The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Berenson, L. S.
Right arrow Articles by Murphy, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berenson, L. S.
Right arrow Articles by Murphy, K. M.
The Journal of Immunology, 2006, 176: 4616-4621.
Copyright © 2006 by The American Association of Immunologists

Selective Requirement of p38{alpha} MAPK in Cytokine-Dependent, but Not Antigen Receptor-Dependent, Th1 Responses1

Lisa S. Berenson*, Jianfei Yang2,*,{dagger}, Barry P. Sleckman*, Theresa L. Murphy* and Kenneth M. Murphy3,*,{dagger}

* Department of Pathology and Center for Immunology, and {dagger} Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of the p38 MAPK pathway in Th1 development has been controversial, because indirect manipulations of either upstream p38 activators or modifiers of p38 activity have had variable effects on IFN-{gamma} production in CD4+ T cells. Uncertainties regarding the specificity of pharmacologic inhibition or p38 dominant negative mutants diminish the strength of conclusions about the role of the p38{alpha} isoform in Th1 development. Also, the effects of some upstream p38 activators, such as MAPK kinase 3, on Th1 development are not as strong as the effects of other manipulations, such as the expression of a dominant negative p38 mutant. Finally, embryonic lethality has prevented a direct examination of p38{alpha}-deficient T cells. To test the requirement for p38{alpha} in Th1 differentiation, we generated Ag-specific p38{alpha}+/– and p38{alpha}–/– CD4+ T cells using RAG2–/– blastocyst complementation and retroviral expression of the DO11.10 TCR. IFN-{gamma} production in response to TCR signaling is normal in p38{alpha}–/– T cells cultured in Th1 conditions, implying normal Th1 development. However, p38{alpha}–/– Th1 cells have a much greater defect in IFN-{gamma} secretion stimulated by IL-12/IL-18 compared with TCR-induced IFN-{gamma} secretion. These results suggest that the activity of p38{alpha} in Th1 cells is relatively restricted to acting in one of two alternative pathways (i.e., cytokine induced) that can induce the production of IFN-{gamma} in differentiated Th1 cells, but that p38{alpha} is not required for the process of Th1 commitment and development itself.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The differentiation of naive CD4 T cells into Th1 cells involves signaling of IFN-{gamma} and IL-12 and the transcription factors T-bet and STAT4 (1, 2, 3, 4). Differentiated Th1 cells can produce large amounts of IFN-{gamma} in response to at least two distinct stimuli, being induced either by signaling through the TCR or by the combination of cytokines IL-12 and IL-18 (5, 6). These stimuli appear to use independent pathways for inducing IFN-{gamma}, based on the differential action of pharmacological inhibitors. First, treatment of Th1 cells with cyclosporin A (CsA)5 blocks IFN-{gamma} production in response to TCR signaling, but not IL-12/IL-18 stimulation (5). In contrast, treatment of Th1 cells with the p38 MAPK inhibitor SB203580 blocks IFN-{gamma} production in response to IL-12/IL-18 (7, 8, 9), but not TCR stimulation.

Previous studies of p38{alpha} in Th1 development and IFN-{gamma} production have used indirect means of examination. Of the four p38 isoforms, {alpha}, beta, {delta}, and {gamma}, all are expressed in T lymphocytes except p38{gamma} (10). The major isoform in T cells is p38{alpha}, and it has been the focus of previous studies regarding regulation of IFN-{gamma} production (8, 9). p38{alpha} is activated by three upstream MAPK kinases (MKKs), MKK3, MKK4, and MKK6, which are activated by MKK kinase 4 (MEKK4) (11, 12, 13). MEKK4 can be regulated by growth arrest and DNA damage 45 (GADD45)beta and GADD45{gamma}, which have been proposed to act in cytokine-induced and TCR-induced IFN-{gamma} production, respectively (7, 14, 15). A dominant negative p38{alpha} (9), p38-specific inhibitors (7, 8), and murine deficiencies in MKK3 (16), MEKK4 (17), GADD45beta (18), and GADD45{gamma} (15) have suggested a role for p38{alpha} in either IFN-{gamma} production or Th1 development, but have not distinguished between an effect on Th1 differentiation or regulation of one of the pathways leading to acute IFN-{gamma} production. Moreover, an actual requirement for p38{alpha} has not been demonstrated for either Th1 development or IFN-{gamma} production due to the embryonic lethality caused by homozygous p38{alpha} disruption (19, 20, 21, 22).

