of September 14, 2018.This information is current as

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

PaulLiying Guo, Joseph F. Urban, Jinfang Zhu and William E.

http://www.jimmunol.org/content/181/6/3984doi: 10.4049/jimmunol.181.6.3984

2008; 181:3984-3993; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/181/6/3984.full#ref-list-1

, 23 of which you can access for free at: cites 51 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Elevating Calcium in Th2 Cells Activates Multiple Pathways toInduce 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, fromin vivo- and in vitro-generated murine Th2 and Th1 cells. Ionomycin-induced cytokine production requires NFAT, p38, andcalmodulin-dependent kinase IV (CaMKIV). Ionomycin induces p38 phosphorylation through a calcium-dependent, cyclosporineA-inhibitable pathway. Knocking down ASK1 inhibits ionomycin-induced p38 phosphorylation and IL-4 production. Ionomycinalso activates CaMKIV, which, together with p38, induces AP-1. Cooperation between AP-1 and NFAT leads to Il4 gene tran-scription. p38 also regulates IL-4 production by mRNA stabilization. TCR stimulation also phosphorylates p38, partially throughthe calcium-dependent pathway; activated p38 is required for optimal IL-4 and IFN-�. The Journal of Immunology, 2008, 181:3984–3993.

O ptimal activation of T cells requires engagement of theTCR-CD3 complex and of costimulatory moleculeswhose ligands are expressed on APCs (1). The binding

of Ag/MHC complexes to the TCR elevates intracellular Ca2�

concentration ([Ca2�]i)3 and activates protein kinase C� (PKC�).

The combination of a calcium ionophore (e.g., ionomycin) andphorbol ester tumor promoters (e.g., PMA), which bypass TCRsignals, results in T cell activation (2) and production of a series ofcytokines, including IL-2, IL-4, and IFN-�.

Elevation of [Ca2�]i is an essential event following TCR stim-ulation (3). Imaging studies reveal that TCR stimulation causessustained elevation in [Ca2�]i over a period of 1–2 h. Such sus-tained signaling allows dephosphorylated NFAT, a key transcrip-tional regulator of cytokine genes, to be maintained in the nucleus(4). The cooperative action of NFAT and AP-1 is generallythought to be necessary for full cytokine transcription (5).

PKC� is a signal regulator of several T cell activation pathways(6). The MAP kinases ERK, JNK, and p38 are among the targetsof PKC�. The ERK and JNK pathways are involved in IL-2 pro-duction, predominantly through activation of AP-1 (7). However,relatively little is known about the role of p38 MAPK in cytokineinduction.

Four p38 MAPK isoforms have been characterized: p38�, p38�,p38�, and p38�. CD4 T cells predominantly express p38� andp38� (8). p38 can be activated by multiple stressors, such as UV

radiation, heat shock, and osmotic shock, as well as by proinflam-matory cytokines, such as TNF-� and IL-1. Ligation of the TCRalso activates p38. TCR stimulation triggers small GTP-bindingproteins such as Ras, Rac-1, and Cdc42, leading to the activationof MAPK cascades (7, 8). p38 is dually phosphorylated at Thr180

and Tyr182 in a highly conserved motif in the p38 activation loop.Salvador and colleagues reported an alternative p38 activationpathway in T cells, in which ZAP70 phosphorylates p38� onTyr323, leading to autophosphorylation of the Thr and Tyr in theactivation loop and to “self-activation” (9). TCR stimulation thusresults in induction of both the classic MAPK cascade and thisalternative p38 inhibitor-sensitive self-activation pathway; the rel-ative contributions of the two pathways and their physiologicalfunctions remain to be determined.

The importance of p38 activity to cytokine production is stillcontroversial. Herein we report that ionomycin alone induces IL-4and IFN-� production from in vivo- and in vitro-generated Th2and Th1 cells, in contrast to IL-2, for which both PMA and iono-mycin are essential for production. The production of IL-4 requireselevation of [Ca2�]i, NFAT dephosphorylation, and activation ofp38 and CaMKIV. Phospho-p38 generated through elevation of[Ca2�]i plays a crucial role in ionomycin-induced IL-4 productionby promoting Il4 gene transcription and stabilizing its mRNA. Fur-thermore, we show that phospho-p38 is essential for optimal IL-4production induced through TCR stimulation.

Materials and MethodsMice and cell culture

Cells from C57BL/6 mice infected with Schistosoma mansoni cercariae for8 wk and splenocytes from C57BL/6 mice infected with Toxoplasma gon-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 micewere inoculated with third-stage Heligmosomoides polygyrus per os, andspleens were isolated 14 days later, as previously described (12).

CD4 T cells were purified from 5C.C7 transgenic Rag2�/� mice bynegative selection. Cells were cultured under Th1 and TH2 conditions asdescribed before (13). CD4 T cells were purified from Il4/Gfp heterozy-gous mice by negative selection. The cells were primed with APC, anti-CD3 (3 �g/ml), and anti-CD28 (3 �g/ml) under Th2 conditions.

Intracellular staining

In vitro differentiated Th1 and Th2 cells were rechallenged under differentconditions for 4 h, in the presence of monensin, to check cytokine produc-tion. The conditions used to rechallenge the cells were: APC � peptide,

*Laboratory of Immunology, National Institute of Allergy and Infectious Dis-eases, National Institutes of Health, Bethesda, MD 20892; and †Nutrient Require-ments & Functions Laboratory, Beltsville Human Nutrition Research Center, Ag-ricultural Research Service, United States Department of Agriculture, Beltsville,MD 20705

Received for publication April 7, 2008. Accepted for publication July 11, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This research was supported by the Intramural Research Program of the NationalInstitutes of Health, National Institute of Allergy and Infectious Diseases.2 Address correspondence and reprint requests to Dr. Liying Guo, Laboratory of Im-munology, National Institute of Allergy and Infectious Diseases, National Institutes ofHealth, Building 10, Room 11N322, 10 Center Drive–MSC 1892, Bethesda, MD20892-1892. E-mail address: lguo@niaid.nih.gov3 Abbreviations used in this paper: [Ca2�]i, intracellular Ca2� concentration; ARE,adenylate/uridylate-rich elements; CsA, cyclosporine A; LN, lymph node; MFI, meanfluorescence intensity; PB, plate-bound; PKC�, protein kinase C�; qPCR, quantitativePCR; shRNA, small hairpin RNA; TG, thapsigargin; UTR, untranslated region.

The Journal of Immunology

www.jimmunol.org

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

cognate cytochrome C peptide (1 �M)-loaded APC; anti-CD3/CD28,plate-bound (PB) anti-CD3 (3 �g/ml) and anti-CD28 (3 �g/ml); P�I, PMA(10 ng/ml) and ionomycin (1 �M), and ionomycin (1 �M) alone.

Harvested cells were fixed with 4% paraformadehyde, washed with0.1% BSA-containing PBS, and stored at 4°C. For staining, cells wereincubated with permeabilization buffer (PBS supplemented with 0.1%BSA/0.1% Triton X-100) and various Abs for 20 min. All Abs were pur-chased from BD Pharmingen.

To test p38 phosphorylation in PB anti-CD3 and/or anti-CD28 stimu-lated Th2 cells, cells were put in Ab-coated 6-well plates and were spun at1600 rpm for 20 s to allow cells to attach to the bottom. Paraformadehyde(4% final concentration) was added to cell culture to fix the cells imme-diately after the cells had been cultured in the plates for the indicatedperiods.

For phospho-p38 staining, fixed cells were incubated with a rabbitmonoclonal phospho-p38 Ab (1/25) (336 ng/ml) (Cell Signaling Technol-ogy) or a nonspecific rabbit isotype control Ab (336 ng/ml) (Imgenex) for30 min at room temperature. After extensive washing, cells were stainedwith a FITC-conjugated donkey anti-rabbit Ab (1/200) (Jackson Immuno-Research Laboratories).

