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The Journal of Immunology, 2008, 180, 4523-4529
Copyright © 2008 by The American Association of Immunologists, Inc.
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Selective Roles of MAPKs during the Macrophage Response to IFN-{gamma}1

Annabel F. Valledor2,*, Ester Sánchez-Tilló2,{dagger}, Luis Arpa{dagger}, Jin Mo Park§, Carme Caelles{ddagger}, Jorge Lloberas{dagger} and Antonio Celada3,{dagger}

* Nuclear Receptors Group, Department of Physiology, School of Biology, {dagger} Macrophage Biology Group, and {ddagger} Cell Signaling Group, Institute for Research in Biomedicine (IRB), and University of Barcelona, Barcelona, Spain; and § Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages perform essential functions in the infection and resolution of inflammation. IFN-{gamma} is the main endogenous macrophage Th1 type activator. The classical IFN-{gamma} signaling pathway involves activation of Stat-1. However, IFN-{gamma} has also the capability to activate members of the MAPK family. In primary bone marrow-derived macrophages, we have observed strong activation of p38 at early time points of IFN-{gamma} stimulation, whereas weak activation of ERK-1/2 and JNK-1 was detected at a more delayed stage. In parallel, IFN-{gamma} exerted repressive effects on the expression of a number of MAPK phosphatases. By using selective inhibitors and knockout models, we have explored the contributions of MAPK activation to the macrophage response to IFN-{gamma}. Our findings indicate that these kinases regulate IFN-{gamma}-mediated gene expression in a rather selective way: p38 participates mainly in the regulation of the expression of genes required for the innate immune response, including chemokines such as CCL5, CXCL9, and CXCL10; cytokines such as TNF-{alpha}; and inducible NO synthase, whereas JNK-1 acts on genes involved in Ag presentation, including CIITA and genes encoding MHC class II molecules. Modest effects were observed for ERK-1/2 in these studies. Interestingly, some of the MAPK-dependent changes in gene expression observed in these studies are based on posttranscriptional regulation of mRNA stability.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages perform essential functions in homeostasis, infection, tissue repair, and resolution of inflammation (1). Macrophages originate in the bone marrow and, through the blood stream, reach all the tissues in the organism. Macrophage activation is characterized by a series of biochemical and morphological modifications that allow these cells to perform their professional functions. IFN-{gamma}, a cytokine mainly secreted by activated Th1 T lymphocytes and NK cells, is a strong endogenous macrophage activator. Target genes for IFN-{gamma} in macrophages include MHC class I and II, which are involved in Ag presentation, immunomodulatory cytokines such as TNF-{alpha}, chemokines, and antiviral proteins (2).

Upon ligand binding, the IFN-{gamma} receptor oligomerizes, and JAK-1 and -2 are subsequently activated, leading to the phosphorylation of the IFN-{gamma} receptor subunit 1. This provides a docking site for Stat-1 (3), which is then phosphorylated on Tyr701, leading to its dimerization, translocation to the nucleus and binding to specific response elements on downstream target genes (4). Full transcriptional activity of Stat-1 homodimers is manifested only when Ser727 in the transcription activation domain is also phosphorylated (5), which enhances binding of the transcription activation domain to several nuclear proteins, including the coactivator CREB-binding protein/p300 (6).

Several reports have shown that members of the MAPK family can be also activated by IFN-{gamma} in a number of cellular models (7, 8). MAPKs are evolutionarily conserved serine/threonine kinases involved in the transduction of externally derived signals that regulate cell growth, differentiation, and apoptosis (9). These kinases include ERK-1 and -2, JNK-1 and -2, and p38. MAPKs directly regulate downstream targets by phosphorylation, including additional protein kinases, components of the cytoskeleton, phospholipase A2, and transcription factors/complexes, such as Ets-1, Elk/TCF, and AP-1, which in turn promote immediate early gene expression.