To test whether p38{alpha} is required for either Th1 development or acute induction of IFN-{gamma}, we used RAG2–/– blastocyst complementation coupled with retroviral expression of {alpha}beta TCR subunits to generate p38{alpha}–/– CD4+ T cells. Our results indicate that p38{alpha} is not required for Th1 development per se or for TCR-induced IFN-{gamma} production, but appears to be largely involved in the ability of differentiated Th1 cells to secrete large amounts of IFN-{gamma} in response to IL-12 and IL-18.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c, DO11.10 transgenic (23), and p38{alpha}+/– and p38{alpha}–/– mice (24) were previously described. All animal studies were approved by the Washington University animal studies committee.

Abs and reagents

PE-conjugated anti-mouse IFN-{gamma}, allophycocyanin-conjugated anti-mouse IFN-{gamma}, PE-Cy5-conjugated anti-mouse CD4, allophycocyanin-conjugated anti-mouse CD4, and purified anti-CD3{epsilon} were purchased from BD Biosciences. PE-conjugated anti-human CD4, Tri-Color-conjugated anti-human CD4, and PE-conjugated KJ1–26 were purchased from Caltag Laboratories.

Cell purifications and activation

Purified splenic CD4+ DO11.10 T cells were stimulated with irradiated BALB/c splenocytes and 0.3 µM OVA. Purified splenic CD4+ p38{alpha}+/– and p38{alpha}–/– T cells were plated onto anti-CD3-coated plates (5 µg/ml) with anti-CD28 (2 µg/ml) at 1 x 106 cells/ml. All cultures received IFN-{gamma} (200 U/ml; gift from R. D. Schreiber, Washington University, St. Louis, MO), anti-IL-4 (11B11 hybridoma supernatant), IL-12 (10 U/ml), and IL-2 (40 U/ml; Takeda). All cells were restimulated on day 7 with irradiated BALB/c splenocytes, 0.3 µM OVA, IL-2, and Th1-inducing cytokines. After 2 wk of Th1 differentiation, cells were assayed for IFN-{gamma} production and passed weekly with irradiated BALB/c splenocytes, 0.3 µM OVA, IL-2, and anti-IL-4.

Retroviral constructs and infection

The {alpha}- and beta-chains of DO11.10 TCR were cloned by PCR from DO11.10 splenic cDNA. DO{alpha}-internal ribosome entry site (IRES)-human (h) CD4-retrovirus (RV) was generated through cloning the DO11.10 {alpha}-chain using the primers Xho-DO{alpha}, 5'-CCG CTC GAG AGA GCA ATG AAA ACA TAC GCT C and 3'-CCG CTC GAG TCA ACT GGA CCA CAG CCT CA, and digestion of the PCR product and IRES-hCD4-RV (25) with XhoI. The beta-chain was cloned by removing the insert from mouse IL-12Rbeta2-IRES-green fluorescence protein (GFP)-RV by BglII and XhoI digestion and subsequent ligation of a similarly digested insert using the primers BglII-DObeta (5'-GAA GAT CTC CCA AGA TGG GCT CCA GGC T) and Xho-DObeta (3'-CCG CTC GAG TCA TGA ATT CTT TCT TTT GAC CAT A) to create DObeta-IRES-GFP-RV. After 36 h of stimulation with anti-CD3, the cells were infected with retroviral supernatant in the presence of polybrene (2 µg/ml) by spin infection. At 60 h, cells were harvested from anti-CD3-coated plates and expanded in the presence of IL-2 (40 U/ml) before a second infection. Cells were passaged weekly as described above and sorted by MoFlo (DakoCytomation) to >85% DO{alpha}-IRES-hCD4+, DObeta-IRES-GFP+ before analysis.

Cytokine quantification

Resting T cells (days 10–12 after stimulation) were stimulated at 1 x 106 cells/ml in 48-well plates for 8 h unless otherwise indicated. Cells were pretreated for 30 min with SB203580 (10 µM) or CsA (100 ng/ml) before addition of cytokines or transfer to anti-CD3 (5 µg/ml)-coated plates. For intracellular cytokine staining (ICS), brefeldin A (1 µg/ml; Epicentre Technology) was added for the final 4 h. For Ag stimulation, 5 x 106 irradiated BALB/c APCs/ml were used with either OVA or hemagglutinin (HA)518–528; when indicated that IL-12 was neutralized, anti-IL-12 (TOSH hybridoma supernatant) was used. ICS and ELISA were performed as previously described (26). For ICS, cells were gated on live CD4+ populations; p38{alpha} lines were also gated on DObeta-IRES-GFP and DO{alpha}-IRES-hCD4 expression.