Small hairpin (sh)RNA lentiviral infection

p38�, ASK1, CaMKII, and CaMKIV shRNA and nonsilencing-controlshRNA constructs were purchased from Sigma-Aldrich. All the plasmidswere purified with EndoFree plasmid purification kit (Qiagen). 293FT“packaging” cells were transfected with shRNA plasmids or nonsilencingcontrol shRNA plasmids, together with the packaging plasmid pCMV�8.9and the envelope plasmid pHCMV-G using the Fugene 6 transfection re-agent (Roche Diagnostics) according to the manufacturer’s protocol. Pack-aging plasmid pCMV�8.9 and envelope plasmid pHCMV-G were kindlyprovided by Dr. N. Hacohen at Harvard Medical School. Lentivirus-con-taining supernatant was collected twice, at 48 and 72 h after transfection,respectively.

To “knockdown” p38, Th2 cells were cultured for a second round underTh2 conditions. Two days later, the lentivirus, collected as describedabove, and polybrene (8 �g/ml) (Sigma-Aldrich) were added and the cellswere centrifuged at 2500 rpm for 90 min at room temperature. The super-natant was removed immediately and fresh medium was added to the cul-ture, which continued under Th2 conditions. Forty-eight hours after infec-tion, cells were placed in medium containing IL-2 and puromycin (5 �g/ml) (Sigma-Aldrich). One week later, puromycin-resistant cells werepurified by cell sorting and cultured with cytochrome C peptide (1 �M)-loaded APC in the presence of anti-IL-4 (10 �g/ml), anti-IFN-� (10 �g/ml), and anti-IL-12 (10 �g/ml) (null Th cell conditions) for 4 days.

To knockdown ASK1, CaMKII, and CaMKIV, two round-primed Th2cells that had just finished 4-day priming under Th2 condition were used.Infection was performed as described above. Forty-eight hours after infec-

tion, cells were maintained in medium containing IL-2 and puromycin (5�g/ml) (Sigma-Aldrich). Another 2 days later, puromycin-resistant cellswere purified by cell sorting.

Retroviral infection

GFP-Cre viral supernatant was prepared as described before (14). SplenicCD4 T cells were purified from mice homozygous for a “floxed” TAK1gene (15) and cultured under Th2 conditions as described above. Two daysafter a third round of Th2 priming, cells were incubated with a GFP-Creretrovirus-containing supernatant. Two days after infection, cells wereplaced in IL-2-containing medium. Twenty-four to 48 h later, cells werestimulated to check cytokine production or p38 phosphorylation inresponse to ionomycin.

RNA purification and quantitative PCR (qPCR)

Total RNA was isolated using RNeasy Mini kits (Qiagen) (treated withRNase-free DNase I); first-strand cDNA was prepared using SuperScriptIII (Invitrogen). All PCR was performed on a 7900HT sequence detectionsystems (Applied Biosystems). The TaqMan universal PCR SuperMix andall the primer and probe sets were purchased from Applied Biosystems. Totest Gfp mRNA level, SYBR Green technology was utilized with the prim-ers as reported before (13). To exclude the possible false signal introducedby binding of SYBR Green to likely primer dimer, the PCR conditionswere set as 95°C for 15 s, 60°C for 1 min, and 78°C for 30 s for 40 cycles,and the increase in fluorescence was detected only at the 78°Cincubation step.

Immunoblotting

Stimulated cells were lysed in 1� SDS sample buffer (0.05 M Tris-HCl(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�g/ml aprotinin, leupeptin, pepstatin, and 1� phosphatase inhibitor cock-tail 1 and cocktail 2 (Sigma-Aldrich). Cell lysates were sonicated for 10 sto shear DNA.

Proteins were separated on 12% pre-made tricine-glycine gels (Invitro-gen) and then transferred onto nitrocellulose membranes (Millipore) by theTrans-Blot SD semidry electrophoretic transfer cell (Bio-Rad). Membraneswere probed with phospho-p38 MAPK Ab (Cell Signaling Technology)(1/1000), followed by a HRP-labeled anti-rabbit Ab (Pierce) (1/1000). Thesignal was visualized with ECL. Membranes was stripped by RestoreWestern blot stripping buffer (Pierce) and then reprobed with p38 MAPKAb (Cell Signaling Technology) (1/1000).

Nuclear extraction and EMSAs

Cells were stimulated with ionomycin for 50 min. Nuclear protein extractswere prepared as previously described (16), and protein concentration was

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 CD44brightCD4T cells was measured by intracellular staining. B, Splenocytes were isolated from C57BL/6 mice 2 wk after H. polygyrus infection. IL-4 (upper panel) andIL-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 vitroexperiments multiple times.

3985The Journal of Immunology

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

determined with the BCA kit (Pierce). EMSAs were conducted with theEMSA kit (Promega). The oligonucleotides corresponding to sense andantisense sequences within the Il4 promoter (17) were 5�-biotin labeled(Bio-Synthesis), HPLC purified, and annealed for use as probe. Sampleswere resolved on a 6% DNA retardation gel (Invitrogen) in 0.5� Tris-borate-EDTA buffer. The protein-DNA binding complex was detected bythe LightShift chemiluminescent EMSA kit (Pierce). The sequences of theoligonucleotides were: sense, 5�-TAATGTAAACTCATTTTCCCTTG-3�and antisense, 5�-CAAGGGAAAATGAGTTTACATTA-3�.

AP-1 ELISA

An ELISA-based TransAM AP-1 kit, in which oligonucleotide containingAP-1 binding motif has been immobilized to 96-well plate, was used toquantify activated AP-1 components (Active Motif). Five-microgram nu-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 bind-ing assay was performed according to the instructions of the manufacturer.

ResultsIonomycin induces IL-4 and IFN-�, but not IL-2, productionfrom in vivo- and in vitro-differentiated Th cells

Mesenteric lymph nodes (LNs) were isolated from mice 8 wk afterS. mansoni infection, which strikingly induces a Th2 response. LNcells were either unstimulated, immediately stimulated with PBanti-CD3/CD28, with a combination of PMA and ionomycin(P�I), or with ionomycin alone. The percentage ofCD44brightCD4 T cells from infected mice that produced IL-4 inresponse to ionomycin was similar to that in response to P�I orto PB anti-CD3/CD28; mean fluorescence intensities (MFI) ofthe IL-4-expressing cells induced by P�I or by ionomycinalone were also similar (Fig. 1A).

Splenocytes isolated from mice 2 wk after H. polygyrus infec-tion, another typical Th2 response inducer, showed only slight dif-ferences in the percentage of IL-4-producing cells and in their MFIin response to P�I or to ionomycin. No IL-2 production was ob-served when cells were stimulated with ionomycin, whereas IL-2expression was induced in 36% of the cells stimulated with P�I(Fig. 1B).

Ionomycin-induced IL-4 production was also observed in Th2cells primed in vitro. A similar percentage of cells produced IL-4when stimulated with peptide/APC, anti-CD3/CD28, P�I, or iono-mycin alone (Fig. 1C). The MFI of IL-4-producing cells inducedby ionomycin alone was about half that of the IL-4-producing cellsinduced by PB anti-CD3/CD28 or by P�I.

