MAPK Kinase 3 Specifically Regulates FcεRI-Mediated IL-4 Production by Mast Cells

Mast cells play a central role in IgE-dependent allergic diseases and are important immune effector cells. Through constitutive expression of the high-affinity receptor, FcεRI, mast cells are sensitive to, and activated by, Ag-dependent cross-linking of IgE on the cell membrane (1). Activation through FcεRI induces an immediate signaling cascade that culminates in the release of various proinflammatory mediators, including potent granule-bound preformed components (e.g., histamine), as well as in the activation of transcription factors that direct gene expression of key cytokines and chemokines (2, 3). Allergic diseases are the manifestation of excessive release and production of these mast cell-derived inflammatory mediators (3). Mast cell-signaling mechanisms that drive the production of specific mediators remain incompletely defined. Elucidation of the signaling mechanisms regulating specific mediator release is the focus of this study.

FcεRI is a multisubunit receptor that includes an IgE-binding α-chain, a signal-amplifying β-chain, and two signal-initiating γ-chains (4). Upon FcεRI cross-linking, Src family protein tyrosine kinases Lyn and Fyn are activated, which then cooperatively activate several important pathways, including the MAPK-signaling pathway, as well as PI3K, IκB–NF-κB and NFAT, resulting in mast cell degranulation and the production of inflammatory cytokines (5–8). The MAPK family includes subgroups p38, ERK, and JNK. Specific MAPK activation is controlled by upstream MAPK kinases (MKKs), which generally act on specific MAPKs. The p38 subfamily is activated by MKK3 and MKK6, with MKK4 also having some specificity for this subfamily (9).

The impact of activation of many of these signaling pathways, including p38 MAPK, is generally at the gene-expression level, because downstream transcription factors are activated (10). These transcription factors then bind to the promoter of inflammatory cytokine genes and drive gene expression. Mast cells secrete many important cytokines following IgE-dependent signaling, including IL-6, IL-13, IL-4, and TNF. We recently described the role of the de novo-expressed transcription factor early growth response-1 (Egr1) in the full responsiveness of mast cell cytokine production following FcεRI signaling (11). The concerted activity of inducible transcription factors like Egr1, with others, including NF-κB and NFAT, can direct mast cell mediator production and shape the cytokine milieu at the site of inflammation (12, 13).

The significance of the MKK3–p38 MAPK pathway in mast cell function has not been examined. In this study, we identified MKK3 as a proinflammatory mediator of allergic inflammation in vivo and characterized the mechanism of MKK3-dependent events in activated mast cells. MKK3 deficiency led to impaired inflammatory response in an in vivo model of cutaneous allergic reaction. Mast cells from MKK3-deficient mice displayed no defect in maturation or degranulation, but they displayed impaired production of proinflammatory cytokine IL-4 in vitro and in vivo and produced an impaired IgE-dependent allergic inflammatory response in vivo in reconstituted mast cell-deficient mice. MKK3 was activated rapidly (5 min) following IgE-dependent signaling in mast cells and persisted for >6 h. MKK3 was required for full p38 activity, as well as for full activation of Egr1. Furthermore, Egr1 bound to the IL-4 promoter in activated mast cells, demonstrating that this MKK3-dependent transcription factor can directly drive IL-4 gene transcription. Thus, MKK3 is an important component of FcεRI signaling and a specific mediator of IL-4 production in allergic inflammation.

MKK3 knockout mice were generated, as previously described (14), and obtained from The Jackson Laboratory (B6.129S1-Map2k3tm1Flv/J). Mast cell-deficient W-sh mice were also obtained from The Jackson Laboratory (B6Cg-kit W-sh/HNihJacBsmJ NistltF4). The protocols were approved by the University Committee on Laboratory Animals, Dalhousie University, in accordance with the guidelines of the Canadian Council on Animal Care.

Abs to phospho-MKK3/6, phospho-p38 MAPK (Thr 180/Tyr 182), phospho-JNK (Thr 183/Tyr 185), JNK, phospho-ERK1/2, and ERK were purchased from Cell Signaling Technology. Abs to MKK3, p38 MAPK, Egr1, Oct-1, and actin and HRP-linked secondary Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated rat anti-mouse c-Kit mAb and FITC-rat IgG2a were purchased from Cedarlane Laboratories (Hornby, ON, Canada). FITC-conjugated rat anti-mouse IgE (IgG1) and FITC-rat IgG1 were purchased from BD Biosciences (San Jose, CA). Alexa Fluor 594 goat and rabbit secondary Ab was from Invitrogen (Eugene, OR). Abs for IL-4 (clone 11B11) and isotype control rat IgG1κ, as well as DyLight 594 goat anti-rat secondary Ab were from BioLegend (San Diego, CA).

Mouse bone marrow-derived mast cells (BMMCs) were cultured, as previously described (11). Mast cells were confirmed by toluidine blue staining and flow cytometry analysis for c-Kit and IgE receptor expression (FACSAria). Following 5–6 wk in culture, mast cell purity was >98%. BMMCs were passively sensitized with IgE from TIB-141 cells (American Type Culture Collection). Cells were then activated by stimulation with 10 ng/ml trinitrophenyl (TNP)-BSA (Biosearch Technologies, Novato, CA). Mast cell degranulation was determined by measuring β-hexosaminidase release.

BMMCs were lysed in radioimmunoprecipitation assay buffer supplemented with a mixture of protease and phosphatase inhibitors. Cleared lysates (30 μg protein) were subjected to electrophoresis in 10% SDS-polyacrylamide gels. Gels were transferred to polyvinylidene difluoride membrane, blocked with 5% nonfat milk powder, probed with primary and secondary Abs, and detected by an ECL-detection system (Western Lightning Plus-ECL; PerkinElmer) on BioMax film (Kodak). Blots were scanned and quantified using ImageJ software.

Protein arrays (Mouse Inflammation Array 1; RayBiotech, Norcross, GA) were conducted according to the manufacturer’s protocol. Mast cells were either unstimulated or stimulated with 10 ng/ml TNP-BSA for 6 h. ELISAs were used to measure cytokine concentrations in cell culture supernatant from mast cells stimulated with TNP-BSA and were conducted according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Abs and standards (DuoSet) were from R&D Systems (Minneapolis, MN). For serum IgE, whole blood was collected from MKK3^+/+ and MKK3^−/− mice, and cleared serum was assayed for total IgE using a mouse Total IgE ELISA kit, according to the manufacturer’s protocol (MD Biosciences, St. Paul, MN).

Mast cells were incubated for 30 min with 2 μM fura 2-AM. After washing, mast cells were resuspended in phosphate buffer with 1.5 mM CaCl₂ at a concentration of 1 × 10⁶ cells/ml. Fluorescence was measured by placing 2 ml mast cell suspension in a 37°C thermostated quartz cuvette with magnetic stirring in an RF-1501 spectrofluorophotometer (Shimadzu, Tokyo, Japan).

Mice were passively sensitized by i.v. injection of 2 μg anti-DNP IgE mAb (Sigma-Aldrich, St. Louis, MO). After 24 h, a cutaneous reaction was elicited by the application of 20 μl dinitrofluorobenzene (DNFB, 0.3% w/v; Sigma-Aldrich) in acetone/olive oil (4:1) to both sides of the right hind paw or right ear and 20 μl of acetone/olive oil to the left hind paw or left ear as a control. The thickness of the footpad or ear was measured using a digital micrometer after 24 h. The weight of the hind paw or ear punch (8 mm) was also determined. The thickness and weight of the right ear or right hind paw (treated with acetone/olive oil only) were used as baseline values. The DNFB-induced change in tissue thickness and weight was expressed as a percentage of the baseline values.

Mice were sensitized by intradermal injection of 20 ng anti-DNP IgE mAb (Sigma-Aldrich) into ear tissue. After 24 h, mice were challenged by i.v. injection of 100 μg DNP-BSA in 200 μl Evan’s blue dye (1% w/v; Sigma-Aldrich). Thirty minutes later, ear tissue was collected in 300 μl formamide and incubated at 80°C for 2 h in a water bath to extract the Evan’s blue dye. The absorbance was determined at 620 nm.

W-sh mice were reconstituted by injection of 20 μl BMMCs at a density of 25 × 10⁶ cells/ml in RPMI 1640 (5 × 10⁵ cells/injection) into the ear and footpad. MKK3^+/+ BMMCs were injected into the left side ear and footpad, whereas MKK3^−/− BMMCs were injected into the right side ear and footpad. Five weeks later, mice received 2 μg anti-DNP IgE mAb via tail vein, and late-phase cutaneous reactions were elicited as described above, with both the left and right side receiving the DNFB application.

Ear tissues from late-phase cutaneous reactions in MKK3^+/+ and MKK3^−/− mice were collected and fixed in 10% formalin. Specimens were embedded in paraffin and sectioned onto microscope slides. Briefly, specimens were deparaffinized in xylene, rehydrated in decreasing concentrations of alcohol, and incubated in 10 mM citrate-Tween buffer at 95°C for 30 min for Ag unmasking. Specimens were blocked in 2% goat serum/5% BSA in phosphate buffer, incubated overnight with primary Ab (rat anti-mouse IL-4, clone 11B11, 20 μg/ml) or isotype control (rat IgG1κ) diluted in Perm/Wash buffer (BD Biosciences), washed in Perm/Wash buffer, incubated in secondary DyLight 594 goat anti-rat IgG (1:1000), and mounted in DAPI (VECTASHIELD). All images were taken using equivalent settings on a Nikon E600 microscope equipped with a 40× objective lens using ACT-1 software (Nikon).

BMMCs from wild-type and MKK3^−/− mice were treated or not with TNP-BSA (10 ng/ml) for 1 h, fixed, and permeabilized using Cytofix/Cytoperm solution (BD Biosciences, San Diego, CA) for 20 min on ice. Cells were then blocked with 5% goat serum (Cedarlane Laboratories) for 1 h, incubated with rabbit polyclonal anti-Egr1 Ab for 3 h, washed, and stained for 1 h with Alexa Fluor 594-conjugated secondary Ab. Fluorescence-labeled mast cells were cytocentrifuged (Cytospin 4; Thermo Shandon, Cheshire, U.K.) onto slides and mounted with DAPI (VECTASHIELD). All images were taken using equivalent settings on a Nikon E600 fluorescence microscope equipped with a 100× objective lens (Nikon, Tokyo, Japan).

Nuclear protein extracts were isolated using a nuclear extract kit (Active Motif, Carlsbad, CA), according to the manufacturer’s protocol. EMSA was performed as previously described (11). Briefly, samples (8 μg) were electrophoresed on native polyacrylamide gels with radiolabeled ([³²P]) dsDNA probe for Egr (Santa Cruz Biotechnology), dried on filter paper for 2 h at 80°C, and exposed to BioMax film (Kodak). Results were scanned and quantified using ImageJ software.

Assays were performed using the ChIP-IT Express Enzymatic kit (Active Motif), according to the manufacturer’s protocol. Briefly, BMMCs were left unstimulated or were stimulated for 1 h with TNP-BSA, fixed with 1% formaldehyde, and the nuclei were subjected to an enzymatic digestion with 5 U enzymatic shearing mixture solution for 25 min at 37°C. Sheared chromatin was immunoprecipitated with 4 μg Egr1, Oct-1 (Santa Cruz Biotechnology), or control IgG (Active Motif). Input DNA and 5% of the precipitated DNA were used as templates for each PCR, consisting of 36 cycles of 20 s at 94°C, 30 s at 54°C, and 30 s at 72°C. PCR products were separated by a 2% agarose gel. Primers for amplification of the IL-4 promoter region were 5′-GAGGGGTGTTTCATTTTCCA-3′ (forward) and 5′-TGCTGGCAGAGGTCTCTCTAT-3′ (reverse); for IL-13, 5′-GCCTTCTGCTTGTCTTGAGG-3′ (forward) and 5′-CCAGCTCCCTTCCCACTG-3′ (reverse); and for TNF, 5′-GGGGAGGAGATTCCTTGATG-3′ (forward) and 5′-TCGCTGAGGGAGCTTCTG-3′ (reverse).

The paired Student t test was used for statistical evaluation of data. Results were considered significant when p < 0.05. Data are expressed as mean ± SEM.

Activated mast cells play a role in the development of late-phase allergic reactions through the production and release of cytokines (15). To examine the role of MKK3 in late-phase allergic inflammation, MKK3-deficient and wild-type mice were sensitized with anti-DNP IgE i.v. 24 h prior to epicutaneous application of Ag, a 0.3% solution of DNFB, or a control solution on the ear and foot for 24 h. MKK3-deficient mice displayed a deficit in IgE/Ag-dependent inflammation, as assessed by ear and foot weight (Fig. 1A, 1C). This inflammatory deficit was also evident when measured by tissue thickness for the ear and foot (Fig. 1B, 1D), suggesting a regulatory role for MKK3 in allergic reaction. We also examined MKK3-deficient mice for any defect in basal serum IgE levels and found no difference compared with wild-type mice (1.04 ± 1.03 versus 1.00 ± 0.64 μg/ml, respectively; n = 3 mice).

The response of MKK3^+/− mice resembled that of MKK3^+/+ mice at the ear, yet it seemed to be more compromised at the foot, falling between the response for MKK3^+/+ and MKK3^−/− mice, although no statistical significance was observed (Fig. 1). To examine MKK3 expression in MKK3^+/− mice, skin tissue and splenic tissue were analyzed by Western blotting for MKK3. MKK3^+/− mice were found to have partially impaired MKK3 expression (Supplemental Fig. 1A).

These results indicated that MKK3 is an important regulator in the development of IgE-dependent late-phase cutaneous anaphylaxis. This finding prompted us to investigate the mechanism through which MKK3 regulates FcεRI-mediated mast cell activation.

To exclude the possibility that the inflammatory deficiency in MKK3-deficient mice is the result of abnormal mast cell development in vivo (e.g., reduced mast cell numbers), ear, back skin, and tongue tissue were collected and analyzed for mast cell numbers by Alcian blue histological staining. Mast cells were present in similar number and morphology in MKK3-deficient mice compared with a wild-type control (Supplemental Fig. 1B, 1C).

To examine the role of MKK3 in FcεRI-mediated mast cell activation, we cultured BMMCs from MKK3-deficient mice. Bone marrow was obtained from wild-type and MKK3-deficient mice and cultured in IL-3 and PGE₂-conditioned media for 5 wk. Toluidine blue staining showed similar metachromatic staining and morphology of mast cells from wild-type and MKK-deficient mice (Fig. 2A). MKK3-deficient mice have a targeted disruption removing exons 8 and 9 from the MKK3 gene, including the dual-phosphorylation site required for MKK3 protein activation (14). To confirm the absence of MKK3 protein in BMMCs, Western blotting was performed on total-protein lysates from MKK3-deficient and wild-type BMMCs. MKK3 protein was not detected in MKK3-deficient BMMCs (Fig. 2B). To further examine the development of MKK3-deficient BMMCs in vitro, BMMCs were analyzed by flow cytometry for c-Kit and IgE receptor expression; no difference was observed (Fig. 2C). These results suggested that mast cells develop normally in vivo and in vitro in the absence of MKK3.

Calcium influx following FcεRI-mediated signaling enhances and sustains mast cell-activation signals (16). To examine whether MKK3 plays a role in mast cell calcium mobilization, MKK3-deficient BMMCs were sensitized with Ag-specific anti-TNP IgE and preloaded with fura 2-AM, followed by stimulation with TNP-BSA. Calcium influx following FcεRI-mediated signaling was unaffected in MKK3-deficient BMMCs compared with wild-type control BMMCs (Fig. 2D).

To examine whether MKK3 plays a role in FcεRI-mediated mast cell degranulation, wild-type and MKK3-deficient BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for 20 min. Mast cell degranulation was assessed by β-hexosaminidase release. No effect on mast cell degranulation in vitro was detected in MKK3-deficient BMMCs (Fig. 2E). This finding was consistent with an in vivo model of mast cell degranulation in which MKK3-deficient and wild-type mice were sensitized with anti-DNP IgE intradermally in ear tissue for 24 h, followed by i.v. challenge with DNP-BSA in a 1% solution of Evan’s blue dye at the tail. Thirty minutes later, ear tissue was collected, and Evan’s blue dye was extracted to assess vascular permeability by spectrophotometry. MKK3-deficient mice did not incur a significant difference in vascular permeability in ear tissue compared with wild-type mice (Fig. 2F). These results indicated that MKK3 does not play a role in FcεRI-mediated mast cell degranulation in vitro or in vivo.

To examine the activation of MKK3 following FcεRI-mediated mast cell activation, wild-type BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA. Cell lysates were analyzed by Western blotting to examine the dynamics of MKK3 and p38 MAPK activation. MKK3 was rapidly activated by phosphorylation (by 5 min) following FcεRI-mediated activation. This activation persisted for up to 3 h, followed by a reduction to near basal levels by 6 h (Fig. 3). This early activation of MKK3 was consistent with p38 (p-p38) activation, but although p38 activity was diminished by 1 h, followed by a rebound in phosphorylation, MKK3 activity was not diminished (Fig. 3A). These findings indicated that MKK3 is a signaling target downstream of FcεRI in activated mast cells.

To examine the impact of MKK3 deficiency on the MAPK pathway, wild-type and MKK3-deficient BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA. Cell lysates were analyzed by Western blotting for activation of MAPKs p38, ERK, and JNK. Activation of p38 was specifically reduced at both early (5 min) and later (3–6 h) time points in MKK3-deficient BMMCs (Fig. 4A), whereas ERK and JNK activation was consistent with that of wild-type BMMCs (Fig. 4B, 4C). This finding demonstrated that MKK3 activity is specific to p38 in mast cells activated through FcεRI and that MKK3 is required for full activation of p38.

Mast cell-derived cytokines contribute to late-phase allergic reactions. To determine whether MKK3 signaling plays a role in FcεRI-dependent mast cell cytokine production, wild-type and MKK3-deficient BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for 6 h. Cell-free supernatants were collected and analyzed for cytokine content using a protein array to assess the cytokine profile of MKK3-deficient BMMCs. Of 40 inflammatory cytokines tested, only IL-4 production was markedly reduced in MKK3-deficient BMMCs compared with wild-type BMMCs (Supplemental Fig. 2A, 2B). To more specifically examine the dynamics of mast cell-derived cytokine production in MKK-deficient BMMCs, cell-free supernatants were prepared for various time points and analyzed by ELISA for IL-4, IL-6, IL-13, TNF, and CCL3 production. Production of IL-4 was significantly reduced at 1, 3, and 6 h following TNP-BSA stimulation (Fig. 5A), with production levels at 45, 40, and 38% of wild-type levels, respectively. IL-6, IL-13, TNF (Fig. 5B–D), and CCL3 (Supplemental Fig. 2C) production was not significantly altered in MKK3-deficient BMMCs. These data indicated that MKK3 activity in activated mast cells contributes to specific signals that promote the production of the proinflammatory cytokine IL-4 and are consistent with MKK3 playing a proinflammatory role in late-phase allergic reactions in vivo (Fig. 1).

To determine whether the in vitro deficit in BMMC IL-4 production following FcεRI-dependent signaling was consistent with in vivo IL-4 production during IgE-dependent late-phase reactions, we examined mouse ear tissues for IL-4 expression by immunohistochemistry. Treatment with Ag (DNFB) displayed an inducible expression of IL-4 in the tissue of wild-type (^+/+) mice (Fig. 6). However, a markedly impaired induction of IL-4 expression was observed in the tissue from MKK3-deficient mice compared with that from wild-type mice (Fig. 6). These data supported a role for MKK3 in IgE-dependent IL-4 production in vivo and are consistent with our in vitro data (Fig. 5A) demonstrating impaired IL-4 production in BMMCs stimulated through FcεRI.

To determine the significance of mast cell MKK3 signaling in IgE-dependent late-phase cutaneous anaphylaxis, mast cell-deficient (W-sh) mice were reconstituted at the ear and footpad with either wild-type (left side) or MKK3-deficient (right side) BMMCs. Reconstituted W-sh mice then received IgE via tail vein and were analyzed for responsiveness in IgE-dependent late-phase cutaneous anaphylaxis with a solution of 0.3% DNFB applied to both sides. Ear and foot swelling was significantly impaired in tissues reconstituted with MKK3^−/− BMMCs compared with tissues reconstituted with MKK^+/+ BMMCs (Fig. 7). This finding underscores the significance of MKK3 signaling in mast cells following IgE-dependent late-phase reactions in vivo.

Among the downstream targets of the MAPK-signaling pathway are various transcription factors that differ based on the activating cell stimuli and cell type. To examine potential transcription factors that might regulate IL-4 transcription in mast cells, the IL-4 gene promoter was analyzed for predicted transcription factor binding sites. Among those candidate transcription factors were NFAT and Egr1. To examine transcription factor activity, nuclear protein extracts from TNP-BSA–stimulated wild-type and MKK3-deficient BMMCs were analyzed by EMSA using ³²P-labeled DNA probes based on transcription factor consensus binding sites. Although NFAT activation was relatively unaffected in MKK3-deficient BMMCs (Supplemental Fig. 3), Egr1 activation was significantly reduced from 20 min to 6 h following FcεRI-mediated mast cell activation (Fig. 8A, 8B). The specificity of the Egr probe was confirmed using unlabeled or mutant probes as competitors for the labeled probe. Supershift assay was also performed to further verify specificity, and nuclear Egr protein was partially blocked by Egr1-specific Ab (Fig. 8C). Egr1 expression was also examined by immunofluorescence and found to be partially impaired in MKK3^−/− BMMCs activated through FcεRI for 1 h (Supplemental Fig. 4), suggesting a role for MKK3 signaling in the de novo production of Egr1 protein. These data indicated that Egr1 is specifically expressed and activated through an MKK3-dependent signaling mechanism in FcεRI-activated mast cells.

We previously documented impaired mast cell IL-13, TNF, and IL-4 production using Egr1-deficient mice (11, 17). A chromatin immunoprecipitation assay was performed to determine whether Egr1 interacts with the promoters of IL-4, IL-13, and TNF following FcεRI-mediated mast cell activation and to examine the impact of partial Egr-1 impairment through MKK3 deficiency. Nuclei from wild-type BMMCs either untreated or activated through FcεRI for 1 h (maximal for Egr1 activity) were enzymatically digested, followed by immunoprecipitation with anti-Egr1 Abs. Immunoprecipitates were analyzed by PCR using primers specific to the gene promoters (Fig. 9A). As shown in Fig. 9B, TNP stimulation specifically induced Egr1 binding to the promoters of all three cytokine genes. In MKK3-deficient cells, relative detection of binding to all three was clearly impaired (Fig. 9B). These results demonstrated that Egr1 is recruited to the IL-4 promoter, as well as the promoters of IL-13 and TNF following FcεRI-dependent signaling in mast cells. However, production of IL-4, but not IL-13 or TNF, was impaired in MKK3-deficient BMMCs (Fig. 5A, 5C, 5D) suggesting that, by comparison, IL-4 gene transcription may be more sensitive to partially impaired Egr1 activity.

The current study elucidated a previously unknown role for MKK3 as a regulator of FcεRI-mediated allergic inflammation and demonstrated that MKK3 can specifically regulate IL-4 production by mast cells. MKK3 is rapidly activated following FcεRI signaling in mast cells, and its activity is required for full p38 MAPK activation. MKK3 is required for the induction of an important mast cell transcription factor, Egr1, following FcεRI activation. Mast cell activation through FcεRI induces binding of Egr1 to the promoter of IL-4, suggesting a mechanism linking MKK3 signaling as a specific regulator of IL-4 production in activated mast cells via Egr1 activity (Fig. 9C).

Cytokine expression must be strictly controlled, because inappropriate expression is associated with many diseases (18). Mast cells secrete a multitude of factors that can contribute to important immune functions; however, when misregulated, they can also contribute to inflammatory diseases, such as allergy. IL-4, in particular, is associated with allergic diseases and autoimmunity (19, 20). Immune cell types of several distinct lineages can express IL-4, including CD4⁺ and CD8⁺ T cells, NKT cells, eosinophils, basophils, and mast cells (21). The most extensively studied functions for IL-4 are its role in directing Th2 cell differentiation and promotion of B cell Ab isotype class switch to IgE, a contribution that has significant implications for allergy (19). Mast cell-derived IL-4 contributes to the promotion of IgE synthesis by B cells (22). IL-4Rs are expressed in various cell types, including T cells, B cells, monocytes, epithelial cells, endothelial cells, mast cells, and synovial fibroblasts (19, 20). Thus, mast cell-derived IL-4 may have multiple targets in allergy. Mechanisms involved in FcεRI-mediated mast cell IL-4 production are unclear. It is likely that distinct mechanisms are involved in the regulation of specific mast cell mediators. In this study, we identified MKK3 as a specific regulator of mast cell-derived IL-4 in vitro and in vivo and showed that MKK3 is a proinflammatory mediator of allergic inflammation. We determined a mechanism for a mast cell-derived contribution of IL-4 in allergic inflammation, dependent on MKK3 signaling, in an IgE-dependent inflammatory response. Furthermore, we established that MKK3 signaling, even when only impaired in mast cells, plays a significant role in shaping the phenotype of proinflammatory late-phase allergic reactions (Fig. 7).

The proximal signaling pathways that regulate mast cell function following FcεRI-dependent signaling are reasonably well defined, but divergent downstream signaling events in the regulation of specific mast cell mediators warrant further study. It is notable that of a panel containing 40 proinflammatory cytokines, only IL-4 production was impaired in MKK3-deficient mast cells. This deficiency was not a general feature of MKK3-deficient cells, because calcium ionophore (A23187)-treated BMMC IL-4 production was not impaired (data not shown) (23). Therefore, MKK3-dependent IL-4 production in mast cells is specific to the FcεRI-signaling pathway and is not required for alternative mechanisms promoting IL-4 production. This finding underscores the specificity of MKK3-dependent events in FcεRI-activated mast cells. Furthermore, MKK3 was shown to play a role in IL-6 expression in synoviocytes in a model of rheumatoid arthritis (24). By contrast, IL-6 production was not affected in MKK3-deficient mast cells, further highlighting the cell-type–specific nature of MKK3 signaling in mast cells.

In a model of rheumatoid arthritis, Inoue et al. (24) characterized an MKK3-dependent defect in TNF signaling in fibroblast-like synoviocytes. The MKK3-deficient synoviocytes are deficient in IL-6 and IL-1β production following TNF treatment. MKK3-dependent TNF signaling at the joint synovium is an example of a potential secondary inflammatory deficiency in our IgE-dependent model using the footpad. IgE-dependent mast cell-produced TNF could activate synoviocytes and further perpetuate the inflammatory response through the secretion of IL-6 and IL-1β. In an MKK3-deficient mouse, the response to secreted TNF from the IgE-mediated inflammatory response is very likely compromised at the foot joint. Thus, MKK3 could play a role in IgE-mediated allergic inflammation in vivo through direct regulation of mast cell function and through regulation of the secondary response of local tissue cells to mast cell-produced cytokines.

MKK3^+/− mice seem to have reduced expression of MKK3. These results suggested that partial expression of MKK3 is sufficient to initiate an allergic response. A trend of reduced inflammation in the footpad was observed in MKK3^+/− mice, which may be potentially reflective of the more complex structure of the foot compared with that of the ear. Additional cell types that can participate in the immune response present in the foot (e.g., synoviocytes at the joints) might contribute to the secondary inflammatory response following IgE-dependent cutaneous reactions.

The specificity of MKK3 for the p38 MAPK pathway is well described (9, 25, 26). It is clear from the current study that MKK3 plays a role in p38 activation downstream of FcεRI in mast cells, but it is worth noting that MKK3 deficiency does not abrogate p38 activation completely (Fig. 4A). This suggests that other kinases with substrate specificity for p38 play a role in FcεRI signaling and raises the questions of what role MKK6 and/or MKK4 may play in mast cell activation and whether interruption of their function may exacerbate the phenotype seen in late-phase allergic reactions in MKK3-deficient mice (Fig. 9C).

Our findings defined a critical role for full Egr1 expression in the regulation of FcεRI-dependent IL-4 production by mast cells. We showed that Egr1 can bind to the promoter of the IL-4 gene in mast cells and that binding is inducible upon signaling through FcεRI (Fig. 9). This finding is consistent with a recent report that Egr1 contributes to IL-4 transcription in TCR-stimulated Th2 cells through synergistic binding activity with NFAT and NF-κB (27). Furthermore, we found that MKK3 deficiency significantly reduced Egr1 expression and activity in the nucleus of activated mast cells (Fig. 8). Thus, MKK3 likely regulates FcεRI-dependent IL-4 transcription through Egr1 (Fig. 9C).

Interestingly, FcεRI-dependent Egr1 binding to the promoters of IL-13 and TNF was also impaired in MKK3-deficient mast cells, whereas IL-13 and TNF expression was not affected, suggesting that IL-4 expression is more sensitive to Egr1 regulation. It is possible that Egr1 may interact with other transcription factors at the IL-4 promoter in a way that is distinct from that at IL-13 and TNF promoters. Alternatively, MKK3 may regulate additional transcription factors that may play a role in the regulation of IL-4 expression. We analyzed NFAT and NF-κB activation in MKK3^−/− BMMCs and found no major deficiency in these transcription factors following IgE activation. We also analyzed the IL-4 promoter for further impairment of potential transcription factors, including Oct-1, SRF, and the GATA family, but found no deficiency by EMSA and Western blot analysis (data not shown). Thus, the exact mechanism of MKK3–Egr1 pathway on the specific regulation of IL-4 expression requires further study.

Given that Egr1 is expressed de novo (11, 28), it stands to reason that MKK3 signaling likely plays a role in a mechanism supporting Egr1 gene transcription. It seems probable that MKK3 may direct downstream signaling, most likely through p38 (10, 29), which acts on the Egr1 promoter itself, which may then lead to IL-4 production through de novo-expressed Egr1 activity in the nucleus of activated mast cells. Without full Egr1 expression, IL-4 transcription is downregulated, likely as a result of this missing key transcription factor. However, the mechanism of MKK3-dependent signaling on the regulation of Egr1 expression remains to be determined.

Collectively, we demonstrated a novel finding that MKK3 signaling contributes to allergic inflammation via mast cell IL-4 production in vitro and in vivo. Given the significance of IL-4 in allergic inflammatory disorders, MKK3 offers a highly specific and unique pathway affecting mast cell IL-4 production. This finding offers a new signaling mechanism to target in the development of therapies for allergic disease. IL-4 is a strong candidate target in the development of therapeutics for allergy, as evidenced by current clinical trials testing the effectiveness of soluble IL-4R in the treatment of patients with allergic diseases (20, 30). The p38 MAPK pathway has drawn attention in the development of therapies for inflammatory conditions, such as rheumatoid arthritis, but inhibitors of p38 have been hampered by organ toxicity and other undesirable side effects (31). Targeting upstream proteins with specificity for the p38 subfamily may offer improved therapies for inflammatory disorders, including allergic diseases.

We thank Sandy Edgar and Fang Liu for excellent technical work.

The authors have no financial conflicts of interest.