Polymerase chain reaction

PCR was performed on p38{alpha}+/–, p38{alpha}–/–, and BALB/c genomic DNA using the primers p38-forward (TGA TAA GCA GGT GTT GTG GC), p38-reverse (AGA TTC ACT CAC ACG TCA ATT G), and Neo2 (TAT CAG GAC ATA GCG TTG GC).

Western blotting

Cellular lysates of p38{alpha}+/–, p38{alpha}–/–, and DO11.10 Th1 cells were analyzed by 10% acrylamide gel, blotted with anti-p38 (1/1000; Santa Cruz Biotechnology), and reprobed with anti-actin (1/1000; Santa Cruz Biotechnology). HRP-conjugated goat anti-rabbit (1/50,000; Jackson ImmunoResearch Laboratories) was used as a secondary Ab for both Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
p38 MAPK inhibition selectively reduces cytokine-induced IFN-{gamma} production compared with TCR-induced IFN-{gamma} production in differentiated Th1 cells

We and others have found p38{alpha} to be selectively required for cytokine-induced IFN-{gamma} production in Th1 cells, but not for TCR-induced IFN-{gamma} production (7, 8). Because we previously examined only the effects of p38 inhibitors on IFN-{gamma} secretion as measured by ELISA, we began in this study by comparing IFN-{gamma} production measured by both ELISA and ICS (Fig. 1). The secretion of IFN-{gamma} in DO11.10 Th1 cells induced by IL-12 and IL-18 was inhibited by the p38 inhibitor SB203580, but not by CsA (Fig. 1, A and B, left panels), as found previously (7). Secretion of IFN-{gamma} induced by anti-CD3 stimulation was not inhibited by SB203580, but was strongly inhibited by CsA (Fig. 1), also consistent with our previous analysis based on IFN-{gamma} ELISA. ICS analysis confirmed the selective action of the p38 inhibitor SB203580 on IL-12/IL-18-induced IFN-{gamma} production (Fig. 1B). Treatment of Th1 cells with SB203580 led to a large reduction in the mean fluorescence intensity (MFI) of ICS for IFN-{gamma} induced by IL-12/IL-18. Interestingly, a slight reduction in the MFI of ICS for IFN-{gamma} induced by TCR signaling was observed, but this was less dramatic than the reduction seen with IL-12/IL-18 stimulation and was not associated with a reduction in secreted IFN-{gamma} measured by ELISA.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1. A p38 inhibitor blocks cytokine-induced, but not TCR-induced, IFN-{gamma} production in DO11.10 Th1 cells. DO11.10 Th1 cells were stimulated with either IL-12/IL-18 or anti-CD3 with no inhibitor, SB203580, or CsA. A, Supernatants from the indicated stimulations were used for ELISA analysis. B, ICS was performed. Shaded histograms represent IFN-{gamma} staining of unstimulated cells, and numbers indicate the percentage of cells staining positively for IFN-{gamma}. Data are representative of four experiments.

 
Because SB203580 has a similar IC50 for p38{alpha} or p38beta (27), the results of these and previous studies cannot identify which p38 isoform might be involved. Also, SB203580 blocks cellular proliferation by a non-p38{alpha}-dependent mechanism (8, 24) and thus cannot be used to examine Th1 development, which requires cellular expansion (28). Therefore, we wanted to generate Ag-specific T cells that were either heterozygous for p38{alpha} or deficient in p38{alpha} to allow analysis of the role of p38{alpha} in Th1 development.

Generation of p38{alpha}+/– and p38{alpha}–/– T cell lines expressing DO11.10 TCR

Briefly, p38{alpha}+/– or p38{alpha}–/– CD4+ T cells generated by RAG2–/– blastocyst complementation were stimulated with anti-CD3 and anti-CD28 in vitro for 3 days under Th1-inducing conditions and infected with retroviruses, DO{alpha}-IRES-hCD4 and DObeta-IRES-GFP, expressing the {alpha}- and beta-chains of DO11.10 TCR, after 36 and 60 h of stimulation, as recently described (29) (Fig. 2A). We assessed retroviral infection by FACS before restimulation with irradiated APCs and OVA323–339 peptide (OVA; Fig. 2B). Initially, ~4% of T cells expressed both viral markers, and an additional 9% each expressed only one of the markers (Fig. 2B, first panel). After 1 wk of Ag-specific stimulation, ~40% of T cells expressed both viral markers (Fig. 2B, second panel), and an additional week of passage with OVA and APCs led to 87% of T cells expressing both markers (Fig. 2B, third panel). Finally, we purified these cells by cell sorting (Fig. 2B, fourth panel).