JNK inhibitorP38 inhibitor

MEK inhibitor

1µM 3µM 10µM1µM 3µM 10µMinhibitors concentration

P+I I

P+I

I

IL-4

Inhibitors: MEK JNK p38

RNA: 1 : 0.38 : 0.13

Protein:

non-silencing control p38shRNA-1 p38shRNA-2

P+I

I

IL-4

CD4

gnicnelis-non lortnoc

1-ANRhs83p

2-ANRhs83p

P+I

I

TG

mock SB203580 SB202190IL-4

CD4

A B C80%

46%

24%

54%

7%

2%

59%

4%

2%

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

80% 52% 88% 66%

54% 11%49% 66%

CD4

(10µM)

52% 34% 23%

28% 12% 5%

MFI:433 349 250

MFI:219 178 139

two p38 inhibitors:

noitcudorp 4-LI

MFI:343 232 240

173 108 103

147 104 103MFI:

MFI:

mock

FIGURE 2. Ionomycin and TG induce IL-4 production through a p38-dependent pathway. A, Ionomycin-induced IL-4 production by Th2 cells is highlysensitive 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 byDMSO-treated cells was set as “1.” B, Knocking down p38 inhibits ionomycin-induced IL-4 production. Th2 cells were lentivirally infected with nonsi-lencing 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 P�I or ionomycin (upper panel), one portion to analyze p38 mRNA by qPCR, and one portion to checkp38 protein by immunoblotting (lower panel). C, TG similarly induces IL-4 production through a p38-dependent pathway. Th2 cells were pretreated thenstimulated as described. SB203580, 5 �M; SB202190, 5 �M; TG, 10 nM. The experiments were conducted three times.

FIGURE 3. p38 is required for optimal IL-4 and IFN-� production, butis not necessary for IL-2 production. A, Splenocytes isolated fromC57BL/6 mice 2 wk after H. polygyrus infection were pretreated for 30 minand then stimulated. The cells shown were gated on CD44brightCD4 T cells.B, Spleens were isolated from C57BL/6 mice 10 days after T. gondii in-fection, and CD4 T cells were purified with CD4 beads by positive selec-tion. The cells shown were gated on CD44brightCD4 T cells. C, Two round-primed Th1 cells were pretreated for 30 min and then stimulated to checkIL-2 production. The in vivo experiments were conducted twice and the invitro experiments multiple times.

3986 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Most in vitro-primed Th1 cells produced IFN-� (Fig. 1C). Basedon the percentage of IFN-�-producing cells and MFI of these cells,the amount of IFN-� induced by ionomycin was �21.5% of thatinduced by P�I. Ionomycin-induced IFN-� production by in vivo-generated Th1 cells was also observed, which will be discussed indetail subsequently.

Neither in vitro-generated Th1 or Th2 cells produced IL-2 whenstimulated with ionomycin (Fig. 1D).

Inhibiting p38 blocks ionomycin-induced IL-4 production

Ionomycin has been shown to activate calcineurin, which in turndephosphorylates NFAT, causing the latter to enter the nucleus andpromote gene transcription. Since AP-1, a target of MAPKs, isgenerally thought to cooperate with NFAT to induce gene tran-scription, we asked whether ionomycin activated a MAPK path-way. We found that 1 �M of the p38 inhibitor SB203580 almostcompletely abolished IL-4 production, whereas little inhibitionwas observed in cells pretreated with 1–10 �M of a JNK inhibitoror 1–3 �M of a MEK inhibitor (Fig. 2A). MEK is upstream of theERK; thus, the inhibitor blocks ERK activation. Also, 10 �M ofthe MEK inhibitor partially inhibited ionomycin-induced IL-4 pro-duction. However, it is possible that chemical toxicity accountedfor this decrease since we could detect no phosphorylation ofERK1/2 in the cells stimulated by ionomycin alone (data notshown).

The p38 inhibitor diminished IL-4 production induced by P�Iby 50% (Fig. 2A). MEK inhibitor pretreatment also decreasedP�I-induced IL-4 production, presumably due to diminished ac-tivation of AP-1. The striking inhibition of ionomycin-inducedIL-4 production by the p38 inhibitor compared with the partial

inhibition of P�I-induced IL-4 production suggests that P�I ac-tivates multiple pathways. By contrast, ionomycin appears to se-lectively activate the p38 pathway.

Knocking down p38 inhibits ionomycin-induced IL-4 production

p38 was knocked down by introducing p38 shRNAs into Th2 cells.The shRNA lentiviruses contain a puromycin resistance gene, al-lowing for selection of cells that express the viral-encoded se-quences. Puromycin-resistant cells were purified and challengedwith P�I or ionomycin. Cells infected with either of the two dif-ferent p38 shRNA lentiviruses expressed less p38 mRNA and pro-tein and produced less IL-4 than did cells infected with the non-silencing control shRNA (Fig. 2B). p38 shRNA construct-2, whichcaused more efficient diminution of p38 mRNA and protein, re-sulted in greater inhibition of IL-4 production. p38 shRNA con-struct-2 reduced the response to P�I by 75%, suggesting that theimportance of p38 in IL-4 production in response to P�I wasunderestimated by experiments relying on the p38 inhibitor.

Thapsigargin (TG) induces IL-4 production through ap38-dependent pathway

We also tested TG for its capacity to stimulate IL-4 production. TGis an endoplasmic reticulum calcium-ATPase inhibitor that in-duces calcium depletion from endoplasmic reticulum stores and, inturn, causes extracellular calcium influx through Ca2� release-ac-tivated Ca2� channels. TG caused 24% of Th2 cells to produceIL-4, which was almost completely abolished by pretreatment withp38 inhibitors (Fig. 2C). This strongly supports the concept thatelevating [Ca2�]i activates p38, which, together with NFAT, playsa critical role in IL-4 production.

B

C E

F

D

FIGURE 4. Calcium is important in p38 phosphoryla-tion mediated by ionomycin and by PB anti-CD3. A, Iono-mycin induces p38 phosphorylation. Th2 (left panel) andTh1 (right panel) cells were stimulated for 15 min and thenp38 phosphorylation was examined by intracellular stain-ing. C and E, PB anti-CD3 but not anti-CD28 induces p38phosphorylation. Th2 cells were stimulated with PB Absfor indicated periods and analyzed with intracellular stain-ing (C) and immunoblotting (E). EGTA, CsA, and FK520abolish p38 phosphorylation induced by ionomycin (B andF) and by anti-CD3 (D and F). Th2 cells were preincu-bated with indicated inhibitors for 30 min to 1 h and stim-ulated with ionomycin for 20 min (B and F) or with PBanti-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.

3987The Journal of Immunology

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

p38 is required for optimal TCR-stimulated effector cytokineproduction

A p38 inhibitor caused a 50% reduction in PB anti-CD3/CD28- orP�I-stimulated IL-4 production by CD44brightCD4 T cells from H.polygyrus infected-mice; the response to ionomycin alone was re-duced by 80% (Fig. 3A). The p38 inhibitor similarly blocked theIL-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 Tcells from T. gondii-infected mice (Fig. 3B). Similarly, p38 inhi-bition reduced IFN-� production by in vitro-primed Th1 cells inresponse to peptide-loaded APC or PB anti-CD3/CD28 (data notshown).

p38 is dispensable for IL-2 production

The p38 inhibitor failed to diminish TCR- or P�I-mediated IL-2production by in vitro-primed Th1 cells (Fig. 3C). A similar failureof inhibition of IL-2 production by in vivo-generated Th1 cells andin vitro-primed Th2 cells in response to TCR-mediated stimuli wasobserved (data not shown). Thus, p38 is required for optimal IL-4and 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 highlyconserved Thr-Gly-Tyr motif of the activation loop. To check theability of ionomycin to induce p38 phosphorylation, we stainedTh2 cells with an Ab that recognizes p38 only when it is duallyphosphorylated. Cells stimulated with ionomycin or P�I for 15min showed an equal increase in staining intensity, involving theentire population of stimulated cells (Fig. 4A). PMA alone hadlittle or no effect on p38 phosphorylation. Ionomycin-induced p38phosphorylation in Th1 cells was similarly observed (Fig. 4A).

