhibitors for 30 min and then stimulated http://www.jimmunol.org/ with ionomycin for 25 min. mRNA in unstimulated cells was set as “1.” D, Th2 cells were pretreated with indi- 0.4 cated inhibitors for 30 min and then stimulated with ionomycin for 50 min. Nuclear extracts were prepared 0 g 1 2 3 4 5 and AP-1 binding was performed by using an ELISA- 1 2 3 4 5 g in ni based AP-1 family transcription factor assay kit. The cne lo CaMKII shRNA c l CaMKIV shRNA n or experiments were conducted at least twice. l rtnoc constructs l e t constructs isn isn no o o c n n by guest on October 2, 2021 C fosB fosl2 fosl1 junB

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anti-CD3/CD28 is more effective than is ionomycin alone in acti- its absence is compensated for by other MAPKs activated by PB vating p38 and is less completely blocked by the p38 inhibitor. anti-CD3/CD28. p38 has been reported to have a stimulatory effect on NFAT p38 plays a role in promoting Il4 transcription activation (20). An EMSA using the NFAT binding site in the Il4 The absence of an ARE in the Gfp 3ЈUTR is associated with sta- promoter as a probe was performed. The capacity of NFAT to bind bility of Gfp mRNA. Thus, the degree of Gfp mRNA induction by to the Il4 promoter in ionomycin-stimulated cells was unchanged ionomycin and its sensitivity to p38 inhibition is a measure of in the presence of a p38 inhibitor (Fig. 6E). Thus, p38 function in ionomycin-induced transcription and of whether p38 plays a role Il4 transcription cannot be accounted for by enhanced activation in such transcription. p38 inhibitor pretreatment caused a 75% in- of NFAT. hibition of Gfp mRNA levels and an 80% inhibition of Il4 mRNA (Fig. 6C). Thus, we conclude that p38 plays an important role in Ionomycin-induced Il4 transcription requires CaMKIV Gfp and, presumably, Il4 transcription. Although KN-93 failed to prevent ionomycin-induced p38 phos- In TCR-induced Il4/Gfp heterozygous Th2 cells, the p38 inhibitor phorylation (Fig. 5D), it strikingly diminished ionomycin-induced led to a ϳ50% diminution in Gfp mRNA induction at 4 h but to less IL-4 production (Fig. 7A). Knocking down CaMK proteins by len- inhibition at 2 and 3 h after stimulation. Although cells pretreated with tiviral shRNAs speciﬁc for CaMKIV but not CaMKII diminished the MEK and JNK inhibitors showed no diminution of Gfp mRNA ionomycin-induced IL-4 production, implying that CaMKIV is the upon stimulation, pretreatment with all three MAPK pathways inhib- major CaMK involved in the ionomycin pathway (Fig. 7B). Con- itors decreased Gfp mRNA by 75% at all time points (Fig. 6D). This sistent with the results from the CaMK inhibitor (Fig. 5D), iono- implies that p38 plays an important role in promoting Il4 gene tran- mycin-induced p38 phosphorylation remained normal in CaMKIV scription in response to TCR engagement that is underestimated since shRNA-infected cells (data not shown). The Journal of Immunology 3991

FIGURE 8. Three pathways involved in ionomycin- induced cytokine production. Calcium activates three pathways that contribute to IL-4 induction: to dephosphorylate NFAT through calcineurin; to activate ASK1, likely through calcineurin, leading to p38 activation; and to induce AP-1 protein synthesis through CaM and CaMKIV. Activated p38 may induce AP-1 production and phosphorylation. Cooperation between AP-1 and NFAT leads to Il4 gene transcription and activated p38 also exerts a role in stabilization of Il4 mRNA half-life. Downloaded from

CaMKIV contributes to cytokine production by inducing CREB function in ionomycin-induced IL-4 production through a mecha- phosphorylation and thereafter, transcription of the CREB-depen- nism other than modification of NFAT binding to Il4 promoter. dent immediate-early genes, including fosB, fosl2, and junB (21, We observed that ionomycin induces p38 phosphorylation in http://www.jimmunol.org/ 22). Similarly, p38 phosphorylation of CREB and induction of Th2 and Th1 cells. Intracellular and extracellular calcium chelators AP-1 transcription has been reported (21). Indeed, fosB and fosl2 completely abolished p38 phosphorylation induced by ionomycin mRNA levels were increased Ͼ15-fold by