Figure 2
View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 2. Generation of Th1 cells with retroviral expression of the DO11.10 TCR. A, CD4+ T cells from p38{alpha}–/– and p38{alpha}+/– spleens were purified and stimulated with anti-CD3, anti-CD28, IL-2, IL-12, IFN-{gamma}, and anti-IL-4 for 36 h before spin infection with concentrated supernatants containing retroviruses for the DO11.10 TCR {alpha}- and beta-chains. Spin infection was repeated at 60 h, and cells were harvested and restimulated with irradiated APCs, OVA, IL-2, IL-12, IFN-{gamma}, and anti-IL-4 on day 7. B, T cells were stained weekly to observe the outgrowth of DO11.10 TCR-expressing cells. Cells were gated on live CD4+ T cells. Data are representative of 12 independently generated Th1 cell lines.

 
We observed outgrowth of T cells expressing both retroviruses in 12 lines generated in this manner (data not shown), indicating that dual retroviral infection generated a functional OVA-specific TCR. We confirmed by FACS that these lines expressed the KJ1–26 clonotype of the DO11.10 TCR (Fig. 3A). KJ1–26 staining on p38{alpha}+/– and p38{alpha}–/– T cell lines was equivalent, but was lower than the level expressed by DO11.10 CD4+ T cells (Fig. 3A). We also verified that each line expressed the expected pattern of p38{alpha} alleles (Fig. 3B) and confirmed the presence or absence of p38 protein by Western analysis (Fig. 3C). Finally, we verified that these cell lines were each responsive to OVA by stimulating Th1 cell lines with APCs and OVA or an irrelevant cognate peptide, HA518–528 (Fig. 3D). Both p38{alpha}+/– and p38{alpha}–/– Th1 cell lines produced IFN-{gamma} in response to OVA, but not in response to HA518–528 (Fig. 3D, upper rows). As a positive control, wild-type DO11.10 Th1 cells were selectively OVA responsive (Fig. 3D, bottom row). Wild-type DO11.10 Th1 cells produced slightly higher levels of IFN-{gamma} in response to OVA/APCs than p38{alpha}+/– or p38{alpha}–/– cells (Fig. 3D). This small difference might be due to the higher level of DO11.10 TCR expressed by the wild-type cells (Fig. 3A). In addition, we observed that p38{alpha}+/– cells exhibited slightly higher IFN-{gamma} production than p38{alpha}–/– cells, but because this condition contains APCs, which can secrete IL-12 and IL-18, this difference could be due to differences in either the TCR-induced or the cytokine-dependent pathway of IFN-{gamma} production. In subsequent analysis, we therefore used anti-CD3 stimulation to selectively activate the TCR pathway without introducing cytokines produced by APCs. These results demonstrate that retroviral expression of the DO11.10 TCR can sustain both T cell passage by OVA/APCs in culture and confer selective Ag-specific cytokine production.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3. Characterization of generated DO11.10 TCR-expressing cell lines. A, Naive BALB/c (dark peak), resting p38{alpha}–/– (solid line), resting p38{alpha}+/– (dashed line), and resting DO11.10 (dotted line) T cells were stained with KJ1–26 to determine the relative levels of DO11.10 TCR expression. Data are representative of 12 independently generated Th1 cell lines. B, Genomic PCR performed on purified T cell populations of the indicated genotypes. C, Western blot of total cell lysates made from purified T cell populations of the indicated genotypes. D, Th1 cells were stimulated overnight with the indicated concentration of peptide and irradiated BALB/c APCs. Shaded histograms represent IFN-{gamma} production with the addition of APCs only. Data are representative of five experiments.

 
p38{alpha} MAPK deficiency does not block Th1 development

This system allowed us to address the role of p38{alpha} in Th1 differentiation. Using lines derived under Th1-inducing conditions, we assessed the ability of p38{alpha}+/– and p38{alpha}–/– T cells to acquire a Th1 phenotype (Fig. 4). First, both p38{alpha}+/– and p38{alpha}–/– T cells were able to produce IFN-{gamma} in response to PMA/ionomycin stimulation. Additionally, p38{alpha}+/– and p38{alpha}–/– cell lines were equally capable of inducing Ag-specific IFN-{gamma} production (Fig. 3D). Both p38{alpha}+/– and p38{alpha}–/– Th1 cells were <5% positive for production of IL-4 by ICS (data not shown), indicating that p38{alpha} is not required to prevent Th2 development under these conditions. In summary, these results indicate that p38{alpha} is not required for Th1 development in CD4+ T cells.