Ionomycin-induced p38 phosphorylation was dependent on cal-cium. Pretreatment of the cells with extracellular calcium chelator,EGTA, completely abolished p38 phosphorylation (Fig. 4B). Iono-mycin-induced p38 phosphorylation was also inhibited by cyclo-sporine A (CsA) or ascomycin (FK520), a chemical analog of ta-crolimus (FK506), suggesting a linkage between calcineurin andthe p38 pathway. Ionomycin-induced p38 activation was not af-fected by p38 inhibition, consistent with the concept that p38 au-tophosphorylation is mediated through ZAP70, which is bypassedby ionomycin.

In contrast to the rapid induction of phospho-p38 in cells stim-ulated with ionomycin, only a portion of cells showed p38 phos-phorylation in the first 15 min after stimulation with anti-CD3 oranti-CD3/CD28 (Fig. 4C). The proportion of phospho-p38-ex-pressing cells increased with time, reaching essentially 100% by90 min. Cells stimulated with PB anti-CD28 alone showed no p38activation, and anti-CD3/CD28-stimulated cells showed no greaterstaining with anti-phospho-p38 at any time point than did cellstreated with anti-CD3 alone, suggesting that CD28 does not play arole in mediating p38 activation.

To determine whether calcium plays an important role in anti-CD3-stimulated p38 activation, cells were pretreated BAPTA, anextracellular calcium chelator, and BAPTA/AM, an intracellularcalcium chelator. Each dramatically decreased p38 phosphoryla-tion in response to anti-CD3 stimulation (Fig. 4D), implying thatcalcium also plays a critical role in TCR-stimulated p38 activation.Anti-CD3-induced p38 phosphorylation was also inhibited by CsAand FK520. By contrast, anti-CD3-induced ERK phosphorylationwas not inhibited by calcium chelators or by CsA (data not shown).Pretreatment with the p38 inhibitor resulted in partial decrease in

anti-CD3-dependent p38 phosphorylation, which may representthe previously reported p38 autophosphorylation pathway.

p38 phosphorylation was also examined by immunoblotting. Itwas minimal at 15 min, but easily detectable at 30 min after anti-CD3 stimulation, peaked at 45–60 min, and decreased at 90 min(Fig. 4E). p38 phosphorylation was not observed in cells stimu-lated by anti-CD28 at any time.

Immunoblotting was also performed to confirm calcium-medi-ated p38 phosphorylation (Fig. 4F). Phosphorylation was observedin cells stimulated by ionomycin for 20 min and abolished in stim-ulated cells pretreated with EGTA, CsA, or FK520. More p38phosphorylation was observed in cells stimulated by anti-CD3 for

FIGURE 5. ASK1 is the major kinase upstream of p38. A, Ionomycininduces phosphorylation of p38, MKK3/6, and TAK1 in a CsA-sensitivemanner. After pretreatment for 30 min, cells were stimulated with iono-mycin for 20 min and lysed in 1� SDS sample buffer to perform immu-noblotting. B, Ionomycin-induced IL-4 production and p38 phosphoryla-tion show modest decrease in TAK1-deficient Th2 cells. Th2 cells derivedfrom TAK1-floxed splenocytes were retrovirally infected with GFP-Creviral supernatant. They were stimulated to check cytokine production andionomycin-induced p38 phosphorylation. C, Knocking down ASK1 strik-ingly diminishes ionomycin-induced IL-4 production and p38 phosphory-lation. Th2 cells were lentivirally infected with ASK1 shRNA or nonsi-lencing control shRNA viral supernatant. Puromycin-resistant cells weresorted and then divided into three groups: to check cytokine production, tocheck ionomycin-induced p38 phosphorylation, and to check Ask1 mRNAlevel. D, Ionomycin-induced p38 phosphorylation is CaMK independent.Th2 cells were pretreated with KN-93 (5 �M) or KN-92 (5 �M) for 30 minand then challenged to detect ionomycin-induced p38 phosphorylation. Theexperiments were conducted at least twice.

3988 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

40 min. Such phosphorylation was partially decreased in cells pre-treated with SB203580, almost completely abolished in the cellspretreated with CsA or FK520, and partially diminished by pre-treatment with calcium chelators BAPTA or BAPTA/AM. Thus,intracellular staining and immunoblotting correlated well, and bothshowed that calcium plays an important role for both ionomycinand anti-CD3-induced p38 phosphorylation.

ASK1 is the upstream kinase of p38

To understand the upstream signaling pathway that leads to[Ca2�]-dependent p38 activation, we tested the phosphorylation ofupstream MAP kinases. Immunoblotting showed that ionomycincaused phosphorylation of MKK3/6 (map2k3/6) and TGF-�-acti-vated kinase 1 (TAK1, map3k7), which was diminished in thepresence of CsA (Fig. 5A). However, retrovirally introducing GFP-Cre into Th2 cells derived from TAK1-floxed mice (15) resulted inonly a subtle decrease in IL-4 production in response to P�I orionomycin alone (Fig. 5B). TAK1-deficient (GFP�) cells demon-strated the same degree of ionomycin-induced p38 phosphoryla-tion as did controls (GFP�, TAK1fl/fl) cells (Fig. 5B). Thus, despiteits ionomycin-induced phosphorylation, TAK1 is not responsiblefor p38 phosphorylation in ionomycin-treated cells.

ASK1 (map3k5), another member of the MKKK family, acti-vates both the JNK and p38 pathways through Map2k3/4/6. Ofthree ASK1 shRNAs introduced into Th2 cells, two caused dim-inution in both ASK1 and in ionomycin-induced p38 phosphory-lation and IL-4 production. Construct 2 diminished Ask1 mRNAby 70%, almost completely inhibited ionomycin-induced p38phosphorylation, and diminished ionomycin-induced IL-4 produc-tion by �60%. Construct 1 was less effective for all three param-

eters, and construct 3 and the nonsilencing control were ineffective(Fig. 5C).

The CaMK inhibitor KN-93 and its inactive analog KN-92failed to inhibit ionomycin-induced p38 phosphorylation (Fig.5D), suggesting that calcium activation of ASK1 is mediatedthrough calcineurin rather than through CaMK. We conclude thatASK1 is a major upstream kinase of p38 phosphorylation in theionomycin-activated pathway.

p38 stabilizes Il4 mRNA

Many cytokine mRNAs are under tight stability regulation throughproteins that bind to adenylate/uridylate (AU)-rich elements(AREs) in their 3� untranslated regions (UTR) (18). Ionomycincould induce IL-4 through message stabilization, new transcrip-tion, or both. Th2 cells heterozygous for an Il4 allele in which Gfphas replaced the first exon and a portion of the first intron of Il4were analyzed to take advantage of the absence of AREs in the Gfp3�UTR (19) and of their presence in the Il4 3�UTR. The half-lifeof Il4 mRNA induced by TCR was 27 min in the presence of thep38 inhibitor and 145 min in its absence (Fig. 6A). The half-life ofIl4 mRNA induced by ionomycin was 4 min in the presence of thep38 inhibitor and 55 min in its absence (Fig. 6B). TCR- and iono-mycin-induced Gfp mRNA were stable in actinomycin D-treatedcells regardless of whether p38 inhibitor was added. Thus, onemechanism through which p38 controls IL-4 production is bymRNA stabilization. Il4 mRNA half-life is longer in cells stimu-lated with TCR than that in cells stimulated with ionomycin alone.This could reflect two possibilities: PB anti-CD3/CD28 activatesother pathway(s) that contribute to Il4 mRNA stabilization, or PB

)tnuoma

AN

Rm 4lI(nl

)tnuoma

AN

Rm pf

G(nl

1

2

3

456

7

8

9

0

1

2

3

4

5

6

7

82/3DC-itna

rh5.1

actinomycin Dactinomycin D+p38 inhibitorA

T1/2=145min

T1/2=27min

020

40

60

90

21U

additional (min)