In this study, we have analyzed the time course of activation of members of the MAPK family upon macrophage stimulation with IFN-{gamma}. Strong activation of p38 was observed early during the response to IFN-{gamma}, whereas weak activation of ERKs and JNK-1 was detected at later stages. We used selective inhibitors and knockout models to study the relative contributions of these MAPKs to regulation of gene expression in response to IFN-{gamma} in macrophages. Interestingly, inhibition of p38 activation resulted in decreased expression of a number of genes involved in chemotaxis and inflammation, without altering Stat-1 serine phosphorylation. ERKs had modest effects on proinflammatory genes, whereas JNK-1 activity mainly regulated the expression of genes involved in Ag presentation. Altogether, our data suggest that each MAPK family member exerts selective roles in the macrophage response to IFN-{gamma}. Recent work has demonstrated a role for p38 in stabilization of the transcripts encoding TNF-{alpha} and CXCL10 (IP-10) (10). In this study, we confirm that p38 plays an important role in the stabilization of a number of IFN-{gamma}-induced transcripts, although not all the p38-mediated effects can be explained by increased mRNA stability. Moreover, ERK and JNK-1 can also modulate the stability of selective transcripts in our studies, suggesting that all three MAPKs provide modulatory mechanisms to enhance the macrophage response to IFN-{gamma}.


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

Recombinant IFN-{gamma} and M-CSF were purchased from R&D Systems. PD98059, SB203580, and SP600125 were purchased from Calbiochem. Actinomycin D, and 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR)4 were obtained from Sigma-Aldrich.

Cell culture and animal models

Bone marrow-derived macrophages were obtained from 7-wk-old BALB/c mice (Charles River Laboratories) as described (11). Macrophages were cultured in DMEM (Sigma-Aldrich), supplemented with 20% FCS (Sigma-Aldrich) and 30% L cell conditioned medium as a source of M-CSF. All experiments were performed at 80% confluency, after 6 days of culture. At this time, the cells were deprived of M-CSF for 18 h and then stimulated with IFN-{gamma} during different periods of time. JNK-1-deficient mice (12) were kindly donated by Dr. R. A. Flavell (Yale University School of Medicine, New Haven, CT). Macrophages deficient in p38{alpha} were obtained from mice generated by crossing p38{alpha} flox/flox mice (13) with LysM-Cre mice (14).

RNA extraction and Northern blot analysis

The cells were washed twice in cold PBS and extraction of total RNA was performed as described (15). Total RNA samples (15–20 µg) were separated on 1.2% agarose gels containing formaldehyde and transferred to nylon membranes (Genescreen; NEN Life Science Products). Specific probes were obtained by labeling full-length cDNAs with [{alpha}-32P]dCTP (ICN). After hybridization at 65°C, membranes were exposed to Kodak X-AR films.

Quantitative real-time PCR analysis

Cells were washed twice with cold PBS, and total RNA was extracted with the EZ-RNA system following the manufacturer’s recommendations (Biological Industries). RNA was treated with DNase (Roche) as described (16). For cDNA synthesis, 1 µg of RNA was subjected to reverse transcription using M-MLV reverse transcriptase RNase H Minus, Point Mutant, oligo(dT)15 primer, and PCR Nucleotide mix (Promega). real-time PCR was performed using the Power SYBR Green Reagent Kit (Applied Biosystems) following the manufacturer’s recommendations with the exception that the final volume was 12.5 µl of SYBR Green Reaction Mix. The sequences of the primers used in this study are shown in Table I. Annealing for all primers was performed at 60°C for 30 s. Real-time monitoring of PCR amplification was performed using the ABI Prism 7900 Sequence Detection System (Applied Biosystems). Data were expressed as relative mRNA levels normalized to L14 expression. A control sample without RNA was included in each experiment. For statistical determinations, a nonparametric Wilcoxon test for paired differences (17) was used (*, p < 0.05; **, p < 0.01).


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  		Table I. Primers used for real-time PCR

 
Protein extraction and Western blot analysis

The cells were washed twice in cold PBS and lysed on ice with lysis solution (1% Triton X-100, 10% glycerol, 50 mM HEPES (pH 7.5), 250 mM NaCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml iodoacetamide, 1 mM PMSF, 1 mM sodium orthovanadate) as described (18). Insoluble material was removed by centrifugation at 13,000 x g for 8 min at 4°C. Cell lysates (50–100 µg) were boiled at 95°C in Laemli SDS-loading buffer and separated by 10% SDS-PAGE (unless otherwise stated), and electrophoretically transferred to nitrocellulose membranes (Hybond-ECL; Amersham). Membranes were blocked in 5% milk in TBS-0.1% Tween 20 (TBS-T) overnight at 4°C and then incubated with primary Ab for 2 h at room temperature. We used the following Abs: monoclonal anti-diphospho-ERK-1/2 (clone MAPK-YT; Sigma-Aldrich); rabbit IgG anti-phospho-p38 (Thr180/Tyr182; Cell Signaling Technology); anti-I-A{alpha} (BD Pharmingen), anti-phosphoStat-1 (Ser727; Cell Signaling Technology); anti-p38{alpha} (Cell Signaling Technology); anti-p38β (Zymed); anti-p38{gamma} (Cell Signaling Technology); anti-p38{delta} (Cell Signaling Technology); and monoclonal anti-mouse β-actin (Sigma-Aldrich). In general, membranes were washed three times in TBS-T and then incubated for 1 h with peroxidase-conjugated secondary Abs (Jackson Immunoresearch and Sigma-Aldrich). After three 15-min washes with TBS-T, ECL detection was performed (Amersham), and the membranes were exposed to x-ray films (Amersham).

JNK activity assay

JNK activity was measured as described (19). Briefly, cells were lysed with nuclear extract protocols and immunoprecipitated with protein A-Sepharose and anti-JNK-1 Ab. After several washes, the reaction was performed with 1 µg of GST-c-jun1–169 (human mannose-binding lectin) as JNK substrate, 20 µM ATP, and 1 µCi [{gamma}-32P]ATP. SDS-PAGE electrophoresis was performed, and the gel was exposed to Agfa x-ray films.

FACS analysis of surface expression

The cells were fixed in PBS-4% formaldehyde, for 30 min at 4°C. After centrifugation, Fc{gamma}III/II receptors were blocked using anti-CD16/CD32 Abs (BD Pharmingen) in PBS, 5% FBS for 15 min at room temperature. The cells were then incubated with anti-I-Aβ Ab (Chemicon) for 1 h at 4°C. The cells were washed by centrifugation through a FBS cushion and incubated with FITC-conjugated anti-rabbit/mouse IgG (Sigma-Aldrich) for 1 h at 4°C. After a second wash, the cells were resuspended in PBS and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
On the basis of previous reports that show that IFN-{gamma} activates MAPK family members in several cell types (7, 8), we studied whether these effects also occur in primary macrophages. For our studies, we used homogeneous populations of primary bone marrow-derived macrophages, which become activated under the effect of IFN-{gamma} (11, 20). The cells were deprived of their specific growth factor, M-CSF, for 18 h to eliminate any growth factor-induced MAPK activity, and then stimulated with IFN-{gamma} for different periods of time (Fig. 1). p38 phosphorylation was observed within 30 min of IFN-{gamma} treatment (Fig. 1A). Analysis of the expression of p38 isoforms in murine bone marrow-derived macrophages revealed that p38{alpha} was abundantly expressed at both mRNA (data not shown) and protein levels (Fig. 1B). We were able to detect very low expression levels of the other three isoforms, p38β, -{gamma}, and -{delta} by real-time PCR (data not shown), but not by immunoblotting (Fig. 1B). These data confirm previous reports on p38 isoform expression in human peripheral blood monocytes (21), which in the majority express p38{alpha} protein. Our data also suggest that the changes in the phosphorylation status of p38 shown in Fig. 1A are most probably due to phosphorylation of p38{alpha}.


Figure 1
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  		FIGURE 1. Effects of IFN-{gamma} on MAPK activation and MKP expression. Macrophages were obtained after 7 days of culture in the presence of M-CSF. The cells were rendered quiescent by incubating them in medium supplemented with 10% FCS (without M-CSF) for 18 h before the start of the experiment. To study direct effects of IFN-{gamma} on MAPK activation, quiescent macrophages were stimulated with IFN-{gamma} (10 ng/ml; in the absence of M-CSF) during the indicated periods of time. Control cells were treated with vehicle (DMSO). A, p38 activation was determined by Western blotting using anti-phospho-p38 Abs. B, p38 isoform expression was determined by Western blotting using Abs specific against each isoform. C, Activation of JNK-1 was studied by immunoprecipitating JNK-1 and then performing an in vitro kinase assay on recombinant c-Jun. D, Activation of ERK-1 and -2 was analyzed by Western blotting using Abs against diphospho-ERK-1/2. These experiments were confirmed at least twice. E, Macrophages were treated with M-CSF (10 ng/ml), IFN-{gamma} (10 ng/ml) or vehicle (DMSO) for 30 min (top), 2 h (middle), and 4 h (bottom). MKP expression was analyzed by real-time PCR In all real-time PCR experiments, the expression values were normalized to the expression levels of the ribosomal gene L14. Error bars were determined from two independent experiments.

 
In contrast to strong activation of p38, activation of ERK-1/2 and JNK-1 was very weak and only slightly detected after prolonged IFN-{gamma} exposure (over 2 h of treatment) (Fig. 1, C and D). JNK-2 activation was almost undetectable (data not shown). We next analyzed the effects of IFN-{gamma} signaling on the expression of members of the MAPK phosphatase (MKP) family, which are negative regulators of MAPKs. In contrast to other stimuli that signal through MAPK activation, such as M-CSF, there was no induction of the expression of a number of phosphatases, including MKP-1, -2, and -4, at any time point after IFN-{gamma} stimulation (Fig. 1E and data not shown). Indeed, lower levels of basal MKP-1 and -4 expression were observed after IFN-{gamma} treatment (Fig. 1E). It is possible that active repression of MAPK phosphatases may be required for prolonged activation of p38 and/or late activation of ERK and JNK.

We next evaluated the specific contributions of each MAPK in the macrophage response to IFN-{gamma}. Different parameters were analyzed throughout these studies, including the expression of the chemokines CXCL9 (monokine induced by IFN-{gamma}), CXCL10 (IFN-{gamma}-inducible protein 10), CCL5 (RANTES), CCL2 (MCP-1), the chemokine receptor CCR5, the cytokine TNF-{alpha}, inducible NO synthase (NOS2), and the genes CIITA and I-Aβ involved in Ag presentation. We had previously characterized the time course of induction of these genes in response to IFN-{gamma} (data not shown). Significant levels of expression of most of these genes was detected within 2 h of treatment with IFN-{gamma}. Maximal levels of induction of NOS2, CCL5, CCR5, and CXCL10 were observed after 6 h of treatment with the cytokine. In contrast, expression of CIITA and I-Aβ is more delayed, with maximal induction observed after 6 or 12 h of treatment, respectively. The use of selective inhibitors and knockout models allowed us to evaluate the specific contributions of MAPKs in IFN-{gamma}-induced responses. Selective inhibitors were used at doses that are effective at blocking their target kinase activities without resulting in general cellular toxicity (22). PD98059, an inhibitor of MEK, was used to block activation of ERK-1/2. SB203580 was used as a p38 inhibitor, based on the fact that p38{alpha}, the main isoform expressed in bone marrow-derived macrophages, is sensitive to this inhibitor. p38{alpha}-deficient macrophages were used in some experiments to confirm that the effects obtained upon treatment with SB203580 were due to specific inhibition of p38{alpha}. The role of JNK-1 was evaluated by using JNK-1-deficient macrophages. In some experiments, we also used the JNK inhibitor SP600125.

Interestingly, blockage of p38 activity resulted in strong and rather generalized inhibitory effects on the expression of several proinflammatory mediators, including the chemokines CCL5, CXCL10, and CXCL9; the chemokine receptor CCR5; the cytokines TNF-{alpha} and IL-1β; and NOS2 (Fig. 2 and data not shown). These generalized effects correlate with the fact that IFN-{gamma} induces strong p38 activation in macrophages (Fig. 1). We next analyzed the specific relevance of the p38{alpha} isoform in the proinflammatory response to IFN-{gamma} (Fig. 2B). The levels of induction of CCL5, CXCL10, and CCR5 were strongly inhibited in macrophages deficient for p38{alpha}, correlating with the effects of the p38 inhibitor. For CXCL9, TNF-{alpha}, and NOS2, we could only observe partial inhibitory effects in the p38{alpha}-deficient cells, which may be due to compensatory mechanisms in the knockout cells. Blockage of ERK-1/2 phosphorylation with PD98059 resulted in partial inhibition of the expression of some but not all IFN-{gamma}-responsive genes (Fig. 2A), including CCL5, CCR5, and CCL2. In contrast, expression of the chemokines and cytokines tested did not undergo significant inhibition in the absence of functional JNK-1 (Fig. 2C). Indeed, higher CCL5 expression levels were observed in JNK-1-deficient cells. Taken together, these results show some sort of selectivity in the contribution of MAPKs to the IFN-{gamma}-mediated inflammatory response, with p38 playing a major role in this context.


Figure 2
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  		FIGURE 2. Effects of MAPK inhibition on IFN-{gamma}-mediated chemokine and cytokine expression. A, Macrophages were preincubated for 1 h with inhibitors of MAPK signaling: PD98059 (50 µM; to block ERK activation), SB203580 (5 µM) (to inhibit p38), or vehicle (DMSO, as control). The cells were then stimulated for 2 or 6 h with IFN-{gamma}. B, Macrophages derived from WT or myeloid-specific p38{alpha}-deficient mice (p38{alpha}–/–) were stimulated for 6 h with IFN-{gamma}. C, Macrophages derived from WT or JNK-1-deficient mice (JNK1–/–) were stimulated for 6 h with IFN-{gamma}. B and C, control cells from each genotype were left untreated. In all these experiments, changes in gene expression were monitored by real-time PCR and normalized to the expression values of the ribosomal gene L14. A and C, error bars were determined from three independent experiments. B, duplicate experiments were analyzed. For statistical determinations, a nonparametric Wilcoxon test for paired differences (17 ) was used. *, p < 0.05; **, p < 0.01.

 
We next studied the involvement of MAPKs in the induction of genes that mediate Ag presentation by macrophages (Fig. 3). IFN-{gamma} induced the expression of the trans activator CIITA in macrophages. In contrast to the effects on proinflammatory gene expression, inhibition of ERK or p38 did not result in changes in IFN-{gamma}-mediated induction of CIITA (Fig. 3A). Likewise, induction of this gene was not altered in p38{alpha}-deficient macrophages in comparison with wild-type (WT) cells (data not shown). In correlation with this, inhibition of ERK or p38 did not result in significant changes in the expression of the class II MHC molecule I-Aβ, a main direct target gene of CIITA (Fig. 3A). However, the absence of functional JNK-1 did compromise the induction of CIITA in response to IFN-{gamma}, which in turn resulted in decreased induced levels of I-Aβ mRNA (Fig. 3B). These data were corroborated with the use of the JNK inhibitor SP600125 (Fig. 3C). Reduced I-Aβ mRNA levels correlated with decreased surface expression of the protein in JNK-1-deficient macrophages in comparison with WT or JNK-2 deficient mice, as demonstrated by flow cytometry studies (Fig. 3D). Similar results were obtained when WT cells were pretreated with the synthetic JNK inhibitor. Furthermore, decreased induction of the {alpha}-chain of I-A (I-A{alpha}) in response to IFN-{gamma} was also observed upon JNK inhibition (Fig. 3E).


Figure 3
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  		FIGURE 3. Effects of MAPK inhibition on IFN-{gamma}-mediated CIITA and MHC class II induction. A, Macrophages were preincubated or not with PD98059 (50 µM) or SB203580 (5 µM) for 1 h and then stimulated with IFN-{gamma} for the indicated periods of time. B, Macrophages derived from WT or JNK-1-deficient mice (JNK1–/–) were stimulated with IFN-{gamma} for 12 h (for I-Aβ) or 6 h (for CIITA) or left unstimulated. C, Macrophages were preincubated with SP600125 (5 µM) for 1 h and then stimulated with IFN-{gamma} for the indicated periods of time. Control cells were left unstimulated. In A–C, the expression of I-Aβ and CIITA was analyzed by real-time PCR, using the expression levels of L14 for normalization. D, Macrophages from WT, JNK-1-deficient or JNK-2-deficient mice were stimulated with IFN-{gamma} for 24 h. Control cells from each genotype were left unstimulated. A set of WT cells was incubated with the JNK inhibitor SP600125 (5 µM). Surface expression of the protein I-Aβ was evaluated by flow cytometry. E, The expression of I-A{alpha} protein in cells stimulated with IFN-{gamma} in the presence of SP600125 was evaluated by Western blotting. The expression of β-actin was monitored as a control. In each panel, error bars were determined from three independent experiments. For statistical determinations, a nonparametric Wilcoxon test for paired differences (17 ) was used. *, p < 0.05; **, p < 0.01.

 
In certain cell types, it has been suggested that p38 mediates Stat-1 phosphorylation on Ser727 (23, 24). However, in our experiments in macrophages Stat-1 serine phosphorylation by IFN-{gamma} was not reduced in the presence of the p38 inhibitor (Fig. 4A). Similar results were obtained by inhibiting the MEK-ERK pathway with PD98059 or by using JNK-1-deficient cells (Fig. 4), which altogether suggests that in macrophages MAPKs modulate selective events during the macrophage response to IFN-{gamma} without affecting Stat-1 serine phosphorylation.


Figure 4
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  		FIGURE 4. IFN-{gamma}-induced Stat-1 phosphorylation on Ser727 does not require MAPK activation. A, Macrophages were preincubated with MAPK inhibitors PD98059 (50 µM) and SB203580 (5 µM) for 1 h and then stimulated with IFN-{gamma} for the indicated periods of time. B, Macrophages from WT or JNK-1-deficient mice were stimulated with IFN-{gamma} for the indicated times. A and B, phosphorylation of Stat-1 on Ser727 was analyzed by Western blotting using specific Abs. β-Actin expression was evaluated as a control of protein loading and transfer. These results were confirmed in two independent experiments.

 
On the basis of the fact that p38 has been shown to affect the stability of the mRNAs for TNF-{alpha} and CXCL10 (10), we next evaluated whether the MAPK-mediated changes observed throughout this study were indeed posttranscriptional effects on mRNA stability. Target gene expression was induced upon treatment with IFN-{gamma} for 2 or 6 h (depending on early or late gene expression patterns) in the presence or absence of MAPK inhibitors or in JNK-1-deficient cells. A mixture of actinomycin D and DBR was used to block further transcriptional activity. For each treatment, the levels of expression of each gene in the absence of the RNA synthesis inhibitor were set at 100%. The remaining amounts of mRNA for each gene of interest were analyzed over a time course. p38 inhibition resulted in significant decrease of the stability of transcripts encoding CCR5, CXCL10, TNF-{alpha}, and CCL5 (Fig. 5A). Inhibition of the MEK-ERK pathway led to partial effects on the mRNA stability of some of the genes tested (Fig. 5A). However, no increase in CCL2 or CXCL9 mRNA stability was observed upon inhibition of the p38 or the ERK cascade (data not shown), which suggests that not all the changes in gene expression reported in Fig. 2 are due to stabilization of target gene mRNA. It is possible that MAPK are also involved in the regulation of transcription of some of these genes. In contrast, JNK-1 deficiency in macrophages resulted in decreased stability of CIITA expression (Fig. 5B), which suggests that all three MAPKs exert posttranscriptional modifications that lead to increased mRNA stability of selective target transcripts.


Figure 5
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  		FIGURE 5. Effects of MAPKs on mRNA stability of IFN-{gamma}-induced genes. A, Macrophages were stimulated with IFN-{gamma} for 2 h in the presence of inhibitors of the MEK-ERK cascade (PD98059, 50 µM) or p38 (SB203580, 5 µM). The cells were then treated for the indicated periods of time with a combination of RNA synthesis inhibitors, actinomycin D (Act D; 5 µg/ml) and DBR (20 µg/ml). B, Macrophages from WT or JNK-1-deficient mice were incubated with IFN-{gamma} for 6 h. Inhibition of RNA synthesis was performed as described in A. In all these experiments, the levels of gene expression were evaluated by real-time PCR. Expression of acidic ribosomal phosphoprotein P0 (36B4) was used for normalization. To evaluate the rate of mRNA degradation, the remaining mRNA after treatment with inhibitors of RNA synthesis was calculated as a percentage of the expression levels of that gene in cells stimulated with IFN-{gamma} (±inhibitors of MAPK signaling; in the absence of RNA synthesis inhibitors). Therefore, the graphics do not reflect differences in gene expression between treatments before the addition of the RNA synthesis inhibitors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IFN-{gamma} regulates innate and acquired immune responses by inducing a variety of mediators (2). Studies in mice that lack an intact Stat-1 gene (25, 26) demonstrated that Stat-1 is used in a highly restricted manner for signaling by IFN-{gamma} and plays a critical role in promoting many of the responses induced by this cytokine. Recently, the use of microarray technology has allowed the identification of a few transcriptional responses that are induced by IFN-{gamma} independently of Stat-1 (27, 28). In our study, we have explored the activation of MAPKs in macrophages stimulated with IFN-{gamma} and how these kinases modulate the macrophage response to this cytokine. Our results indicate that p38, ERK, and JNK-1 become activated upon treatment with IFN-{gamma}, although at very distinct time points and intensity. p38 was strongly activated early upon IFN-{gamma} stimulation, whereas ERK-1/2 and JNK-1 were detected only weakly and late. p38 activation has been previously documented for type I IFNs (IFN-{alpha}β) (reviewed in Ref. 29) and for IFN-{gamma} in HeLa cells (23) and, while this work was in progress, in peritoneal macrophages (10). Delayed activation of ERK by IFN-{gamma} has been reported in the macrophage cell lines J774 (30) and Raw264.7 (31), whereas to our knowledge this is the first time that JNK-1 is shown to be activated during the macrophage response to IFN-{gamma}. We have not explored the upstream regulators of each of these MAPKs in response to IFN-{gamma}. However, it has been recently proposed that IFN-{gamma} triggers the physical association of its receptor and the adaptor protein MyD88, which activated a signalosome involving mixed-lineage kinase 3 and MEK 3, leading to activation of at least p38 (10). As for ERKs and JNK-1, we cannot discard the theory that weak and late activation of these kinases is indeed induced by secondary mediators of the macrophage response to IFN-{gamma}.

In our studies, activation of p38 appears to play a critical role in regulation of the expression of a number of chemokines and cytokines induced by IFN-{gamma}, as demonstrated by the relatively broad effects of the p38 inhibitor SB203580. Some of these effects can be explained by modulation of the mRNA stability of target genes, based on our own results and those of another group (10) using synthetic blockers of RNA synthesis. However, we cannot discard the notion that p38 triggers transcriptional mechanisms to enhance the expression on selective targets, such as CXCL9, based on the fact that inhibition of p38 compromised the expression of this gene without affecting the rate of degradation of its mRNA. Interestingly, ERK activity contributed also to chemokine mRNA stabilization. The mechanism underlying combined p38 and ERK stabilizing actions on common target transcripts deserves further attention. Among the different chemokines tested, CCL2 was only modulated by ERK activation, not by p38{alpha}. In this case, regulation of the levels of expression of CCL2 did not correlate with changes in its mRNA stability, which suggests that ERK may also trigger transcriptional mechanisms to enhance IFN-{gamma}-mediated gene expression. CCL2 has indeed been shown to be induced by IFN-{gamma} in a Stat-1-independent manner (27). Our results may well indicate that the signal required for CCL2 expression is also provided by the ERK cascade in agreement with recent findings showing that c-Jun is required for expression of a number of IFN-{gamma}-stimulated genes independently of Stat-1 in mouse embryonic fibroblasts (32).