Figure 4
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 4. p38{alpha} is not essential for Th1 differentiation. CD4+ DO11.10, p38{alpha}+/–, and p38{alpha}–/– cells that had been differentiated for 2 wk in the presence of Th1-inducing factors (IL-12, IFN-{gamma}, and anti-IL-4) were stimulated with PMA and ionomycin for 4 h before ICS for IFN-{gamma} production. Shaded histograms represent IFN-{gamma} production of unstimulated cells. Data are representative of eight experiments.

 
p38{alpha} MAPK is preferentially involved in cytokine-induced IFN-{gamma} production compared with TCR-induced IFN-{gamma} production

Finally, we examined the requirements for p38{alpha} in differential IFN-{gamma} production induced by IL-12/IL-18 compared with that produced by TCR signaling. Our use of the DO11.10 TCR in the derivation of these cell lines provided us with the ability to stimulate cells with OVA/APCs to evaluate the role of p38{alpha} in TCR-induced IFN-{gamma} production. Thus, we first compared p38{alpha}+/– and p38{alpha}–/– cells for IFN-{gamma} production in response to APCs and several concentrations of the OVA peptide Ag to which DO11.10 cells react (Fig. 5, A and D). We found that p38{alpha}+/– and p38{alpha}–/– cells produced equivalent amounts of IFN-{gamma} at each concentration of OVA peptide, as measured by both ELISA (Fig. 5A) and ICS (Fig. 5D). These results suggest that p38{alpha} is not a critical component of the pathway linking TCR signaling to the production of IFN-{gamma}.


Figure 5
View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 5. p38{alpha} is required for cytokine-induced, but not TCR-induced, IFN-{gamma} production. A–C, p38{alpha}+/– and p38{alpha}–/– Th1 cells were stimulated for 14 h with OVA/APCs and anti-IL-12 (A) or for 24 h with IL-12/IL-18 and the indicated inhibitors (B) or with anti-CD3 and the indicated inhibitors (C) before supernatant collection and ELISA. {blacksquare}, p38{alpha}+/– cells; {square}, p38{alpha}–/– cells. Data are representative of three experiments. B, p38{alpha}+/– and p38{alpha}–/– Th1 cells were stimulated for 24 h with IL-12/IL-18 and the indicated inhibitors before supernatant collection and ELISA. {blacksquare}, p38{alpha}+/– cells; {square}, p38{alpha}–/– cells. Data are representative of three experiments. C, p38{alpha}+/– and p38{alpha}–/– Th1 cells were stimulated with anti-CD3 and the indicated inhibitors before supernatant collection and ELISA. {blacksquare}, p38{alpha}+/– cells; {square}, p38{alpha}–/– cells. Data are representative of three experiments. D–F, ICS was performed on p38{alpha}+/– and p38{alpha}–/– Th1 cells that were stimulated with the indicated cytokines; shaded histograms represent unstimulated cells. OVA/APC stimulation was performed for 18 h, and IL-12/IL-18 and anti-CD3 stimulations were performed for 8 h. Data are representative of three (D) or six (E and F) experiments.

 
We next compared p38{alpha}+/– and p38{alpha}–/– cells for IFN-{gamma} production in response to stimulation with IL-12 and IL-18. In this study, p38{alpha}–/– T cells secreted 10-fold less IFN-{gamma} than p38{alpha}+/– cells (Fig. 5B). After IL-12/IL-18 stimulation, CsA treatment did not inhibit IFN-{gamma} secretion by p38{alpha}+/– T cells, but SB203580 abolished IFN-{gamma} secretion, consistent with the pattern of inhibition described for wild-type Th1 cells. As a final control, we examined these same cells for their response to TCR signaling induced by anti-CD3 treatment. Again, similar to stimulation using OVA/APCs, we found that p38{alpha}–/– T cells secreted an equivalent amount of IFN-{gamma} as that secreted by p38{alpha}+/– cells. IFN-{gamma} production induced by anti-CD3 treatment in both p38{alpha}–/– and p38{alpha}+/– cells was abolished by CsA treatment, but was unaffected by SB203580 treatment (Fig. 5C). Thus, results based on ELISA measurements indicate a selective requirement for p38{alpha} activity in IL-12/IL-18-induced, but not TCR-induced, IFN-{gamma} secretion.