82/3DC-itna

rh5.1

02

04

06

09

02

1Uadditional (min)

0

0.2

0.4

0.6

0.8

1

1.2 Gfp Il4level A

NR

m evit al er U ”I“ 83p+”I“rotibihni

C

U ”I“ 83p+”I“rotibihni

0

1

2

3

4

5

6

0.0

0.5

1.0

1.5

2.0

2.5

BT1/2=55min

T1/2=4min

)tnuoma

AN

Rm 4lI(nl

)tnuoma

AN

Rm pf

G(nl rh2 ”I“

525

40

75

90

21

U

additional (min)

rh2 ”I“

525

40

75

90

21

U

additional (min)

actinomycin Dactinomycin D+p38 inhibitor

MEK, JNK and p38 inhibitorsMEK, JNK inhibitorsp38 inhibitormockD

anti-CD3/28 stimulation (hrs)

AN

Rm pf

G evi ta lertnuo

ma

0.0

5.0

10.0

15.0

20.0

0 1 2 3 4

U I I+p38

inhib

itorE

FIGURE 6. p38 controls IL-4 production by promoting Il4 transcription and mRNA stability. A and B, p38 extends Il4, but not Gfp, mRNA half-life.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 for 2 h (B). ActinomycinD (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-lifewas calculated by linear regression analysis of a plot of natural log Il4 (Gfp) mRNA (in arbitrary units) against time. C and D, p38 activation promotesIl4 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) forindicated 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 andEMSA was performed. The experiments were conducted three times.

3989The Journal of Immunology

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

anti-CD3/CD28 is more effective than is ionomycin alone in acti-vating p38 and is less completely blocked by the p38 inhibitor.

p38 plays a role in promoting Il4 transcription

The absence of an ARE in the Gfp 3�UTR is associated with sta-bility of Gfp mRNA. Thus, the degree of Gfp mRNA induction byionomycin and its sensitivity to p38 inhibition is a measure ofionomycin-induced transcription and of whether p38 plays a rolein such transcription. p38 inhibitor pretreatment caused a 75% in-hibition of Gfp mRNA levels and an 80% inhibition of Il4 mRNA(Fig. 6C). Thus, we conclude that p38 plays an important role inGfp and, presumably, Il4 transcription.

In TCR-induced Il4/Gfp heterozygous Th2 cells, the p38 inhibitorled to a �50% diminution in Gfp mRNA induction at 4 h but to lessinhibition at 2 and 3 h after stimulation. Although cells pretreated withthe MEK and JNK inhibitors showed no diminution of Gfp mRNAupon stimulation, pretreatment with all three MAPK pathways inhib-itors decreased Gfp mRNA by 75% at all time points (Fig. 6D). Thisimplies that p38 plays an important role in promoting Il4 gene tran-scription in response to TCR engagement that is underestimated since

its absence is compensated for by other MAPKs activated by PBanti-CD3/CD28.

p38 has been reported to have a stimulatory effect on NFATactivation (20). An EMSA using the NFAT binding site in the Il4promoter as a probe was performed. The capacity of NFAT to bindto the Il4 promoter in ionomycin-stimulated cells was unchangedin the presence of a p38 inhibitor (Fig. 6E). Thus, p38 function inIl4 transcription cannot be accounted for by enhanced activationof NFAT.

Ionomycin-induced Il4 transcription requires CaMKIV

Although KN-93 failed to prevent ionomycin-induced p38 phos-phorylation (Fig. 5D), it strikingly diminished ionomycin-inducedIL-4 production (Fig. 7A). Knocking down CaMK proteins by len-tiviral shRNAs specific for CaMKIV but not CaMKII diminishedionomycin-induced IL-4 production, implying that CaMKIV is themajor CaMK involved in the ionomycin pathway (Fig. 7B). Con-sistent with the results from the CaMK inhibitor (Fig. 5D), iono-mycin-induced p38 phosphorylation remained normal in CaMKIVshRNA-infected cells (data not shown).

37 8.339.6mock KN-92 KN-93

CaMK inhibitor

IL-4

CD4

32.5 39.2 37.8 36.3 35.3

10.8 31.5 29.6 21.7 20.5

38.1

CaMKII shRNA constructsnonsilencing control

IL-4

CD4

A

B

C

0

0.4

0.8

1.2

gnicnelisnon

lortnoc

gnicnelisnon

lortnoc

1 2 3 4 5 1 2 3 4 5 CaMKIV shRNA

constructsCaMKII shRNA

constructs

CaMKII mRNA CaMKIV mRNAAN

Rm evitaler

tnuoma

U I 83p+I29NK+I

39NK+I

AN

Rm evitaler

tnuoma 0

4

8

12

16

20

0

4

8

12

16

20

0

1

2

3

0

1

2

3fosB fosl2 fosl1 junB

U I 83p+I29NK+I

39NK+IU I 83p+I

29NK+I

39NK+IU I 83p+I

29NK+I

39NK+I

CaMKIV shRNA constructs

I

I

00.20.40.60.811.2

U I 83p+I29NK+I

39NK+I

AP

-1 E

LIS

A

D

FIGURE 7. CaMKIV and p38 induce AP-1 familygene transcription. A, A CaMK inhibitor markedly di-minishes ionomycin-induced IL-4 production. Tworound-primed Th2 cells were pretreated for 30 min andthen stimulated to check ionomycin-induced IL-4 pro-duction. B, CaMKIV but not CaMKII is involved inionomycin-induced IL-4 production. Th2 cells were len-tivirally infected with the indicated shRNA viral super-natant. Puromycin-resistant cells were sorted and thenstimulated with ionomycin for 4 h to check IL-4 pro-duction, or collected to check Camk2 or Camk4 mRNAlevel. The mRNA amount in nonsilencing controlshRNA infected cells was set as “1.” C, fosB and fosl2,but not fosl1 or junB, mRNA are induced by ionomycinand significantly decreased in the presence of p38 in-hibitor and CaMK inhibitor. Th2 cells were pretreatedwith indicated inhibitors for 30 min and then stimulatedwith ionomycin for 25 min. mRNA in unstimulated cellswas set as “1.” D, Th2 cells were pretreated with indi-cated inhibitors for 30 min and then stimulated withionomycin for 50 min. Nuclear extracts were preparedand AP-1 binding was performed by using an ELISA-based AP-1 family transcription factor assay kit. Theexperiments were conducted at least twice.

3990 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

CaMKIV contributes to cytokine production by inducing CREBphosphorylation and thereafter, transcription of the CREB-depen-dent immediate-early genes, including fosB, fosl2, and junB (21,22). Similarly, p38 phosphorylation of CREB and induction ofAP-1 transcription has been reported (21). Indeed, fosB and fosl2mRNA levels were increased �15-fold by ionomycin and signif-icantly decreased in cells pretreated with CaMK inhibitors or p38inhibitor (Fig. 7C). Fosl1 and junB were only modestly induced byionomycin. Ionomycin induced increased AP-1 binding activitythat was blocked by a p38 inhibitor and by the CaMK inhibitor(Fig. 7D). Thus, p38 and CaMKIV control the amount and activityof AP-1 family transcription factors. This, together with NFAT,explains ionomycin-induced Il4 transcription. Together with iono-mycin-induced p38 prolongation of Il4 mRNA half-life, this ex-plains the increase in Il4 mRNA (Fig. 8).

