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The Journal of Immunology, 2007, 178: 2549-2555.
Copyright © 2007 by The American Association of Immunologists, Inc.

Selective Activation of Fyn/PI3K and p38 MAPK Regulates IL-4 Production in BMMC under Nontoxic Stress Condition1

Barbara Frossi*,{dagger}, Juan Rivera{ddagger}, Emilio Hirsch§ and Carlo Pucillo2,*,{dagger}

* Dipartimento di Scienze e Tecnologie Biomediche; {dagger} Microgravity, Ageing, Training, Immobility, Center of Excellence, Università di Udine, Udine, Italy; {ddagger} Molecular Inflammation Section Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; and § Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, Torino, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells have the ability to react to multiple stimuli, implicating these cells in many immune responses. Specific signals from the microenvironment in which mast cells reside can activate different molecular events that govern distinct mast cells responses. We previously demonstrated that hydrogen peroxide (H2O2) promotes IL-4 and IL-6 mRNA production and potentates Fc{epsilon}RI-induced cytokine release in rat basophilic leukemia RBL-2H3 cells. To further evaluate the effect of an oxidative microenvironment (which is physiologically present in an inflammatory site) on mast cell function and the molecular events responsible for mast cell cytokine production in this environment, we analyzed the effect of H2O2 treatment on IL-4 production in bone marrow-derived, cultured mast cells. Our findings show that nanomolar concentrations of H2O2 induce cytokine secretion and enhance IL-4 production upon Fc{epsilon}RI triggering. Oxidative stimulation activates a distinct signal transduction pathway that induces Fyn/PI3K/Akt activation and the selective phosphorylation of p38 MAP kinase. Moreover, H2O2 induces AP-1 and NFAT complexes that recognize the IL-4 promoter. The absence of Fyn and PI3K or the inhibition of p38 MAPK activity demonstrated that they are essential for H2O2-driven IL-4 production. These findings show that mast cells can respond to an oxidative microenvironment by initiating specific signals capable of eliciting a selective response. The findings also demonstrate the dominance of the Fyn/p38 MAPK pathway in driving IL-4 production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells exhibit an array of adhesion molecules, immune response receptors, and other surface molecules that empower these cells with the ability to react to multiple stimuli and function both in innate and adaptive immunity (1, 2). Although much is known about the role of Fc receptors (such as the high affinity receptor for IgE, Fc{epsilon}RI), TLRs, and a G protein-coupled receptors (2) on mast cell activation, the influence of the microenvironment in promoting mast cell responses is just beginning to be explored (3, 4). Whether a stimulus induces mast cell granule exocytosis (5) and cytokine production or selectively induces just the latter has been demonstrated to be partly dependent on the strength of the stimulus (6, 7). These studies showed that the cell’s response is governed by kinetic proofreading parameters (molecular events) that must occur to achieve the particular response. However, they also demonstrated that the molecular requirements differ for responses. In fact, bone marrow-derived cultured mast cells (BMMC)3 were found to release a different profile of cytokine mRNA with varying concentrations of Ag or varying receptor occupancy with IgE. The mRNA accumulations of MIP-1{alpha}, MIP-1beta, MCP-1, IL-2, and IL-4 reach maximal levels at significantly lower concentrations of Ag than IL-3, IL-6, IL-10, LIF, MIP-2, and IFN-{gamma} (7). These different patterns of cytokine secretion reflect the activation of specific signaling events after Fc{epsilon}RI aggregation.

In mast cells, Lyn and Fyn are Fc{epsilon}RI-proximal kinases that propagate signals respectively through the adaptors LAT (linker of activated T cells) and Gab2 (8, 9). Fyn and Gab2 are required for the activation of PI3K and Akt (9, 10), whereas LAT is essential for calcium responses (11). Low receptor occupancy is substantially effective in stimulating the Fyn-initiated signaling that uses the adaptor Gab2, while the adaptor LAT is fully engaged with a strong stimulus (7). The selective profile of cytokine production seen at low receptor occupancy is accompanied by p38 MAP kinase phosphorylation, whereas intermediate and high receptor occupancy are required for JNK1 and ERK2 activation (7). These different signals, which collectively are required for a maximal mast cell effector response, individually seem to represent a mechanism by which mast cells can modulate gene expression based on the strength of the observed stimulus.