Similar conclusions were supported by ICS analysis of IFN-{gamma} production. First, in response to IL-12/IL-18 treatment, the MFI of IFN-{gamma} was greatly reduced in p38{alpha}–/– cells compared with p38{alpha}+/– cells (Fig. 5E, left panels). After IL-12/IL-18 stimulation, SB203580 greatly decreased the MFI of IFN-{gamma} in p38{alpha}+/– T cells, whereas CsA treatment did not reduce the MFI of IFN-{gamma} in these cells. Second, in response to TCR signaling, the MFI of IFN-{gamma} was slightly decreased in p38{alpha}–/– compared with p38{alpha}+/– cells, consistent with the change in MFI seen previously in SB203580-treated wild-type Th1 cells (Fig. 1B). However, the MFI of IFN-{gamma} induced by TCR signaling in p38{alpha}–/– cells (Fig. 5F) was substantially greater than that induced by IL-12/IL-18 treatment (Fig. 5E), again supporting a selective role for p38{alpha} in IL-12/IL-18-induced IFN-{gamma} production. The effects of CsA and SB203580 were similar to those determined by ELISA. In summary, both ELISA and ICS analyses support a selective requirement for p38{alpha} activity in IL-12/IL-18-induced, but not TCR-induced, IFN-{gamma} production.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study excludes a strict requirement for p38{alpha} in Th1 differentiation. Because no previous study has directly examined p38{alpha}–/– T cells for Th1 development, our results are consistent with reports that have inferred a potential role for p38{alpha} based on the effects of MEKK4 or GADD45 proteins (7, 15, 17), but suggest a tempering of their interpretations. A possible explanation for the discrepancies between our study and others is their use of Con A to nonspecifically activate or restimulate cells (9, 16), a pitfall we avoided through the use of Ag-specific T cell lines and anti-CD3 stimulation.

Additionally, our production of p38{alpha}–/– Ag-specific T cell lines allowed us to re-examine the role of p38{alpha} in the two pathways for IFN-{gamma} induction. We found that p38{alpha}–/– T cells have nearly absent IFN-{gamma} secretion in response to IL-12/IL-18, but normal IFN-{gamma} secretion in response to TCR signaling, consistent with previous observations based on inhibitor studies (7). These studies do not, however, rule out the possibility of the involvement of other p38 isoforms in IFN-{gamma} production in response to TCR signaling. p38{alpha}–/– T cells also show drastically reduced MFI of intracellular IFN-{gamma} in response to IL-12/IL-18. We observed a slight reduction in the MFI of intracellular IFN-{gamma} in response to TCR signaling in p38{alpha}–/– T cells and wild-type T cells treated with SB203580 compared with controls, suggesting that p38{alpha} might have some role in IFN-{gamma} production independent of cytokine secretion.

A recent study suggests that p38 may participate in both transcriptional and post-transcriptional processes in IFN-{gamma} production, leading to selective stabilization of IFN-{gamma} mRNA when stimulated by IL-12/IL-18 in human PBLs and NK cells (30). Although our results indicate that p38{alpha} is required for cytokine-induced IFN-{gamma} production in Th1 cells, our study does not distinguish whether this effect is acting at the level of transcription or at some subsequent step in cytokine production. Consistent with previous findings based on pharmacologic inhibitors, we confirm a selective requirement for p38{alpha} in IFN-{gamma} production induced by IL-12 and IL-18 compared with induction via TCR signaling.


   Acknowledgments
 
We thank J. M. White for generation of the RAG2–/– blastocyst chimeras, and M. Himmelmann for secretarial support.