DiscussionFull activation of T cells requires signaling through the TCR/CD3complex and the CD28 costimulatory receptor. P�I are often uti-lized to bypass TCR stimulation, through the action of PMA onPKC� and the capacity of ionomycin to elevate [Ca2�]i. Unex-pectedly, we found that ex vivo CD44brightCD4 T cells isolatedfrom either S. mansoni- or H. polygyrus-infected mice producedsimilar amounts of IL-4 when stimulated with ionomycin alone orwith P�I. Both ionomycin and a second Ca2� elevating agent, TG,induced IL-4 production from in vitro-primed Th2 cells. Ionomy-cin also induced IFN-� production by CD44brightCD4 T cells fromT. gondii-infected mice and in vitro-primed Th1 cells. Ionomycinalone has been recently reported to induce IL-21 production inpreactivated T cells (23). By contrast, as has been previously re-ported (24), IL-2 was not produced when these cells were stimu-lated with ionomycin, although it was produced in response tocognate peptide-loaded APC, anti-CD3/CD28, or P�I.

Ionomycin activates calcineurin, which in turn dephosphorylatesNFAT, causing the latter to enter the nucleus and promote genetranscription. Our data demonstrate that blocking p38 activity byinhibitors or diminishing p38 expression by RNA interferencestrikingly inhibited ionomycin-induced IL-4 production, althoughthe capacity of NFAT to bind to the Il4 promoter remained normal.Thus, ionomycin-induced NFAT translocation alone is not suffi-cient for IL-4 production; p38 activity is essential. p38 mediates its

function in ionomycin-induced IL-4 production through a mecha-nism other than modification of NFAT binding to Il4 promoter.

We observed that ionomycin induces p38 phosphorylation inTh2 and Th1 cells. Intracellular and extracellular calcium chelatorscompletely abolished p38 phosphorylation induced by ionomycinand greatly decreased phosphorylation in response to PB anti-CD3,indicating that calcium plays an important role in p38 activationeven when cells were stimulated through TCR. Ionomycin-inducedp38 activation was not affected by the p38 inhibitor SB203580,indicating that this calcium-mediated p38 activation represents aseparate pathway from the ZAP70-mediated p38 autophosphory-lation pathway (9). CsA and FK520 completely abolish ionomy-cin-induced p38 phosphorylation and partially blocked p38 phos-phorylation in response to anti-CD3. These results indicate thatcalcineurin activation is required for ionomycin-induced p38 phos-phorylation and contributes to TCR-induced p38 phosphorylation.

Ca2� elevation has been reported to activate the MAPK cascade(25–28). Several MKKKs, such as TAK1 and ASK1, can be ac-tivated by elevated [Ca2�]i and in turn activate the classicalMAPK cascade, leading to p38 phosphorylation (27, 29). The in-volvement of calcineurin in MAPK activation in Jurkat cells wassuggested by the finding that JNK activation by PMA plus A23187was blocked by CsA (30).

Calcineurin-like molecules have been reported to interact withthe MAPK analog. In Caenorhabditis elegans, KIN-29, a Ser/Thrkinase, binds to the calcineurin-like phosphatase Tax6 (31). InArabidopsis, calcineurin B-like proteins (CBLPs) are reported tobind to CBL-interacting Ser/Thr kinases (AtCIPKs) in a calcium-dependent manner (32). Mammalian kinases corresponding to At-CIPKs include Raf-1. Raf-1 possesses an autoinhibitory site that isgenerally phosphorylated in quiescent cells. Activation of Raf-1involves dephosphorylation of this autoinhibitory site (33).

Among the kinases upstream of p38 in the classical MAPKpathway, ASK1 has been reported to be activated by calcium sig-nal in primary neurons and synaptosomes (27, 34) and has a well-conserved autoinhibitory site (35), suggesting that it might be atarget of calcineurin. It has been reported that ASK1 activity isregulated through this autoinhibitory site (36, 37). In a yeast two-hybrid screen, the calcium-binding subunit B of calcineurin andASK1 were found to be direct physical partners (38). Furthermore,

FIGURE 8. Three pathways involved in ionomycin-induced cytokine production. Calcium activates threepathways that contribute to IL-4 induction: to dephos-phorylate NFAT through calcineurin; to activate ASK1,likely through calcineurin, leading to p38 activation;and to induce AP-1 protein synthesis through CaM andCaMKIV. Activated p38 may induce AP-1 productionand phosphorylation. Cooperation between AP-1 andNFAT leads to Il4 gene transcription and activated p38also exerts a role in stabilization of Il4 mRNA half-life.

3991The Journal of Immunology

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

in an in vitro phosphatase assay, calcineurin directly dephospho-rylated ASK1, resulting in its activation. In cardiomyocytes, theendogenous subunit B of calcineurin could be coimmunoprecipi-tated with ASK1, suggesting physiological importance of theirinteraction.

Herein, we showed that knocking down ASK1 resulted in di-minished ionomycin-induced p38 phosphorylation and IL-4 pro-duction. By contrast, ionomycin-induced p38 phosphorylation andIL-4 production were not impaired in TAK1-deficient cells, al-though TAK1 was phosphorylated in ionomycin-stimulated cells.We conclude that ASK1 is the major kinase upstream of p38 in theionomycin-stimulated cells. Inhibiting or knocking down CaMKsdid not affect ionomycin-induced p38 phosphorylation. Thus, iono-mycin does not activate ASK1 through CaMK. Instead, it is likelythat elevated [Ca2�] activates calcineurin, which in turn dephos-phorylates the autoinhibitory site of ASK1 and leads to itsactivation.

The importance of p38 for effector cytokine production is stillcontroversial. IL-4 production from Th2 cells has been reported tobe impaired by a p38 inhibitor (39). However, in another report,p38 phosphorylation was described to be selectively induced byCon A in Th1 cells but not in Th2 cells (40). Our data showed thatp38 is essential for both IL-4 and IFN-� production in response toionomycin and for optimal production in response to anti-CD3 andanti-CD28. Since Con A, like anti-CD3 and anti-CD28, can induceIL-4 by both p38-dependent and independent pathways, the degreeof priming could have had a major impact on the relative use ofthese pathways and may account for the difference between ourresults and those reported by Rincon et al. (40). More recently,Berenson and colleagues using p38��/� and p38��/� CD4 T cellsprepared by RAG2�/� blastocyst complementation, emphasizedthat p38 is required for IL-12/IL-18-stimulated IFN-� productionby Th1 cells (41). However, as the authors pointed out in thediscussion, their data indicated that TCR-stimulated IFN-� pro-duction was also partially inhibited, as judged from the reductionof MFI of IFN-�-expressing cells. Thus, the importance of p38 instimulation of cytokine production may be easily underestimated.

The physiological substrates of p38 MAPK in T cells have notbeen identified. MAPK-activated protein kinase 2 (MAPKAPK2or MK2), the major kinase downstream of p38, has been shown tocause stabilization of many cytokine mRNAs including Il2, Il3,Il6, Il8, Tnf, Inf�, and Gmcsf (42). Herein, we have shown that p38plays an important role lengthening the half-life of Il4 mRNA instimulated Th2 cells. The p38 inhibitor did not destabilize GfpmRNA derived from a gene in which Gfp is knocked into the Il4locus, consistent with the absence of AREs in the Gfp 3�UTR.However, the striking increase in Gfp mRNA in ionomycin-stim-ulated Th2 cells from Il4/Gfp heterozygous donors implies thationomycin’s effect involves substantial induction of transcription.Furthermore, since Gfp induction is also blocked by the p38 inhibitor,it must be concluded that p38 regulates IL-4 production in responseto ionomycin both by a striking induction of transcription as wellas by mRNA stabilization.

Inhibiting or knocking down CaMKs greatly diminished iono-mycin-induced IL-4 production, although p38 activation in re-sponse to ionomycin remained normal. fosl2 and fosB, but notfosl1 and junB, mRNA were induced by ionomycin; p38 andCaMK inhibitors blocked the induction. AP-1 is regulated by phos-phorylation as well as by induction (43); p38 has been reported tomediate such phosphorylation (44). Thus, calcium activates at leastthree pathways that contribute to IL-4 induction: through cal-cineurin to dephosphorylate NFAT; likely through calcineurin toactivate ASK1, leading to p38 activation; through CaM andCaMKIV to induce AP-1 protein synthesis; and activated p38 may

induce AP-1 production and phosphorylation (Fig. 8). Cooperationbetween AP-1 and NFAT leads to Il4 gene transcription, and ac-tivated p38 also exerts a role in stabilization of Il4 mRNA half-life.

AP-1 and NFAT are also critical for Il2 transcription; however,IL-2 production was not detected in Th1 or Th2 cells. This can beaccounted for by the requirement for additional factors, not in-duced by ionomycin, for Il2 transcription. Indeed, the CD28 re-sponse element (CD28RE) is essential for Il2 transcription, andc-Rel homodimers up-regulated in response to CD28 signalingbind to the Il2 CD28RE (45–47). c-Rel-deficient cells showed astriking defect in IL-2 production (48). When cells are stimulatedthrough TCRs, p65/p50 dimers also interact with CD28RE in theIl2 promoter and act as potent transactivators (49). In resting cellsand ionomycin-stimulated CD4T cells, p50/p50 homodimers, po-tent repressors of Il2 transcription, bind to Il2 promoter (24, 50,51). Thus, the absence of c-Rel or p65/p50 in ionomycin-stimu-lated CD4 T cells explains the lack of Il2 transcription.

Do physiologic ligands that lead to robust calcium elevationcause direct effector T cell cytokine production in tissues, or dophysiologic ligands that can activate p38 in Th2 or Th1 cells en-hance the minimal cytokine production that would have occurredin response to limiting amounts of Ag/APC? Such activation mightbe particularly important in secondary responses to pathogens toensure rapid cytokine production even before substantial amountsof Ag are released during infection. Thus, the p38 effect mightconfer on Th cells a type of innate-immune status. The nature ofthe ligands that would act to elevate [Ca2�]i in effector cells res-ident in tissues remains to be determined, but many candidatescould be considered, particularly from the G protein-coupled re-ceptor family of molecules, which would include chemokines.

AcknowledgmentsWe thank Dragana Jankovic (Laboratory of Parasitic Diseases, NationalInstitute of Allergy and Infectious Diseases (NIAID), National Institutes ofHealth) for preparing cells from S. mansoni-infected mice and T. gondii-infected mice, Richard Flavell for Tak1-floxed cells, Sarah Tanksley (Labof Immunology, NIAID) for cell sorting, Ludmila Jirmanova for the de-tailed immunoblotting protocol to check p38 phosphorylation, Shirley Star-nes for editorial assistance, and Jonathan Ashwell, Ludmila Jirmanova,Warren Leonard, and Arthur Weiss for helpful discussions and criticalreviews of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

References1. Call, M. E., and K. W. Wucherpfennig. 2005. The T cell receptor: critical role of

the membrane environment in receptor assembly and function. Annu. Rev. Im-munol. 23: 101–125.

2. Takahama, Y., and H. Nakauchi. 1996. Phorbol ester and calcium ionophore canreplace TCR signals that induce positive selection of CD4 T cells. J. Immunol.157: 1508–1513.

3. Lewis, R. S. 2001. Calcium signaling mechanisms in T lymphocytes. Annu. Rev.Immunol. 19: 497–521.

4. Macian, F. 2005. NFAT proteins: key regulators of T-cell development and func-tion. Nat. Rev. Immunol. 5: 472–484.

5. Rooney, J. W., T. Hoey, and L. H. Glimcher. 1995. Coordinate and cooperativeroles for NF-AT and AP-1 in the regulation of the murine IL-4 gene. Immunity2: 473–483.

6. Isakov, N., and A. Altman. 2002. Protein kinase C(�) in T cell activation. Annu.Rev. Immunol. 20: 761–794.

7. Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immuneresponse. Annu. Rev. Immunol. 20: 55–72.

8. Ashwell, J. D. 2006. The many paths to p38 mitogen-activated protein kinaseactivation in the immune system. Nat. Rev. Immunol. 6: 532–540.

9. Salvador, J. M., P. R. Mittelstadt, T. Guszczynski, T. D. Copeland,H. Yamaguchi, E. Appella, A. J. Fornace, Jr., and J. D. Ashwell. 2005. Alterna-tive p38 activation pathway mediated by T cell receptor-proximal tyrosine ki-nases. Nat. Immunol. 6: 390–395.

10. Grunvald, E., M. Chiaramonte, S. Hieny, M. Wysocka, G. Trinchieri,S. N. Vogel, R. T. Gazzinelli, and A. Sher. 1996. Biochemical characterization

3992 CALCIUM-DEPENDENT p38 PHOSPHORYLATION AND CYTOKINE PRODUCTION

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

and protein kinase C dependency of monokine-inducing activities of Toxoplasmagondii. Infect. Immun. 64: 2010–2018.

11. Jankovic, D., T. A. Wynn, M. C. Kullberg, S. Hieny, P. Caspar, S. James,A. W. Cheever, and A. Sher. 1999. Optimal vaccination against Schistosomamansoni requires the induction of both B cell- and IFN-�-dependent effectormechanisms. J. Immunol. 162: 345–351.

12. Urban, J. F., Jr., I. M. Katona, and F. D. Finkelman. 1991. Heligmosomoidespolygyrus: CD4� but not CD8� T cells regulate the IgE response and protectiveimmunity in mice. Exp. Parasitol. 73: 500–511.

13. Guo, L., J. Hu-Li, J. Zhu, C. J. Watson, M. J. Difilippantonio, C. Pannetier, andW. E. Paul. 2002. In TH2 cells the Il4 gene has a series of accessibility statesassociated with distinctive probabilities of IL-4 production. Proc. Natl. Acad. Sci.USA 99: 10623–10628.

14. Zhu, J., B. Min, J. Hu-Li, C. J. Watson, A. Grinberg, Q. Wang, N. Killeen,J. F. Urban, Jr., L. Guo, and W. E. Paul. 2004. Conditional deletion of Gata3shows its essential function in TH1-TH2 responses. Nat. Immunol. 5: 1157–1165.

15. Wan, Y. Y., H. Chi, M. Xie, M. D. Schneider, and R. A. Flavell. 2006. The kinaseTAK1 integrates antigen and cytokine receptor signaling for T cell development,survival and function. Nat. Immunol. 7: 851–858.

16. Lenardo, M. J., C. M. Fan, T. Maniatis, and D. Baltimore. 1989. The involvementof NF-�B in �-interferon gene regulation reveals its role as widely induciblemediator of signal transduction. Cell 57: 287–294.

17. Szabo, S. J., J. S. Gold, T. L. Murphy, and K. M. Murphy. 1993. Identificationof cis-acting regulatory elements controlling interleukin-4 gene expression in Tcells: roles for NF-Y and NF-ATc. Mol. Cell. Biol. 13: 4793–4805.

18. Dean, J. L., G. Sully, A. R. Clark, and J. Saklatvala. 2004. The involvement ofAU-rich element-binding proteins in p38 mitogen-activated protein kinase path-way-mediated mRNA stabilisation. Cell. Signal. 16: 1113–1121.

19. Hu-Li, J., C. Pannetier, L. Guo, M. Lohning, H. Gu, C. Watson, M. Assenmacher,A. Radbruch, and W. E. Paul. 2001. Regulation of expression of IL-4 alleles:analysis using a chimeric GFP/IL-4 gene. Immunity 14: 1–11.

20. Wu, C. C., S. C. Hsu, H. M. Shih, and M. Z. Lai. 2003. Nuclear factor of activatedT cells c is a target of p38 mitogen-activated protein kinase in T cells. Mol. Cell.Biol. 23: 6442–6454.

21. Mayr, B., and M. Montminy. 2001. Transcriptional regulation by the phospho-rylation-dependent factor CREB. Nat. Rev. Mol. Cell Biol. 2: 599–609.

22. Anderson, K. A., and A. R. Means. 2002. Defective signaling in a subpopulationof CD4� T cells in the absence of Ca2�/calmodulin-dependent protein kinase IV.Mol. Cell. Biol. 22: 23–29.

23. Kim, H. P., L. L. Korn, A. M. Gamero, and W. J. Leonard. 2005. Calcium-dependent activation of interleukin-21 gene expression in T cells. J. Biol. Chem.280: 25291–25297.

24. Macian, F., F. Garcia-Cozar, S. H. Im, H. F. Horton, M. C. Byrne, and A. Rao.2002. Transcriptional mechanisms underlying lymphocyte tolerance. Cell 109:719–731.

25. Graves, J. D., K. E. Draves, A. Craxton, J. Saklatvala, E. G. Krebs, andE. A. Clark. 1996. Involvement of stress-activated protein kinase and p38 mito-gen-activated protein kinase in mIgM-induced apoptosis of human B lympho-cytes. Proc. Natl. Acad. Sci. USA 93: 13814–13818.

26. Elzi, D. J., A. J. Bjornsen, T. MacKenzie, T. H. Wyman, and C. C. Silliman.2001. Ionomycin causes activation of p38 and p42/44 mitogen-activated proteinkinases in human neutrophils. Am. J. Physiol. 281: C350–C360.

27. Takeda, K., A. Matsuzawa, H. Nishitoh, K. Tobiume, S. Kishida,J. Ninomiya-Tsuji, K. Matsumoto, and H. Ichijo. 2004. Involvement of ASK1 inCa2�-induced p38 MAP kinase activation. EMBO Rep. 5: 161–166.

28. Funaba, M., T. Ikeda, K. Ogawa, and M. Abe. 2003. Calcium-regulated expres-sion of activin A in RBL-2H3 mast cells. Cell. Signal. 15: 605–613.

29. Ishitani, T., S. Kishida, J. Hyodo-Miura, N. Ueno, J. Yasuda, M. Waterman,H. Shibuya, R. T. Moon, J. Ninomiya-Tsuji, and K. Matsumoto. 2003. TheTAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca2� pathway to antagonize Wnt/�-catenin signaling. Mol. Cell. Biol. 23:131–139.

30. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, and Y. Ben-Neriah. 1994. JNKis involved in signal integration during costimulation of T lymphocytes. Cell 77:727–736.

31. Singaravelu, G., H. O. Song, Y. J. Ji, C. Jee, B. J. Park, and J. Ahnn. 2007.Calcineurin interacts with KIN-29, a Ser/Thr kinase, in Caenorhabditis elegans.Biochem. Biophys. Res. Commun. 352: 29–35.

32. Luan, S. 2003. Protein phosphatases in plants. Annu. Rev. Plant Biol. 54: 63–92.33. Muslin, A. J., J. W. Tanner, P. M. Allen, and A. S. Shaw. 1996. Interaction of

14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.Cell 84: 889–897.

34. Nishitoh, H., A. Matsuzawa, K. Tobiume, K. Saegusa, K. Takeda, K. Inoue,S. Hori, A. Kakizuka, and H. Ichijo. 2002. ASK1 is essential for endoplasmicreticulum stress-induced neuronal cell death triggered by expanded polyglu-tamine repeats. Genes Dev. 16: 1345–1355.

35. Tobiume, K., T. Inage, K. Takeda, S. Enomoto, K. Miyazono, and H. Ichijo.1997. Molecular cloning and characterization of the mouse apoptosis signal-regulating kinase 1. Biochem. Biophys. Res. Commun. 239: 905–910.

36. Zhang, L., J. Chen, and H. Fu. 1999. Suppression of apoptosis signal-regulatingkinase 1-induced cell death by 14-3-3 proteins. Proc. Natl. Acad. Sci. USA 96:8511–8515.

37. Goldman, E. H., L. Chen, and H. Fu. 2004. Activation of apoptosis signal-reg-ulating kinase 1 by reactive oxygen species through dephosphorylation at serine967 and 14-3-3 dissociation. J. Biol. Chem. 279: 10442–10449.

38. Liu, Q., B. J. Wilkins, Y. J. Lee, H. Ichijo, and J. D. Molkentin. 2006. Directinteraction and reciprocal regulation between ASK1 and calcineurin-NFAT con-trol cardiomyocyte death and growth. Mol. Cell. Biol. 26: 3785–3797.

39. Dodeller, F., A. Skapenko, J. R. Kalden, P. E. Lipsky, and H. Schulze-Koops.2005. The p38 mitogen-activated protein kinase regulates effector functions ofprimary human CD4 T cells. Eur. J. Immunol. 35: 3631–3642.

40. Rincon, M., H. Enslen, J. Raingeaud, M. Recht, T. Zapton, M. S. Su, L. A. Penix,R. J. Davis, and R. A. Flavell. 1998. Interferon-� expression by Th1 effector Tcells mediated by the p38 MAP kinase signaling pathway. EMBO J. 17:2817–2829.

41. Berenson, L. S., J. Yang, B. P. Sleckman, T. L. Murphy, and K. M. Murphy.2006. Selective requirement of p38� MAPK in cytokine-dependent, but not an-tigen receptor-dependent, Th1 responses. J. Immunol. 176: 4616–4621.

42. Winzen, R., M. Kracht, B. Ritter, A. Wilhelm, C. Y. Chen, A. B. Shyu,M. Muller, M. Gaestel, K. Resch, and H. Holtmann. 1999. The p38 MAP kinasepathway signals for cytokine-induced mRNA stabilization via MAP kinase-acti-vated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 18:4969–4980.

43. Pulverer, B. J., J. M. Kyriakis, J. Avruch, E. Nikolakaki, and J. R. Woodgett.1991. Phosphorylation of c-jun mediated by MAP kinases. Nature 353: 670–674.

44. Derijard, B., J. Raingeaud, T. Barrett, I. H. Wu, J. Han, R. J. Ulevitch, andR. J. Davis. 1995. Independent human MAP-kinase signal transduction pathwaysdefined by MEK and MKK isoforms. Science 267: 682–685.

45. Fraser, J. D., B. A. Irving, G. R. Crabtree, and A. Weiss. 1991. Regulation ofinterleukin-2 gene enhancer activity by the T cell accessory molecule CD28.Science 251: 313–316.

46. Huang, D. B., Y. Q. Chen, M. Ruetsche, C. B. Phelps, and G. Ghosh. 2001. X-raycrystal structure of proto-oncogene product c-Rel bound to the CD28 responseelement of IL-2. Structure 9: 669–678.

47. Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF-�B and Rel proteins: evolu-tionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225–260.

48. Kontgen, F., R. J. Grumont, A. Strasser, D. Metcalf, R. Li, D. Tarlinton, andS. Gerondakis. 1995. Mice lacking the c-rel proto-oncogene exhibit defects inlymphocyte proliferation, humoral immunity, and interleukin-2 expression.Genes Dev. 9: 1965–1977.

49. Ghosh, P., T. H. Tan, N. R. Rice, A. Sica, and H. A. Young. 1993. The interleukin2 CD28-responsive complex contains at least three members of the NF�B family:c-Rel, p50, and p65. Proc. Natl. Acad. Sci. USA 90: 1696–1700.

50. Kang, S. M., A. C. Tran, M. Grilli, and M. J. Lenardo. 1992. NF-�B subunitregulation in nontransformed CD4� T lymphocytes. Science 256: 1452–1456.

51. Zhong, H., M. J. May, E. Jimi, and S. Ghosh. 2002. The phosphorylation statusof nuclear NF-�B determines its association with CBP/p300 or HDAC-1. Mol.Cell. 9: 625–636.

3993The Journal of Immunology

by guest on September 14, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from