In the course of allergic and inflammatory reactions mast cells are exposed to an oxidative microenvironment, because reactive oxygen species (ROS) are produced as a consequence of phagocytosis and the killing of bacteria. Hydrogen peroxide (H2O2) is one of the predominant oxidants produced by the respiratory burst reactions of cells such as eosinophils and neutrophils. ROS and H2O2 can function as second messengers and influence many biological processes that can be either beneficial or detrimental for the host (12). The balance of the oxidative/antioxidative environment is well known to play an important role in the modulation of mast cell function, particularly in the context of an inflammatory response (13).

Our previous findings showed that oxidative stimulation can induce a pro-type 2 inflammatory response from rat basophilic leukemia RBL-2H3 cells that is independent of Fc{epsilon}RI stimulation (14). Nonetheless, H2O2 and Fc{epsilon}RI signaling can be additive, enhancing IL-4 production. IL-4 is a pleiotropic cytokine, implicated as a stimulatory and regulatory factor in B cell growth, CD4+ Th2 polarization, isotype switching, and IgE production (15, 16). IL-4 also modulates inflammatory reactions and could be produced by stimulation of BMMC with low doses of Ag (7). In this study we find that H2O2 induces IL-4 production in BMMC through preferential activation of a distinct signal transduction pathway that includes Fyn/PI3K/Akt activation, the selective phosphorylation of p38 MAPK, and the DNA binding of NFAT and AP-1, which are both known to modulate IL-4 gene transcription (17). Moreover, the results show that both Fyn and p38 are critical for H2O2-driven IL-4 production. Thus, as seen with a weak stimulus (7), oxidative treatment evokes specific mast cell responses that cause selective gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Antibodies and reagents

Murine DNP-specific IgE was produced as described (18). DNP-human serum albumin (HSA) (DNP36-HSA; Ag) was from Sigma-Aldrich. Abs used for immunoprecipitation were anti-Lyn, anti-Fyn, anti-PI3K, and anti-Akt, all from BD Biosciences. A peroxidase-conjugated anti-phosphotyrosine Ab (Cell Signaling) was used to detect phosphorylated proteins in the immunoblotting experiments. Rabbit Abs to ERK2, JNK1, and p38 MAPK were from Santa Cruz Biotechnology, and mouse anti-phospho-ERK2, rabbit anti-phospho-JNK and rabbit anti-phospho-p38 MAPK were from Cell Signaling. The secondary Abs used for immunoblotting were goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP from Sigma-Aldrich. PD98059, a specific inhibitor of ERK kinase, and SB203580, a specific inhibitor of p38 kinase, were purchased from Calbiochem. All other reagents were purchased from Sigma-Aldrich.

Mice and cell culture

Fyn–/– mice (strain B6.i29S-Fyn) and Lyn–/– mice (strain c57BL6J-Lyn(N6)) were from The Jackson Laboratory. PI3K-deficient mice, which are on a 129v inbred genetic background, were generated as described previously (19). Wild-type age- and sex-matched littermates were used as controls. All mice were used in accordance with National Institutes of Health guidelines and National Institute of Arthritis and Musculoskeletal and Skin Diseases-approved Animal Study Proposal A001-04-03. BMMC were obtained by in vitro differentiation of bone marrow cells taken from mouse femur by culturing in RPMI 1640 supplemented with 20% FCS, 2 mM L-glutamine, 100U/ml penicillin, 100 µg/ml streptomycin, nonessential amino acids, sodium pyruvate, and HEPES buffer and containing 20 ng/ml stem cell factor (SCF) and 4 ng/ml IL-3 for 4 to 8 wk. All cell cultures were grown at 37°C in a humidified atmosphere with 5% CO2. Cell culture reagents were obtained from BioWhittaker. After 4 wk, 1 x 106/ml BMMC were stained for Fc{epsilon}RI expression with 2.5 µg/ml anti-DNP IgE (SPE-7 mAb; Sigma-Aldrich), followed by PE-conjugated goat anti-mouse Ig (Southern Biotechnology) and analyzed by flow cytofluorometry using a FACScan (BD Biosciences) cytometer. Purity was usually >97%.

All experiments were performed using at least three separate BMMC preparations, each one obtained from two mice.

Cell stimulation

Because SCF and IL-3 induce some cell responses, BMMC were rested before stimulation by the removal of SCF overnight (for 16 h) followed by the removal of IL-3 for 3 h. Then cells were incubated with the appropriate H2O2 concentration in SCF and IL-3-free medium. To stimulate cells via Fc{epsilon}RI, cells were sensitized with 1 µg/ml anti-DNP mouse IgE in SCF and IL-3-free medium for 4 h and then washed twice with PBS. Culture medium containing the 100 ng/ml DNP36-HSA (Ag) was then added. At the indicated times, samples were collected by centrifugation for 5 min at 1200 rpm and RNA or protein was extracted.

RT-PCR

Total RNA was isolated from 5 x 106 cells using TRIzol Reagent (Invitrogen Life Technologies) according to manufacturer’s instructions. cDNA was synthesized from total RNA by reverse transcription using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) and oligo(dT) priming. cDNA was amplified by PCR using Red Taq polymerase (Sigma-Aldrich) in a GeneAmp PCR system 2400 thermal cycler (PerkinElmer). Amplification conditions were as follows: hot start at 94°C for 5 min, denaturation at 94°C (20 s), annealing at:55°C (15 s) for primers IL-4, IL-5, and IL-10 or 60°C (15 s) for GAPDH and IL-6, extension at 72°C (45 s) for 35 cycles, and a final extension step at 72°C for 10 min. All primers for mouse cytokines were purchased from Clontech. The PCR products were separated by agarose gel electrophoresis and were visualized by ethidium bromide staining. A 100-bp DNA ladder (MBI Fermentas) was run as a reference marker. Gel documentation and subsequent densitometric analysis of band intensity was conducted using the Gel Doc 2000 system (Bio-Rad).

Cytokine release detection

Quantitative measurement of IL-4 in cell supernatants was performed using the BioTrak mouse IL-4 ELISA from Amersham Biosciences. Fifty microliter aliquots of cell supernatants were used for the assay according to the manufacturer’s instructions.

Preparation of protein extracts

For total protein extracts, 10 x 106 cells were resuspended into 100 µl of 1% Nonidet P-40 lysis buffer (1% Nonidet p-40, 500 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 5 mM beta-glycerophosphate, 2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A) for 30 min. Lysates were then centrifuged for 20 min at 14,000 rpm at 4°C and the protein concentration was determined (BCA protein assay; Pierce). Nuclear proteins extracts used in EMSA assays were obtained by lysing 5 x 106 cells with 50 µl of ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT). Samples were centrifuged at 800 x g for 10 min at 4°C and the supernatants were collected as cytoplasmic extracts. Then, the pellets were resuspended in 50 µl of buffer B (20 mM HEPES (pH 7.9), 400 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 5% glycerol, 0.5 mM PMSF, and 0.5 mM DTT) to extract nuclear proteins. After incubation for 20 min on ice, the samples were centrifuged at 10,000 x g for 30 min at 4°C and the supernatants were collected as nuclear extracts and stored at –80°C. The amount of protein in the nuclear extracts was quantified by the Bradford method (20).

Immunoprecipitation assay

Briefly, 10 µg of each individual mAb was incubated with 50 µl of protein G-Sepharose (Amersham Biosciences) overnight at 4°C in 1% Nonidet P-40 lysis buffer. Lysates (of equal protein concentration) from unstimulated and stimulated cells were incubated with the Abs prebound to Protein G-Sepharose for 4 h at 4°C. After two washes with 1% Nonidet P-40 lysis buffer and one with 0.01% Nonidet P-40 lysis buffer, proteins were recovered with 50 µl of Tris-glycine SDS sample buffer that contained 1% 2-ME and 1 mM orthovanadate and were resolved by SDS-PAGE and Western blot analysis.

Western blot analysis

The resolved proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell). The nonspecific binding sites on the membranes were blocked by incubation in 5% nonfat milk in PBS with 0.1% Tween 20 and then incubated with the indicated primary Abs for 60 min at room temperature. After three washes with PBS plus 0.1% Tween 20, the membranes were incubated with the appropriate anti Ig coupled to peroxidase. After 60 min of incubation at room temperature the membranes were washed several times with PBS with 0.1% Tween 20. Proteins were detected by ECL chemiluminescence (Amersham Biosciences) using BioMax-Light films (Kodak) and quantitated by GelDoc 2000 (Bio-Rad).

Electromobility shift assay

The sequence of the top strand of double-stranded oligonucleotide probes used in EMSAs were 5'-CTGGTGTAATAAAATTTTCCAATGTAAC-3' for NFAT and 5'-CGCTTGATGAGTCAGCCGGAA-3' for AP-1. Oligonucleotides were end labeled with [{gamma}-32P]dATP by incubation with T4 polynucleotide kinase (MBI Fermentas), annealed, and purified on column SpinX Costar 8160 (Corning) filled with Sephadex G-50 fine (Amersham Biosciences). Nuclear extracts (10 µg) were incubated with 0.5–1 ng of labeled probe (10,000–20,000 cpm) for 30 min at room temperature in binding buffer (200 mM HEPES (pH 7.9), 500 mM KCl, 10 mM MgCl2, 1mMEDTA, 50 mM DTT, and 12.5% glycerol for AP-1; 10 mM Tris-HCl (pH 7.4), 10mM KCl, 1 mM EDTA, 5 mM DTT, and 12.5% glycerol for NFAT) with 1 µg of calf thymus. Unlabeled oligonucleotides, as DNA competitors, were added 10 min before the addition of DNA probe at 100-fold molar excess. The samples were separated on a 5% polyacrylamide Tris borate-EDTA gel, which was dried and then exposed to a Hyperfilm MP (Amersham Biosciences) film at –80°C. To normalize nuclear extract loading, we used a probe specific for the constitutively expressed transcription factor CREB, which is not modified in its binding activity following the cell treatment used in this study (data not shown).

Data analysis

The data shown is a summary from at least three experiments. Data are shown as the mean ± SD. Statistical significance of the data was calculated using a Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
H2O2 treatment induces IL-4 up-regulation

Mast cells are known to produce a variety of cytokines in response to Fc{epsilon}RI stimulation. We previously demonstrated that H2O2 treatment can also cause cytokine gene expression in the rat basophilic leukemia RBL-2H3 cell line (14), but this had not been tested in BMMC.

We first compared the profile of cytokine mRNA from unstimulated and cells stimulated via Fc{epsilon}RI or with 10 nM H2O2. As shown in Fig. 1A, a nanomolar concentration of H2O2 clearly increased IL-4 mRNA while weakly affecting IL-5 and IL-6 mRNA levels and had no effect on IL-10 mRNA levels. The IL-4 response to H2O2 was similar to that of Fc{epsilon}RI stimulation.


Figure 1
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  		FIGURE 1. H2O2 stimulation promotes the release of IL-4 and potentates the IL-4 production induced by Fc{epsilon}RI engagement in BMMC. A, cDNA from BMMC, stimulated via Fc{epsilon}RI or with 10 nM H2O2 or left untreated, was amplified using primers for IL-4, IL-5, IL-6, and IL-10. B, cDNA from BMMC treated with the indicated concentrations of H2O2 was analyzed for IL-4 expression. Densitometric analysis was performed on three independent experiments and normalized to GAPDH, which is shown in the graph. C, BMMC were incubated with the indicated H2O2 concentrations. After 24 h secreted IL-4 was detected by ELISA as described in Materials and Methods. Results are expressed as picograms of cytokine per 1 x 106 cells. Asterisks indicate statistical difference (*, p < 0.005; **, p < 0.001) vs untreated cells. D, The effect of costimulation by Fc{epsilon}RI and H2O2 treatment on IL-4 secretion was evaluated by ELISA after 24 h of stimulation. Asterisks indicate statistical difference (*, p < 0.001) vs Fc{epsilon}RI stimulated cells.

 
Recently, it was proposed that at inflammatory sites oxidative stress might induce IL-4 production by mast cells and basophils and direct Th2 cell differentiation (21). Importantly, non-IgE-dependent modes of maintaining Th2 responses have been demonstrated, as Fc{epsilon}RI-null mice develop a normal Th2 response when infected with Schistosoma mansoni despite lacking Fc{epsilon}RI dependent IL-4 production (22). Thus, the oxidative microenvironment might provide such an alternate mode by promoting IL-4-production by mast cells in an IgE-independent manner. So, BMMC were stimulated with varying concentrations of H2O2, and IL-4 mRNA expression was evaluated by RT-PCR. To also determine whether H2O2 stimulates IL-4 secretion, we measured the IL-4 concentrations in the culture supernatants of BMMC incubated with varying doses of H2O2 for 24 h. The results shown in Fig. 1, B and C, demonstrate that H2O2 treatment (at 10 nM) increased IL-4 mRNA by >3-fold relative to the untreated sample (Fig. 1B). Consistent with this observation, the stimulation of BMMC with varying doses of H2O2 induced the secretion of IL-4 reaching a maximum at 10 nM of 19 ± 2.3 pg/106 cells, an increase approximating 3-fold more than that observed in the medium of untreated cells (Fig. 1C). As a positive control, we stimulated Fc{epsilon}RI-dependent IL-4 secretion and found it to be ~2-fold greater than H2O2-induced secretion (Fig. 1D). Concomitant stimulation via Fc{epsilon}RI and H2O2 treatment demonstrated an additive effect on IL-4 secretion (Fig. 1D), consistent with our previous results (14) and suggesting that the signals leading to IL-4 synthesis and secretion were not saturated by either individual stimulus.

H2O2 treatment causes phosphorylation of multiple signaling proteins used by Fc{epsilon}RI but does not stimulate LAT phosphorylation

To assess which signals are required for H2O2-mediated cytokine production, we studied the tyrosine phosphorylation of various signaling proteins known to function downstream of Fc{epsilon}RI. Lyn, Fyn, LAT, PI3K, and Akt were immunoprecipitated and their phosphorylation states were determined by immunoblotting with a mAb to phosphotyrosine. Equal protein loading was verified by immunoblotting with an Ab to the specific protein and all results were normalized to the total protein following densitometric analysis. As shown in Fig. 2A, Fc{epsilon}RI stimulation of BMMC caused increased tyrosine phosphorylation of Lyn, LAT, Fyn, PI3K, and Akt. The results following H2O2 treatment of BMMC were quite similar with one significant exception, namely the failure of LAT to become tyrosine phosphorylated (Fig. 2A). The kinetic experiments on H2O2-treated cells shown in Fig. 2B demonstrate that Fyn and Lyn tyrosine phosphorylation are rapid (within 5 min of stimulation). Maximal phosphorylation of PI3K and Akt lags behind Lyn and Fyn phosphorylation but appears to still be augmented after 15 min of treatment. H2O2 failed to induce LAT phosphorylation at any time point studied (Fig. 2B). Thus, these results show that unlike Fc{epsilon}RI stimulation, which results in robust LAT phosphorylation and generates downstream signals (see Fig. 4 below), an oxidative stimulus fails to activate LAT. In contrast, Fyn activation and the phosphorylation of PI3K and Akt were robust (Fig. 2B). Interestingly, we previously observed similar results by weak stimulation of BMMC (7).


Figure 2
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  		FIGURE 2. H2O2 treatment induces the phosphorylation of Lyn/Fyn/PI3K and Akt but not that of LAT. A, BMMC were stimulated with 10 nM H2O2 for 10 min. As a positive control, IgE-sensitized BMMC were incubated with 100 ng/ml DNP for 10 min. The indicated proteins were then immunoprecipitated with protein G-Sepharose prebound Ab and immunoblots were probed with anti-phosphotyrosine (p-). Protein levels were determined by the reprobing of blots with Ab to each protein. Asterisk indicates statistical difference (*, p < 0.005; **, p < 0.001) vs untreated cells. B, BMMC were stimulated with 10 nM H2O2 for indicated times and analyzed as in A. IP, Immunoprecipitation; WB, Western blot.

 

Figure 4
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  		FIGURE 4. H2O2 treatment of BMMC induces preferential phosphorylation of p38 MAPK. A, BMMC were stimulated with the indicated concentrations of H2O2 for 30 min. As a positive control, DNP-specific IgE-sensitized BMMC were stimulated with 100 ng/ml DNP-HSA. p38 MAPK, ERK, and JNK phosphorylation (p-) was measured by Western blot using specific anti-phospho-p38, anti-phospho-ERK, and anti-phospho-JNK. B, BMMC from wild-type, Lyn–/–, and Fyn–/– mice were stimulated via Fc{epsilon}RI or with 10 nM H2O2. p38 MAPK phosphorylation was evaluated as in A. Asterisks indicate statistical difference (*, p < 0.006; **, p < 0.002) vs untreated cells.

 
H2O2-induced IL-4 up-regulation requires Fyn

Cytokine production in mast cells as a consequence of Fc{epsilon}RI stimulation seems to be primarily driven by Fyn and not Lyn kinase (23, 24). In fact, Fc{epsilon}RI-dependent stimulation of IL-4 production was demonstrated to be defective in Fyn-deficient BMMC but not in Lyn deficiency (23, 24, 25) To determine whether Lyn and/or Fyn might be required for H2O2-induced IL-4 production, we analyzed IL-4 mRNA up-regulation using BMMC from Lyn–/–, Fyn–/–, and PI3K–/– mice. BMMC from wild type, Lyn-, Fyn-, and PI3K-null mice were treated with 10 nM H2O2 for 3 h and then analyzed together with untreated samples for IL-4 mRNA production. Fig. 3 shows that H2O2 induced up-regulation of IL-4 mRNA only in wild-type and Lyn-null BMMC. In contrast, H2O2 failed to induce a significant increase in IL-4 mRNA in both Fyn- and PI3K-null BMMC. The results were also confirmed by ELISA (data not shown). These findings are consistent with the requirement for Fyn in Fc{epsilon}RI-induced IL-4 production (23) and demonstrate that H2O2-driven IL-4 production preferentially uses Fyn-mediated signals, whereas Lyn-mediated signals are not crucial. Moreover, these results indicate that PI3K is a key regulatory factor for IL-4 expression.


Figure 3
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  		FIGURE 3. IL-4 mRNA up-regulation by H2O2 requires Fyn and PI3K but not Lyn. BMMC from wild-type (WT), Lyn–/–, Fyn–/–, and PI3K–/– mice were placed in medium containing 10 nM H2O2 for 3 h. The total RNA was extracted. IL-4 mRNA expression was evaluated by RT-PCR using specific primers. IL-4 expression was normalized to GAPDH. Quantitations shown in the graph are from three experiments. Asterisks indicate statistical difference (*, p < 0.001) vs unstimulated cells.

 
H2O2 induce preferentially p38 MAPK phosphorylation

H2O2 stimulation is known to promote the activation of MAPK pathways, including ERK, JNK, and p38 kinase in various cell types (26). Because MAPKs play an important role in the regulation of cytokine production in mast cells (27), we sought to determine whether H2O2 treatment of BMMC led to the activation of MAPKs. Cells were stimulated with varying concentrations of H2O2 and the degree of MAPKs phosphorylation was compared with cells stimulated via Fc{epsilon}RI. As shown in Fig. 4A, H2O2 treatment did not induce the phosphorylation of ERK and JNK; however, as expected ERK and JNK were potently activated after Fc{epsilon}RI engagement. In contrast, p38 MAPK phosphorylation was significantly induced in a dose-dependent manner by H2O2 treatment. At 10 nM, H2O2 phosphorylation of p38 MAPK reached levels of ~60% of that seen with Fc{epsilon}RI stimulation. Because Fyn kinase was reported to stimulate p38 MAPK phosphorylation (7, 23), we investigated whether Lyn or Fyn might be required for p38 MAPK phosphorylation following H2O2 treatment of BMMC deficient in these Src kinases. Fig. 4B demonstrates that both Fc{epsilon}RI stimulation and H2O2 treatment induce p38 MAPK activation in wild-type and Lyn-null BMMC. In contrast, p38 MAPK activation was dramatically inhibited in Fyn-null BMMC because both Fc{epsilon}RI stimulation or oxidative treatment failed to induce its phosphorylation. These results establish that H2O2 treatment of BMMC activates Fyn-dependent phosphorylation (activation) of p38 MAPK.

It is well understood that multiple pathways cooperate with the activity of MAPKs in gene expression (28). Thus, to determine the importance of p38 MAPK activity in the mRNA accumulation of IL-4 induced by H2O2 stimulation, we used pharmacological inhibitors of ERK (PD98059) and p38 MAPK (SB203580) activity. Cells were briefly pretreated with these inhibitors as previously described (7) and were then stimulated via Fc{epsilon}RI or by H2O2 treatment. Fig. 5 shows that the treatment of BMMC with the two MAPK inhibitors caused a marked decrease in both Fc{epsilon}RI- and H2O2-induced IL-4 mRNA accumulation. In fact, after treatment with the p38 MAPK inhibitor SB203580 the level of IL-4 mRNA in all samples appeared to be reduced below those of nontreated cells, while the ERK inhibitor PD98059 showed only weak effect on both Fc{epsilon}RI- and H2O2-stimulated samples, but some IL-4 mRNA was still detected (Fig. 5). Results were also confirmed by ELISA (data not shown). These findings indicate that p38 MAPK activation is essential for IL-4 production whereas ERK has a minimal role. The findings are consistent with the preferential signaling of p38 MAPK in the induction of an IL-4 gene in cells exposed to oxidative stimulation.


Figure 5
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  		FIGURE 5. The selective inhibition of p38 MAPK prevents IL-4 mRNA up-regulation following H2O2 treatment of BMMC. BMMC were preincubated with 10 µM PD98059 or 10 µM SB203580 for 30 min followed by IgE/DNP or H2O2 stimulation for 3 h or left untreated. IL-4 mRNA expression was evaluated by RT-PCR after isolation of total RNA. GAPDH was used to normalize the data. The asterisk indicates statistical difference (*, p < 0.001) vs unstimulated cells.

 
H2O2 treatment induces DNA binding of NFAT and AP-1 on the IL-4 promoter

IL-4 gene expression in mast cells is selectively regulated by specific transcription factors (16). In T cells as well as in mast cells the NFAT and AP-1 transcriptional factors bind cooperatively to their cognate binding sites in the IL-4 promoter, and the binding of both is necessary for maximal transcription in T cells (29). Because NAFT and AP-1 are known to be redox-sensitive transcription factors, we assessed their DNA binding activity in unstimulated and H2O2-treated BMMC using EMSAs. As an NFAT probe, the sequence between –88 and –60 in the 5' region of the IL-4 gene was used (the activation-responsive element), which was demonstrated to be a binding site for NFAT but not for AP-1 in mast cells (17). To determine AP-1 DNA binding activity, a commercial probe containing the consensus binding site for AP-1, present in the IL-4 promoter (29, 30), was used. Fig. 6A shows that H2O2 treatment caused an increase in both AP-1 and NFAT DNA binding activity, evidenced by a shift of the NFAT band and the increased intensity of the AP-1 band. The increase in binding activity was similar to that induced by Fc{epsilon}RI stimulation. The specificity of binding was also confirmed by incubation with a 100-fold excess of unlabeled oligonucleotide.


Figure 6
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  		FIGURE 6. H2O2 treatment of BMMC induces DNA binding of NFAT and AP-1. A, Nuclear extracts from unstimulated BMMC or Fc{epsilon}RI- and H2O2-stimulated BMMC were incubated with NFAT and AP-1 32P labeled probes in the absence (–) or presence (+) of a competitor. Arrows indicate the position of protein-DNA complexes. B, To test the role of p38 MAPK in forming these complexes, BMMC were preincubated with p38 MAPK inhibitor (SB203580) and then stimulated with H2O2. Nuclear extracts were used in EMSA to determine protein bound DNA complexes.

 
To further explore whether the H2O2-mediated activation of p38 MAPK plays a role in the DNA binding activity of NFAT and AP-1, BMMC were preincubated with the p38 inhibitor SB203580 and were subsequently stimulated for 1 h in the presence or absence of 10 nM H2O2. The nuclear extracts were then assayed to determine the DNA binding activity of AP-1 and NFAT. Fig. 6B shows that inhibition of p38 MAPK activity prevents the binding of AP-1 and NFAT to their consensus binding domains in the IL-4 promoter. These findings link the H2O2-induced activation of p38 MAPK as essential for IL-4 gene expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
ROS are important determinants in the regulation of cell function. This includes proliferation, apoptosis, transformation, and the mediation of cellular responses by various extracellular stimuli (31). Several lines of evidence suggest a role for ROS in the production of cytokines, growth factors, and hormones and the activation of nuclear transcription, although the mechanisms are still unclear (31). Probably, ROS exerts a direct effect on kinases or redox-sensitive proteins by inducing conformational changes that are required for protein activation and these early signals can then elicit transcriptional activity (32). In all biological systems H2O2 is a physiological product and is well documented as influencing mast cell behavior, although the findings are often contradictory about the beneficial or detrimental effects of H2O2 on mast cell effector responses (33, 34).

In the present study we find that low concentrations of H2O2, known not to alter cell growth or apoptosis (14) or to significantly promote cell degranulation (our unpublished data) can induce IL-4 production in mast cells independently of other stimuli and, together with Fc{epsilon}RI stimulation, further enhance IL-4 production. We demonstrate that H2O2–mediated induction of IL-4 is dependent on Fyn kinase and p38 MAPK activity, which drives both AP-1 and NFAT binding to their respective consensus regions on the IL-4 promoter. This appears to be independent of a requirement for the Lyn kinase activity, LAT phosphorylation, and is primarily independent of ERK and JNK because these MAP kinases were not potently stimulated by H2O2 treatment.

Our findings are in agreement with several previous studies that demonstrated the involvement of PI3K/p38 activated pathway, Fc{epsilon}RI-induced IL-4 production in the mast cell (23, 35). It is known that PI3K regulates the activation of Rac, which, in turn, activates p21-activated kinase resulting in the induction of p38 MAPK and JNK (36). Moreover, the Fyn kinase is known to be required for PI3K activation, and Fyn deficiency results in the reduction of both p38 MAPK and JNK activation but not that of ERK (23). Additionally, the importance of MAPKs for Th2 cytokine production has been reported (37). Thus, the collective findings provide a strong argument for the role of Fyn and PI3K in regulating MAPK activity that promotes the Th2 cytokine response of mast cells.

The physiological production of IL-4 is strictly regulated and is restricted to activated T cells and mast cells/basophils as a result of the coordination and cooperation of NFAT and AP-1 on the IL-4 gene promoter (29). In both T cells and mast cells it was demonstrated that NFAT is associated with the IL-4 transcription complex and that is involved in IL-4 production (17). Although the importance of AP-1 binding is less clear, both NFAT and AP-1 are transcriptional factors known to be redox regulated. In fact, it has been previously demonstrated that AP-1 DNA binding activity was increased after 1 h of exposure to a micromolar concentration of H2O2, probably due to AP-1 phosphorylation by stress-inducible protein kinases like JNK or p38 MAPK (38). NFAT binding to DNA was also demonstrated to occur in an H2O2-dependent manner, because it was impaired by preincubation with the specific H2O2 scavenger catalase (39). Importantly, we now establish a connection between the H2O2 induction of early signaling molecules and the activation of both AP-1 and NFAT activities that promote the consequent IL-4 gene transcription and cytokine production.

In conclusion, our findings demonstrate a strong similarity between the weak stimulation of Fc{epsilon}RI (7) and H2O2 treatment. Both stimuli induced the selective activation of signal molecules, leading to a limited profile of mast cell responses (4, 7). Low doses of Ag preferentially favored the phosphorylation of Gab2, which is a consequence of Fyn activation (8), and p38 MAPK activation resulting in expression of some cytokines, among them IL-4 (7). Similarly, H2O2 stimulation induced the activation of Fyn with the consequent phosphorylation of PI3K and Akt and p38 MAPK activation, both Fyn and p38 MAPK being required for IL-4 production. It should be noted that selective responses as a consequence of weak stimulation is not a phenomenon restricted to mast cells. T cell activation through the TCR results in preferential cytokine production related to the strength of the stimulus. In fact, it was demonstrated that both weak and strong TCR stimuli induce different calcium responses with the amplitude of the calcium signal correlating with IFN-{gamma} production, while IL-4 expression was induced in suboptimal conditions (40). Our findings of IL-4 production by mast cells in an oxidative microenvironment represents another manner in which these cells may contribute to the immune response. Because these cells are an important part of the innate immune response (1), one might envision that, at inflammatory sites, oxidative stress might induce mast cell and basophil IL-4-production that could direct the Th2 cell differentiation (20). The fact that Fc{epsilon}RI-null mice, when infected with Schistosoma mansoni, develop a normal Th2 response suggests that this may be a possibility (21). We now establish that an oxidative microenvironment can activate mast cell responses through selective activation of Fyn-dependent signals. These findings demonstrate a previously unrecognized role for Fyn-dependent signaling in response to oxidative stress. Studies aimed at exploring whether the oxidative microenvironment can lead to mast cell activation in vivo should reveal whether this plays a key role in the mast cell repertoire in pathophysiological conditions.


   Acknowledgment
 
We thank Dr. Marco De Carli for a useful discussion.


   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 a grant from Ministero dell’Istruzione Università e Ricerca (PRIN 2005), Agenzia Spaziale Italiana. (Progetto OSMA), and LR.11 del Friuli Venezia Giulia. Back

2 Address correspondence and reprint requests to Dr. Carlo Pucillo, Immmunology Section, Department of Biomedical Science and Technology, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy. E-mail address: carlo.pucillo{at}uniud.it Back

3 Abbreviations used in this paper: BMMC, bone marrow-derived cultured mast cells; LAT, linker of activated T cells; ROS, reactive oxygen species; SCF, stem cell factor. Back

Received for publication September 22, 2006. Accepted for publication December 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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