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


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Howard Hughes Medical Institute (to K.M.M.) and National Institutes of Health Grants PO1AI31238 and P50HL54619 (to K.M.M.). Back

2 Current address: Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, CT 06877. Back

3 Address correspondence and reprint requests to Dr. Kenneth M. Murphy, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: murphy{at}pathbox.wustl.edu Back

4 Abbreviations used in this paper: CsA, cyclosporin A; GAAD45, growth arrest and DNA damage 45; RV, retrovirus; h, human; HA, hemagglutinin; ICS, intracellular cytokine staining; IRES, internal ribosome entry site; MKK, MAPK kinase; MFI, mean fluorescence intensity. Back

Received for publication October 14, 2005. Accepted for publication January 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Szabo, S. J., S. T. Kim, G. L. Costa, X. K. Zhang, C. G. Fathman, L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655-669. [Medline]
  2. Afkarian, M., J. R. Sedy, J. Yang, N. G. Jacobson, N. Cereb, S. Y. Yang, T. L. Murphy, K. M. Murphy. 2002. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nat. Immunol. 3: 549-557. [Medline]
  3. Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, Jr, K. M. Murphy. 1995. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J. Exp. Med. 181: 1755-1762. [Abstract/Free Full Text]
  4. Thierfelder, W. E., J. M. van Deursen, K. Yamamoto, R. A. Tripp, S. R. Sarawar, R. T. Carson, M. Y. Sangster, D. A. Vignali, P. C. Doherty, G. C. Grosveld, et al 1996. Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells. Nature 382: 171-174. [Medline]
  5. Yang, J., T. L. Murphy, W. Ouyang, K. M. Murphy. 1999. Induction of interferon-{gamma} production in Th1 CD4+ T cells: evidence for two distinct pathways for promoter activation. Eur. J. Immunol. 29: 548-555. [Medline]
  6. Robinson, D., K. Shibuya, A. Mui, F. Zonin, E. Murphy, T. Sana, S. B. Hartley, S. Menon, R. Kastelein, F. Bazan, et al 1997. IGIF does not drive Th1 development but synergizes with IL-12 for interferon-{gamma} production and activates IRAK and NF{kappa}B. Immunity 7: 571-581. [Medline]
  7. Yang, J., H. Zhu, T. L. Murphy, W. J. Ouyang, K. M. Murphy. 2001. IL-18-stimulated GADD45beta required in cytokine-induced, but not TCR-induced, IFN-{gamma} production. Nat. Immunol. 2: 157-164. [Medline]
  8. Yu, J. J., C. S. Tripp, J. H. Russell. 2003. Regulation and phenotype of an innate Th1 cell: role of cytokines and the p38 kinase pathway. J. Immunol. 171: 6112-6118. [Abstract/Free Full Text]
  9. Rincon, M., H. Enslen, J. Raingeaud, M. Recht, T. Zapton, M. S. Su, L. A. Penix, R. J. Davis, R. A. Flavell. 1998. Interferon-{gamma} expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J. 17: 2817-2829. [Medline]
  10. Hale, K. K., D. Trollinger, M. Rihanek, C. L. Manthey. 1999. Differential expression and activation of p38 mitogen-activated protein kinase {alpha}, beta, {gamma}, and {delta} in inflammatory cell lineages. J. Immunol. 162: 4246-4252. [Abstract/Free Full Text]
  11. Rincon, M.. 2001. MAP-kinase signaling pathways in T cells. Curr. Opin. Immunol. 13: 339-345. [Medline]
  12. Brancho, D., N. Tanaka, A. Jaeschke, J. J. Ventura, N. Kelkar, Y. Tanaka, M. Kyuuma, T. Takeshita, R. A. Flavell, R. J. Davis. 2003. Mechanism of p38 MAP kinase activation in vivo. Genes Dev. 17: 1969-1978. [Abstract/Free Full Text]
  13. Dong, C., R. J. Davis, R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20: 55-72. [Medline]
  14. Takekawa, M., H. Saito. 1998. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95: 521-530. [Medline]
  15. Lu, B., H. Yu, C. Chow, B. Li, W. Zheng, R. J. Davis, R. A. Flavell. 2001. GADD45{gamma} mediates the activation of the p38 and JNK MAP kinase pathways and cytokine production in effector TH1 cells. Immunity 14: 583-590. [Medline]
  16. Lu, H. T., D. D. Yang, M. Wysk, E. Gatti, I. Mellman, R. J. Davis, R. A. Flavell. 1999. Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO J. 18: 1845-1857. [Medline]