In contrast with the effects on chemokines and cytokines, expression of the transcriptional activator CIITA and subsequent regulation of the levels of MHCII, did not fall under the control of p38{alpha} or ERKs. Instead, JNK-1 seemed to play a role at this level. It is interesting that JNK-1 does not participate in the regulation of the expression of the chemokines and cytokines tested, whereas it shows a selective involvement in the control of genes that participate in Ag presentation. The mechanism that accounts for this selective contribution also deserves further consideration.

A previous study proposed that p38 can function as a serine kinase for Stat-1 (23), and it is widely accepted that phosphorylation on Ser727 is required for its full transcriptional activity (5) without modifying its ability to bind to DNA (33). Our results suggest that in macrophages, Ser727 phosphorylation occurs independently of activation of p38, in correlation with reports generated by other authors (34); their studies demonstrated a role for this MAPK in Stat-1 phosphorylation only in response to stress signaling. In our studies, we can also conclude that ERK-1/2 and JNK-1 do not participate in Stat-1 serine phosphorylation either. We have not explored other candidate serine kinases that may mediate this effect, such as PI3K, calcium/calmodulin-dependent protein kinase II, or thymoma viral proto-oncogene 1 (AKT) (35).

Of special interest is also the fact that expression of the phosphatase MKP-1 was not up-regulated in coordination with activation of MAPKs during the response to IFN-{gamma}. Members of the MKP family mediate dephosphorylation of threonine/serine and tyrosine residues within the MAPK activation motif, which is sufficient for total enzymatic inactivation (36). Other macrophage activating agents, such as LPS, induce strong levels of expression of MKP-1 (37) which contribute to attenuate proinflammatory responses mediated by LPS-induced MAPK activation (38, 39). We hypothesize that repression of MKPs might be necessary to ensure adequate prolonged MAPK activity necessary for stabilizing transcripts involved in the biological response to IFN-{gamma}.


   Acknowledgments
 
We thank Dr. Richard A. Flavell (Yale University School of Medicine, New Haven, CT) for the JNK-1 knockout mice, Dr. Kinya Otsu (Osaka University Graduate School of Medicine, Osaka, Japan) for the p38{alpha} flox/flox mice used in generating myeloid-specific p38{alpha} knockout mice, and Drs. Eusebio Perdiguero and Pura Muñoz (Centre de Recerca Genòmica, Barcelona, Spain) for primers and Abs specific for p38 isoforms. We also thank Tanya Yates for editorial help.


   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 grants from the Comisión Interministerial de Ciencia y Tecnología (BFU2004-05725/BMC to A.C.) and the European Commission (International Reintegration Grant IRG-31137 to A.F.V.). A.F.V. was a Ramon y Cajal investigator. L.A. was supported by a predoctoral fellowship (Formació Personal Investigador, Generalitat de Catalunya). Back

2 A.F.V. and E.S.T. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Antonio Celada, Macrophage Biology Group, Institute for Biomedical Research, Josep Samitier 1-5, 08028 Barcelona, Spain. E-mail address: acelada{at}ub.edu Back

4 Abbreviations used in this paper: DBR, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside; WT, wild type; TBS-T, TBS, 0.1% Tween 20; MKP, MAPK phosphatase; NOS2, inducible NO synthase. Back

Received for publication July 25, 2007. Accepted for publication January 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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A. F. Valledor, L. Arpa, E. Sanchez-Tillo, M. Comalada, C. Casals, J. Xaus, C. Caelles, J. Lloberas, and A. Celada
IFN-{gamma}-mediated inhibition of MAPK phosphatase expression results in prolonged MAPK activity in response to M-CSF and inhibition of proliferation
Blood, October 15, 2008; 112(8): 3274 - 3282.
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