ionomycin and signif- and greatly decreased phosphorylation in response to PB anti-CD3, icantly decreased in cells pretreated with CaMK inhibitors or p38 indicating that calcium plays an important role in p38 activation inhibitor (Fig. 7C). Fosl1 and junB were only modestly induced by even when cells were stimulated through TCR. Ionomycin-induced ionomycin. Ionomycin induced increased AP-1 binding activity p38 activation was not affected by the p38 inhibitor SB203580, that was blocked by a p38 inhibitor and by the CaMK inhibitor indicating that this calcium-mediated p38 activation represents a (Fig. 7D). Thus, p38 and CaMKIV control the amount and activity separate pathway from the ZAP70-mediated p38 autophosphory- by guest on October 2, 2021 of AP-1 family transcription factors. This, together with NFAT, lation pathway (9). CsA and FK520 completely abolish ionomy- explains ionomycin-induced Il4 transcription. Together with iono- cin-induced p38 phosphorylation and partially blocked p38 phos- mycin-induced p38 prolongation of Il4 mRNA half-life, this ex- phorylation in response to anti-CD3. These results indicate that plains the increase in Il4 mRNA (Fig. 8). calcineurin activation is required for ionomycin-induced p38 phosphorylation and contributes to TCR-induced p38 phosphorylation. Discussion Ca2ϩ elevation has been reported to activate the MAPK cascade Full activation of T cells requires signaling through the TCR/CD3 (25–28). Several MKKKs, such as TAK1 and ASK1, can be ac- complex and the CD28 costimulatory receptor. PϩI are often uti- ϩ tivated by elevated [Ca2 ] and in turn activate the classical lized to bypass TCR stimulation, through the action of PMA on i MAPK cascade, leading to p38 phosphorylation (27, 29). The in- PKC␪ and the capacity of ionomycin to elevate [Ca2ϩ] . Unex- i volvement of calcineurin in MAPK activation in Jurkat cells was pectedly, we found that ex vivo CD44brightCD4 T cells isolated suggested by the finding that JNK activation by PMA plus A23187 from either S. mansoni- or H. polygyrus-infected mice produced was blocked by CsA (30). similar amounts of IL-4 when stimulated with ionomycin alone or ϩ Calcineurin-like molecules have been reported to interact with with PϩI. Both ionomycin and a second Ca2 elevating agent, TG, the MAPK analog. In Caenorhabditis elegans, KIN-29, a Ser/Thr induced IL-4 production from in vitro-primed Th2 cells. Ionomy- cin also induced IFN-␥ production by CD44brightCD4 T cells from kinase, binds to the calcineurin-like phosphatase Tax6 (31). In T. gondii-infected mice and in vitro-primed Th1 cells. Ionomycin Arabidopsis, calcineurin B-like proteins (CBLPs) are reported to alone has been recently reported to induce IL-21 production in bind to CBL-interacting Ser/Thr kinases (AtCIPKs) in a calcium- preactivated T cells (23). By contrast, as has been previously re- dependent manner (32). Mammalian kinases corresponding to At- ported (24), IL-2 was not produced when these cells were stimu- CIPKs include Raf-1. Raf-1 possesses an autoinhibitory site that is lated with ionomycin, although it was produced in response to generally phosphorylated in quiescent cells. Activation of Raf-1 cognate peptide-loaded APC, anti-CD3/CD28, or PϩI. involves dephosphorylation of this autoinhibitory site (33). Ionomycin activates calcineurin, which in turn dephosphorylates Among the kinases upstream of p38 in the classical MAPK NFAT, causing the latter to enter the nucleus and promote gene pathway, ASK1 has been reported to be activated by calcium sig- transcription. Our data demonstrate that blocking p38 activity by nal in primary neurons and synaptosomes (27, 34) and has a well- inhibitors or diminishing p38 expression by RNA interference conserved autoinhibitory site (35), suggesting that it might be a strikingly inhibited ionomycin-induced IL-4 production, although target of calcineurin. It has been reported that ASK1 activity is the capacity of NFAT to bind to the Il4 promoter remained normal. regulated through this autoinhibitory site (36, 37). In a yeast two- Thus, ionomycin-induced NFAT translocation alone is not suffi- hybrid screen, the calcium-binding subunit B of calcineurin and cient for IL-4 production; p38 activity is essential. p38 mediates its ASK1 were found to be direct physical partners (38). Furthermore, 3992 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION in an in vitro phosphatase assay, calcineurin directly dephospho- induce AP-1 production and phosphorylation (Fig. 8). Cooperation rylated ASK1, resulting in its activation. In cardiomyocytes, the between AP-1 and NFAT leads to Il4 gene transcription, and ac- endogenous subunit B of calcineurin could be coimmunoprecipi- tivated p38 also exerts a role in stabilization of Il4 mRNA half-life. tated with ASK1, suggesting physiological importance of their AP-1 and NFAT are also critical for Il2 transcription; however, interaction. IL-2 production was not detected in Th1 or Th2 cells. This can be Herein, we showed that knocking down ASK1 resulted in di- accounted for by the requirement for additional factors, not in- minished ionomycin-induced p38 phosphorylation and IL-4 produced by ionomycin, for Il2 transcription. Indeed, the CD28 reduction. By contrast, ionomycin-induced p38 phosphorylation and sponse element (CD28RE) is essential for Il2 transcription, and IL-4 production were not impaired in TAK1-deficient cells, al- c-Rel homodimers up-regulated in response to CD28 signaling though TAK1 was phosphorylated in ionomycin-stimulated cells. bind to the Il2 CD28RE (45–47). c-Rel-deficient cells showed a We conclude that ASK1 is the major kinase upstream of p38 in the striking defect in IL-2 production (48). When cells are stimulated ionomycin-stimulated cells. Inhibiting or knocking down CaMKs through TCRs, p65/p50 dimers also interact with CD28RE in the did not affect ionomycin-induced p38 phosphorylation. Thus, iono- Il2 promoter and act as potent transactivators (49). In resting cells mycin does not activate ASK1 through CaMK. Instead, it is likely and ionomycin-stimulated CD4T cells, p50/p50 homodimers, po- that elevated [Ca2ϩ] activates calcineurin, which in turn dephos- tent repressors of Il2 transcription, bind to Il2 promoter (24, 50, phorylates the autoinhibitory site of ASK1 and leads to its 51). Thus, the absence of c-Rel or p65/p50 in ionomycin-stimu- activation. lated CD4 T cells explains the lack of Il2 transcription. The importance of p38 for effector cytokine production is still Do physiologic ligands that lead to robust calcium elevation controversial. IL-4 production from Th2 cells has been reported to cause direct effector T cell cytokine production in tissues, or do be impaired by a p38 inhibitor (39). However, in another report, physiologic ligands that can activate p38 in Th2 or Th1 cells en- Downloaded from p38 phosphorylation was described to be selectively induced by hance the minimal cytokine production that would have occurred Con A in Th1 cells but not in Th2 cells (40). Our data showed that in response to limiting amounts of Ag/APC? Such activation might p38 is essential for both IL-4 and IFN-␥ production in response to be particularly important in secondary responses to pathogens to ionomycin and for optimal production in response to anti-CD3 and ensure rapid cytokine production even before substantial amounts anti-CD28. Since Con A, like anti-CD3 and anti-CD28, can induce of Ag are released during infection. Thus, the p38 effect might IL-4 by both p38-dependent and independent pathways, the degree confer on Th cells a type of innate-immune status. The nature of http://www.jimmunol.org/ 2ϩ of priming could have had a major impact on the relative use of the ligands that would act to elevate [Ca ]i in effector cells res- these pathways and may account for the difference between our ident in tissues remains to be determined, but many candidates results and those reported by Rincon et al. (40). More recently, could be considered, particularly from the G protein-coupled re- Berenson and colleagues using p38␣ϩ/Ϫ and p38␣Ϫ/Ϫ CD4 T cells ceptor family of molecules, which would include chemokines. prepared by RAG2Ϫ/Ϫ blastocyst complementation, emphasized that p38 is required for IL-12/IL-18-stimulated IFN-␥ production Acknowledgments by Th1 cells (41). However, as the authors pointed out in the We thank Dragana Jankovic (Laboratory of Parasitic Diseases, National discussion, their data indicated that TCR-stimulated IFN-␥ pro-

Institute of Allergy and Infectious Diseases (NIAID), National Institutes of by guest on October 2, 2021 duction was also partially inhibited, as judged from the reduction Health) for preparing cells from S. mansoni-infected mice and T. gondii- of MFI of IFN-␥-expressing cells. Thus, the importance of p38 in infected mice, Richard Flavell for Tak1-floxed cells, Sarah Tanksley (Lab stimulation of cytokine production may be easily underestimated. of Immunology, NIAID) for cell sorting, Ludmila Jirmanova for the de- The physiological substrates of p38 MAPK in T cells have not tailed immunoblotting protocol to check p38 phosphorylation, Shirley Star- been identified. MAPK-activated protein kinase 2 (MAPKAPK2 nes for editorial assistance, and Jonathan Ashwell, Ludmila Jirmanova, Warren Leonard, and Arthur Weiss for helpful discussions and critical or MK2), the major kinase downstream of p38, has been shown to reviews of the manuscript. cause stabilization of many cytokine mRNAs including Il2, Il3, ␥ Il6, Il8, Tnf, Inf , and Gmcsf (42). Herein, we have shown that p38 Disclosures plays an important role lengthening the half-life of Il4 mRNA in The authors have no financial conflicts of interest. stimulated Th2 cells. The p38 inhibitor did not destabilize Gfp mRNA derived from a gene in which Gfp is knocked into the Il4 References locus, consistent with the absence of AREs in the Gfp 3ЈUTR. 1. Call, M. E., and K. W. Wucherpfennig. 2005. The T cell receptor: critical role of However, the striking increase in Gfp mRNA in ionomycin-stim- the membrane environment in receptor assembly and function. Annu. Rev. Im- ulated Th2 cells from Il4/Gfp heterozygous donors implies that munol. 23: 101–125. ionomycin’s effect involves substantial induction of transcription. 2. Takahama, Y., and H. Nakauchi. 1996. Phorbol ester and calcium ionophore can replace TCR signals that induce positive selection of CD4 T cells. J. Immunol. Furthermore, since Gfp induction is also blocked by the p38 inhibitor, 157: 1508–1513. it must be concluded that p38 regulates IL-4 production in response 3. Lewis, R. S. 2001. Calcium signaling mechanisms in T lymphocytes. Annu. Rev. Immunol. 19: 497–521. to ionomycin both by a striking induction of transcription as well 4. Macian, F. 2005. NFAT proteins: key regulators of T-cell development and func- as by mRNA stabilization. tion. Nat. Rev. Immunol. 5: 472–484. Inhibiting or knocking down CaMKs greatly diminished iono- 5. Rooney, J. W., T. Hoey, and L. H. Glimcher. 1995. Coordinate and cooperative roles for NF-AT and AP-1 in the regulation of the murine IL-4 gene. Immunity mycin-induced IL-4 production, although p38 activation in re- 2: 473–483. sponse to ionomycin remained normal. fosl2 and fosB, but not 6. Isakov, N., and A. Altman. 2002. Protein kinase C(␪) in T cell activation. Annu. fosl1 and junB, mRNA were induced by ionomycin; p38 and Rev. Immunol. 20: 761–794. 7. Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune CaMK inhibitors blocked the induction. AP-1 is regulated by phos- response. Annu. Rev. Immunol. 20: 55–72. phorylation as well as by induction (43); p38 has been reported to 8. Ashwell, J. D. 2006. The many paths to p38 mitogen-activated protein kinase activation in the immune system. Nat. Rev. Immunol. 6: 532–540. mediate such phosphorylation (44). Thus, calcium activates at least 9. Salvador, J. M., P. R. Mittelstadt, T. Guszczynski, T. D. Copeland, three pathways that contribute to IL-4 induction: through cal- H. Yamaguchi, E. Appella, A. J. Fornace, Jr., and J. D. Ashwell. 2005. Alterna- cineurin to dephosphorylate NFAT; likely through calcineurin to tive p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases. Nat. Immunol. 6: 390–395. activate ASK1, leading to p38 activation; through CaM and 10. Grunvald, E., M. Chiaramonte, S. Hieny, M. Wysocka, G. Trinchieri, CaMKIV to induce AP-1 protein synthesis; and activated p38 may S. N. Vogel, R. T. Gazzinelli, and A. Sher. 1996. Biochemical characterization The Journal of Immunology 3993

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