  17. Chi, H., B. Lu, M. Takekawa, R. J. Davis, R. A. Flavell. 2004. GADD45beta/GADD45{gamma} and MEKK4 comprise a genetic pathway mediating STAT4-independent IFN{gamma} production in T cells. EMBO J. 23: 1576-1586. [Medline]
  18. Lu, B., A. F. Ferrandino, R. A. Flavell. 2004. Gadd45beta is important for perpetuating cognate and inflammatory signals in T cells. Nat. Immunol. 5: 38-44. [Medline]
  19. Adams, R. H., A. Porras, G. Alonso, M. Jones, K. Vintersten, S. Panelli, A. Valladares, L. Perez, R. Klein, A. R. Nebreda. 2000. Essential role of p38{alpha} MAP kinase in placental but not embryonic cardiovascular development. Mol. Cell 6: 109-116. [Medline]
  20. Allen, M., L. Svensson, M. Roach, J. Hambor, J. McNeish, C. A. Gabel. 2000. Deficiency of the stress kinase p38{alpha} results in embryonic lethality: characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells. J. Exp. Med. 191: 859-869. [Abstract/Free Full Text]
  21. Mudgett, J. S., J. X. Ding, L. Guh-Siesel, N. A. Chartrain, L. Yang, S. Gopal, M. M. Shen. 2000. Essential role for p38{alpha} mitogen-activated protein kinase in placental angiogenesis. Proc. Natl. Acad. Sci. USA 97: 10454-10459. [Abstract/Free Full Text]
  22. Tamura, K., T. Sudo, U. Senftleben, A. M. Dadak, R. Johnson, M. Karin. 2000. Requirement for p38{alpha} in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell 102: 221-231. [Medline]
  23. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250: 1720-1723. [Abstract/Free Full Text]
  24. Kim, J. M., J. M. White, A. S. Shaw, B. P. Sleckman. 2005. MAPK p38{alpha} is dispensable for lymphocyte development and proliferation. J. Immunol. 174: 1239-1244. [Abstract/Free Full Text]
  25. Ranganath, S., W. Ouyang, D. Bhattarcharya, W. C. Sha, A. Grupe, G. Peltz, K. M. Murphy. 1998. GATA-3-dependent enhancer activity in IL-4 gene regulation. J. Immunol. 161: 3822-3826. [Abstract/Free Full Text]
  26. Berenson, L. S., J. D. Farrar, T. L. Murphy, K. M. Murphy. 2004. Absence of functional STAT4 activation despite detectable tyrosine phosphorylation induced by murine IFN-{alpha}. Eur. J. Immunol. 34: 2365-2374. [Medline]
  27. Lee, J. C., S. Kassis, S. Kumar, A. Badger, J. L. Adams. 1999. p38 mitogen-activated protein kinase inhibitors: mechanisms and therapeutic potentials. Pharmacol. Ther. 82: 389-397. [Medline]
  28. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M. A. Mahowald, J. R. Sider, T. F. Gajewski, C. R. Wang, S. L. Reiner. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9: 229-237. [Medline]
  29. Yang, L. L., X. F. Qin, D. Baltimore, L. Van Parijs. 2002. Generation of functional antigen-specific T cells in defined genetic backgrounds by retrovirus-mediated expression of TCR cDNAs in hematopoietic precursor cells. Proc. Natl. Acad. Sci. USA 99: 6204-6209. [Abstract/Free Full Text]
  30. Mavropoulos, A., G. Sully, A. P. Cope, A. R. Clark. 2005. Stabilization of IFN-{gamma} mRNA by MAPK p38 in IL-12- and IL-18-stimulated human NK cells. Blood 105: 282-288. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
L. Jirmanova, D. Jankovic, A. J. Fornace Jr., and J. D. Ashwell
Gadd45{alpha} Regulates p38-Dependent Dendritic Cell Cytokine Production and Th1 Differentiation
J. Immunol., April 1, 2007; 178(7): 4153 - 4158.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. J. Murray
The JAK-STAT Signaling Pathway: Input and Output Integration
J. Immunol., March 1, 2007; 178(5): 2623 - 2629.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
M. N. Hedrick, C. M. Olson Jr., D. B. Conze, T. C. Bates, M. Rincon, and J. Anguita
Control of Borrelia burgdorferi-Specific CD4+-T-Cell Effector Function by Interleukin-12- and T-Cell Receptor-Induced p38 Mitogen-Activated Protein Kinase Activity.
Infect. Immun., October 1, 2006; 74(10): 5713 - 5717.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Berenson, L. S.
Right arrow Articles by Murphy, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berenson, L. S.
Right arrow Articles by Murphy, K. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS