MK2/3 Are Pivotal for IL-33–Induced and Mast Cell–Dependent Leukocyte Recruitment and the Resulting Skin Inflammation

Mast cells modulate innate and adaptive immune responses. Thereby, mast cells regulate leukocyte recruitment (1–4), dendritic cell maturation and functions (5, 6), promote Th₁ and Th₁₇ responses, and increase the functionality of CD8⁺ T cells (5, 6). Therefore, mast cells are involved in tissue inflammation (1, 6), and contribute to immune responses against pathogens (5). Mast cells are also essential in type I hypersensitivity, and are central to the pathogenesis of allergic diseases (7). The participation of mast cells in the pathogenesis of autoimmune diseases has also been reported (8–10). Furthermore, mast cells regulate the tumor microenvironment (11), and expression of constitutively active c-Kit mutants in mast cells results in the development of mastocytosis and mast cell tumors (12–14).

C-Kit and its ligand stem cell factor (SCF) are essential for mast cell differentiation in vivo (15). The activation of mature mast cells is typically mediated by crosslinking of the FcεRI. This leads to the release of mediators including cytokines, chemokines, histamine, and proteases in an NFAT-dependent manner (16). Mast cells are also activated by Toll/IL-1R family members, such as IL-33R (17). The biological function of IL-33R depends on the IL-1 associated protein (IL-1Racp) (18, 19), and is modulated by activated c-Kit as well as IL-3 (20–22). IL-33, the ligand of IL-33R, is stored as an alarmin, and is released by necrosis (23). Therefore mast cells, via IL-33R, are the sensors of cell injury, and mediate local inflammation (24, 25). Stimulation of mast cells with IL-33 induces an FcεRI-independent production of IL-6 and IL-13 (26) via the canonical NF-κB signaling (27) and p38 (28). P38 is known to mediate the activation of MK2 and MK3, two serine/threonine kinases, which are essential for the stabilization of IL-6 and TNF-α mRNAs (29). Thereby, the MK2/3-dependent stabilization of IL-6 and TNF-α mRNAs is mediated by adenylate–uridylate-rich elements in the 3′ non-translated region of these mRNAs and the MK2/3-mediated exchange of adenylate–uridylate–rich element-binding proteins (30, 31). Furthermore, MK2/3 is involved in the activation of the PI3K-protein kinase B (PKB)/Akt signaling and of ribosomal S6 kinases (RSKs) in neutrophils, macrophages, and dendritic cells (32–34). We found that MK2/3 are essential for the IL-33-induced signaling to mediate cytokine production in vitro and the resulting proinflammatory mast cell effector functions in vivo.

Mice were maintained at the Animal Research Facility of the Universitätsklinikum Jena, the Medizinische Hochschule Hannover, and the Technische Universität Dresden, and were kept under specific pathogen-free conditions. We used sex- and age-matched myd88^−/−, mk2^−/−, mk2/3^−/− (29) and wild-type (wt) littermates. Furthermore, we used mast-cell deficient Mcpt5-cre⁺-RDTA and Mcpt5-cre⁻-RDTA control mice (7). All animal experiments were approved by the appropriate institutional and governmental committees for animal welfare, and by the Landes-direktion Dresden (24-9168.11-1/2012-38).

Bone marrow obtained from femur and tibia was cultured in IMDM (PAA Laboratories GmbH) supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-ME and 20 ng/ml ×63Ag-653 bovine papilloma virus-rmIL-3 supernatant. Cells were used when cultures consisted of 95% bone marrow–derived mast cells (BMMCs) (after 4 wk).

For characterization, BMMCs were washed with PBA (0.25% BSA; 0.02% sodium azide in PBS) and were treated with anti-CD16/CD32 (clone 2.4G2) and rat-IgG (Jackson) to avoid non-specific Ab binding. BMMCs were stained with anti-murine IL-33R [3E10 (35)], CD117 and FcεRI (both from eBioscience). Prior to CD107α staining, cells were treated with PMA/Iono (10 nM/2 μM) or IL-33 (50 ng/ml) for 1 h. BMMCs were analyzed with the LSR II flow cytometer (BD Bioscience) and evaluated data with FlowJo 8.1.1 (Treestar, Ashland, OR). For preparation of cell suspensions and the subsequent analysis by flow cytometry (after 24 h and 22 d), ear skin was cut into pieces and digested in DMEM (PAA Laboratories GmbH) containing 20 mM HEPES, 0.025 mg/ml Liberase TM (Roche), 396 U/ml DNase I (Roche), and 0.5 mg/ml Hyaluronidase (Sigma-Aldrich) at 37°C. Samples were passed through a sieve (40 μm) and were washed with PBS. Subsequently, for the determination of leukocytes, macrophages, mast cells, and neutrophils, cells were resuspended in PBS/2% BSA and stained with anti-murine CD117, FcεRI, CD45, F4/80, CD11b, and Ly6G (all from eBioscience). Cells were washed in PBS/2% BSA and analyzed with the Miltenyi MACSQuant flow cytometer and MACSQuantTM or FlowJo Analysis Software.

BMMCs (10⁶ cells/ml) were washed and seeded in IL-3-free media. After 1 h cells were incubated (30 min) with vehicle (DMSO) or different inhibitors (all Calbiochem) (as indicated in the figures) and stimulated with IL-33 (Peprotech) (50 ng/ml). Cells were lysed (20 mM HEPES, pH7.5; 10 mM EGTA; 40 mM β-glycerophosphate; 2.5 mM MgCl₂; 2 mM orthovanadate; 1 mM DTT; 20 μg/ml aprotinin; 20 μg/ml leupeptin supplemented with 1% Triton) and protein concentration was determined (BCA-kit; Pierce). Samples were boiled in 6× Laemmli buffer, were separated on 10% NaDodSO₄ (SDS) Laemmli gels and were transferred on to nitrocellulose membranes (BioStep) by electroblotting. Membranes were blocked with dry milk and incubated with Abs-detecting phosphorylated/non-phosphorylated proteins. We used anti-pT180/Y182-p38, anti-pS177-IKK2, anti-pT183/Y185-JNK1/2, anti-pT202/Y204-ERK1/2, anti-pS473-PKB/Akt, anti-pS32-IκBα, anti-pT389-p70S6K and the respective anti-total Abs (all from Cell Signaling except anti-IKK1/2 [Santa Cruz]). Membranes were washed in 0.1% Tween/TBS and incubated with HRP-conjugated secondary Abs: anti-rabbit-Ig, anti-goat-Ig (both Santa Cruz), and anti-mouse-Ig (Thermo Scientific). Detection was performed with an ECL reagent (Pierce).

For calcium assays, the FLIPR Calcium 4 Assay Kit and the FlexStation3 microplate reader (Molecular Devices, Sunnyvale, CA) were used. All recordings were done on poly-l-lysine–coated 96-well plates (Greiner). Per well, 1 × 10⁵ BMMCs were resuspended in 80 μl Phenol Red-free DMEM (Life Technologies), and were mixed with 20 μl Ringer’s solution (80 mM KCl; 130 mM NaCl; 1 mM MgCl₂; 1 mM CaCl₂; 10 mM HEPES pH7.3; 20 mM glucose; Probenecid 25 mM) and an equal volume of loading dye. Prior to stimulation with IL-33 cells were pretreated with inhibitors (30 min).

BMMCs (10⁶ cells/ml) were washed and seeded in IL-3-free media. After 1 h cells were incubated with vehicle (DMSO, ethanol) or inhibitors (30 min) (as indicated in the figures) and were stimulated with SCF, IL-3 or IL-33 alone, or IL-33 in combination with SCF or IL-3 (all 50 ng/ml). After 8 h supernatants were collected and analyzed for cytokines by ELISA using matched pair Abs (eBioscience).

Skin inflammation was induced by intradermal (i.d.) injection of IL-33 (500 ng/10 μl) into the ear pinnae every other day for a period of 22 d. In some experiments mice were treated with pyrilamine (10 μg/g bodyweight) prior to IL-33 injection. For reconstitution experiments, mast cell deficient Mcpt5-cre⁺-RDTA mice were locally reconstituted with wt or mk2/3^−/− BMMCs by i.d. injection of 2 × 10⁶ BMMCs in 20 μl sterile saline into the ear pinnae. After reconstitution, IL-33 (500 ng/10 μl) or PBS was i.d. injected into the ear pinnae every other day over a period of 22 d. The inflammation in all experiments was assessed as ear swelling by measuring the ear thickness with a caliper (Mitutoyo) every other day and by quantification of skin infiltrating cell subsets using flow cytometry at day 22. Ear swelling was calculated as percentage of ear thickness increase related to the basal ear thickness.

Blot intensities were quantified with LabImage 1D (Kapelan Bio-Imaging GmbH, Germany). Statistical analysis was performed with IBM SPSS Statistics version 20.0 (IBM, Ehningen, Germany). Significance was assessed by a Mann–Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001). All Western blots were performed at least three times. Proliferation assays and cytokine ELISAs were performed at least three times in at least a six-fold determination (shown as one representative experiment). Results are shown as the mean of measurements ± SD.

We determined the relevance of the p38 targets, MK2 and MK3 (29), for the IL-33-induced signaling and effector functions. Therefore, we used BMMCs from mk2^−/− and from the MK2/MK3 double knockout mk2/3^−/− mice. The surface expression of IL-33R, c-Kit and the FcεRI was similar in wt and mk2/3^−/− BMMCs showing that MK2/3 does not influence mast cell maturation in vitro (Supplemental Fig. 1A). IL-33 induces a MyD88- TGF-β-activated kinase 1 (TAK1)-dependent activation of NF-κB (27), of MAP-kinases as p38, and of PKB/Akt resulting in production of IL-6 and IL-13 (Supplemental Fig. 1B–G). In contrast, IL-33 alone did not induce the production of TNF-α (22). These cytokines are known to mediate inflammatory and allergic responses (36–43). We focused on the p38 targets MK2/3, and their role in the IL-33-induced production of IL-6, IL-13 and TNF-α. MK2/3 deficiency blocked the IL-33-induced production of IL-6 and IL-13 (Fig. 1A). IL-3 enables IL-33 to induce the production of TNF-α [Supplemental Fig. 2A and (22)]. We determined the role of MK2/3 in the TNF-α production induced by costimulation with IL-3 and IL-33. In mk2/3^−/− BMMCs this TNF-α response was blocked (Supplemental Fig. 2A). Furthermore, we speculated that stimulation with SCF also mediates an IL-33-induced and MK2/3-dependent TNF-α response. Indeed, stimulation with SCF enables IL-33 to induce a MK2/3-dependent TNF-α production (Supplemental Fig. 2B). This demonstrates that growth factors are essential to enable IL-33 to induce a MK2/3-dependent TNF-α production. Together, these results show that the p38-MK2/3 signaling axis is essential for the IL-33-induced cytokine production.

Next, we investigated the role of MK2/3 in IL-33-induced signaling. MK deficiencies slightly influence the longitude of IKK activation but did not affect the activation of JNK1/2 (Fig. 1B, 1C). In contrast, activation of PKB/Akt, S6K1 (p70S6K) and ERK1/2 was reduced in mk2^−/− BMMCs and abolished in mk2/3^−/− BMMCs (Fig. 1C). Furthermore, and as described previously (44), MK2/3 deficiency resulted in a strongly reduced expression and activation of p38 (Fig. 1C). These data show that MK2/3 are essential for activation of PKB/Akt, p70S6K and ERK1/2. Next, we investigated the role of PI3K-PKB/Akt and ERK1/2 signaling in IL-33-induced cytokine production using the PI3K-inhibitor LY294002 and the MEK-ERK1/2 inhibitor U0126. PI3K inhibition reduced the production of IL-6 and IL-13 (Fig. 2A), and blocked the activation of p70S6K but did not affect the activation of ERK1/2 (Fig. 2B), IKKs, and p38 (Fig. 2C). MEK inhibition also reduced the production of IL-6 and IL-13 (Fig. 2D), the activation of ERK1/2, and p70S6K (Fig. 2E) whereas the activation of IKKs and p38 were not affected (Fig. 2F). The combination of PI3K- and MEK-inhibition further reduced the IL-33-induced production of IL-6 and IL-13 (Supplemental Fig. 2C).

Next, we tested the role of PI3Ks and MEK-ERK1/2 signaling on TNF-α production. As shown in Supplemental Fig. 2D, inhibition of PI3Ks or MEK reduced TNF-α production. Consequently, inhibition of PI3Ks and MEK blocked the induced production of TNF-α (Supplemental Fig. 2D). This data indicated that ERK1/2, and PI3Ks mediate IL-33-induced cytokine production via the activation of RSKs. To test the role of RSKs we used the RSK-inhibitor SL0101 (45). Preincubation with SL0101 blocked the IL-33-induced cytokine production (Supplemental Fig. 2E, 2F). Together, these data demonstrate that the IL-33-induced cytokine response depends on the MK2/3-mediated activation of the PI3K-PKB/Akt- and on the ERK1/2 signaling resulting in activation of RSKs.

To check the in vivo relevance of IL-33-induced, and MK2/3-dependent mast cell activation, we used the IL-33-induced skin inflammation model, which is known to be mast cell dependent (2). For these experiments, we used mast cell deficient Mcpt5-cre⁺-RDTA mice, which are characterized by a depletion of connective tissue mast cells, and an otherwise unaltered immune system (7). First we tested Mcpt5-cre⁺-RDTA mice in the IL-33-induced skin inflammation model. In control Mcpt5-cre⁻-RDTA mice, which display normal mast cell development (7), IL-33 induced a rapid ear swelling, which decreased to basal levels after 24 h (Fig. 3A). In contrast, in mast cell-deficient Mcpt5-cre⁺-RTDA the rapid IL-33-induced ear swelling was strongly suppressed (Fig. 3A). This demonstrates the functionality of this mouse model and shows that IL-33 induces a rapid and mast cell-dependent skin inflammation. Rapid inflammatory and allergic responses (e.g., stimulation of the FcεRI) are mostly dependent on degranulating and histamine-producing mast cells. These processes are Ca²⁺-dependent. Stimulation of mast cells with IL-33 also induced Ca²⁺ release (Supplemental Fig. 3A), and a Ca²⁺-dependent cytokine production [Supplemental Fig. 3B, 3C and (22)]. However, IL-33 did not induce histamine production (26) or degranulation (Supplemental Fig. 3D). Therefore, we expected that the anti-histamine pyrilamine would not reduce the IL-33-induced inflammatory response and ear swelling. Unexpectedly, treatment with pyrilamine reduced the IL-33-induced rapid response to a similar extent as observed in mast cell-deficient Mcpt5-cre⁺-RTDA mice (Fig. 3A). These data show that IL-33 induces a rapid inflammatory response, which strongly depends on histamine production. To test the relevance of mast cell MK2/3 in rapid IL-33-induced skin inflammation we performed reconstitution experiments. We reconstituted mast cell-deficient Mcpt5-cre⁺-RDTA mice with in vitro generated wt or mk2/3^−/− BMMCs. Compared to Mcpt5-cre⁻-RDTA control mice, the reconstitution of Mcpt5-cre⁺-RDTA mice with wt BMMCs completely recovered the IL-33-induced inflammatory response (Fig. 3B). Reconstitution of Mcpt5-cre⁺-RDTA mice with mk2/3^−/− BMMCs reduced the IL-33-induced ear swelling (Fig. 3B). Because mast cell deficiency completely blocked the IL-33-induced ear swelling, this indicates a partial contribution of mast cell MK2/3 to the IL-33-induced rapid inflammatory response. IL-33 induces leukocyte attraction (2, 3). Given that histamine and mast cells are essential for rapid IL-33-induced inflammatory responses, we determined whether IL-33 induces histamine- and/or mast cell-dependent infiltration of leukocytes into ear skin. In control mice, IL-33 increased the number of CD45⁺ leukocytes and Ly6G⁺/CD11b⁺ neutrophils in the ear skin, which was slightly reduced in pyrilamine-treated control mice but was strongly reduced in mast cell-deficient Mcpt5-cre⁺-RDTA mice (Fig. 3C, 3D). Next, we determined whether reconstitution with either wt or mk2/3^−/− BMMCs leads to an equal mast cell repopulation in Mcpt5-cre⁺-RDTA mice. We found that IL-33 equally increased the number of mast cells in Mcpt5-cre⁺-RDTA mice either reconstituted with wt or mk2/3^−/− BMMCs (Fig. 3E). This is in line with earlier reports showing that IL-33 induces survival and/or proliferation of mast cells (9, 28). Furthermore, this demonstrates an equally efficient reconstitution of wt or mk2/3^−/− BMMCs in Mcpt5-cre⁺-RDTA mice. Together, this shows that the rapid skin inflammation is predominantly mediated by histamine and mast cells.

Next we investigated whether IL-33 induces an MK2/3-dependent late-phase inflammatory response. In Mcpt5-cre⁻-RDTA control mice, IL-33 induced a strong ear swelling, which persisted for up to 3 wk and was strongly reduced in mast cell deficient Mcpt5-cre⁺-RDTA mice (Fig. 4A). Reconstitution of mast cell deficient Mcpt5-cre⁺-RTDA mice with in vitro generated wt BMMCs completely restored the IL-33-induced ear swelling and is comparable with Mcpt5-cre⁻-RDTA control mice (Fig. 4B). In contrast, reconstitution of Mcpt5-cre⁺-5-RDTA mice with in vitro generated mk2/3^−/− BMMCs strongly decreased the IL-33-induced late-phase inflammatory response in the first 16–18 d (Fig. 4B). From day 18 the IL-33-induced ear swelling increased again in Mcpt5-cre⁺-RTDA mice reconstituted mk2/3^−/− BMMCs (Fig. 4B). Together, these data show that in contrast to rapid inflammatory responses, the late-phase response strongly depends on MK2/3. Therefore, we hypothesized that mast cell MK2/3 mediates the IL-33-induced influx of leukocytes. In Mcpt5-cre⁻-RDTA control mice, IL-33 induces a strong influx of CD45⁺ leukocytes (Fig. 4C), neutrophils (Fig. 4D) and Ly6G⁺ macrophages (Supplemental Fig. 4A, 4B), which was reduced in mast cell deficient Mcpt5-cre⁺-RDTA mice (Fig. 4C, 4D, Supplemental Fig. 4A, 4B). Reconstitution of Mcpt5-cre⁺-RDTA mice with wt BMMCs recovered the infiltration of these cells (Fig. 4C, 4D, Supplemental Fig. 4A, 4B). In contrast, reconstitution of Mcpt5-cre⁺-RDTA mice with mk2/3^−/− BMMCs again reduced the influx of CD45⁺ leukocytes (Fig. 4C), neutrophils (Fig. 4D), and macrophages (Supplemental Fig. 4A, 4B). These data demonstrate that mast cell MK2/3 is involved in the IL-33-induced leukocyte attraction, which results in late-phase inflammatory responses.

We found that the MAPK-activated protein kinases MK2 and MK3 are pivotal for IL-33-induced cytokine production in vitro and inflammatory responses in vivo. We show that IL-33 induces the MK2/3-dependent activation of PKB/Akt and ERK1/2, which are both required for p70S6K activation and the resulting cytokine response. How MK2/3 activates the PI3K-PKB/Akt-p70S6K-signaling and ERK1/2 is unknown. Interestingly, McGuire et al. showed that MK2 modulates the availability of PIP₃ (32). We found that pharmacological inhibition of PI3Ks and MK2/3 deficiency reduced the IL-33-induced activation of p70S6K. Therefore, we conclude that IL-33 mediates a MK2/3- and PI3K-dependent activation of the PKB/Akt-p70S6K signaling axis. We also found a complete loss of ERK1/2 activation in mk2/3^−/− BMMCs. ERK1/2 activation is typically mediated by MEK1/2, which phosphorylates ERK1/2 on the T202/Y204 motif (46). We speculate that MK2/3 phosphorylates MEK1 on T292, which fits the MK2/3 consensus phosphorylation site (ØXRXXS/TØ) (47) and is also essential for ERK activation (46). Together our data show that MK2/3 are essential for activation of the PI3K- and MEK-ERK1/2- signaling and the resulting IL-33-induced cytokine production in mast cells. We propose a model in which NF-κB-dependent cytokine production is supported by two signaling pathways: 1) the activation of the p38-MK2/3 module, which promotes mRNA stability (29), and 2) the p38-MK2/3-PKB/Akt(ERK1/2) signaling pathway, which is involved in translational processes by inducing p70S6K activation (48).

Given that MK2/3 are essential for IL-33-induced cytokine production, we tested the in vivo relevance of mast cell MK2/3 in IL-33-induced inflammatory responses. Therefore, we used mast-cell deficient Mcpt5-cre⁺-5-RDTA mice and reconstituted them with wt and mk2/3^−/− BMMCs (7). With this model we demonstrate that the IL-33-induced rapid inflammatory response partially depends on MK2/3 but is surprisingly completely mediated by histamines. Because IL-33 does not induce degranulation and/or the release of histamine (26), this indicates that IL-33 indirectly induces mast cell degranulation. Indeed, in vivo, IL-33 induces B-cell activation, which results in an IgE-dependent but allergen-independent histamine production in mast cells (49). This suggests the IL-33-induced and pyrilamine-sensitive rapid skin inflammation is indirectly mediated via B cell activation. However, compared with pyrilamine treatment, mast cell MK2/3 only modestly contributes to the IL-33-induced early inflammatory response, which indicates an MK2/3-independent pathway leading to mast cell degranulation and/or histamine production (Fig. 5). This is in accordance to recently published data showing that the upstream signaling of MK2/3, the MKK3-p38 module, is not involved in the FcεRI-mediated Ca²⁺ release and degranulation (50, 51). Together, we hypothesize that IL-33 induces two signaling events: 1) the MK2/3-dependent cytokine production leading to leukocyte recruitment (Fig. 5), and 2) the MK2/3-independent degranulation, presumably mediated by B cell activation (Fig. 5).

The IL-33-induced late-phase inflammation strongly depends on MK2/3. Interestingly, from day 18, ear swelling in Mcpt5-cre⁺-RDTA mice reconstituted with mk2/3^−/− BMMCs increased again. This indicates a compensatory effect that reduces the dominant role of MK2/3 in IL-33-induced late-phase reactions. However, the IL-33-induced influx of neutrophils and macrophages depends on mast cell MK2/3. Given that the TNF-α production mediates skin inflammation (36–38), and that the IL-33-induced TNF-α response in mast cells contributes to infiltration of neutrophil into the peritoneal cavity (3), this indicates an important role of TNF-α in IL-33-induced leukocyte recruitment. Importantly, we found that the presence of SCF is the precondition for IL-33 to induce an MK2/3-dependent TNF-α production in BMMCs. These data support a model in which the permanent interaction of membrane-bound SCF with c-Kit in the periphery not only mediates mast cell survival but also enables IL-33 to induce TNF-α production, and therefore to attract neutrophils in vivo (Fig. 5). Consequently, we suggest that the p38-MK2/3-TNF-α axis induced by costimulation with SCF and IL-33 is essential to attract neutrophils to the side of inflammation (Fig. 5). In addition we show that the IL-33-induced production of IL-6 and IL-13 also depends on MK2/3. Interestingly, IL-6R and IL-13R signaling are crucial for the development of skin inflammation by inducing increased numbers of T cells, macrophages, and mast cells (39–43). Together with these previously published data, our results indicate that the MK2/3-dependent signaling and cytokine production in mast cells contributes to the IL-33-induced inflammatory responses. Therefore, we hypothesize that the release of the alarmin IL-33 by damage of endothelial cells leads to a strong MK2/3-dependent mast cell activation resulting in cytokine production and thus to attraction of leukocytes (Fig. 5). Consequently, targeting MK2/3 by pharmacological inhibitors might be an alternative and novel approach to combat inflammation induced by the alarmin IL-33.

We are grateful to Claudia Küchler, Karin Müller, and Freya Rost for excellent technical support.

The authors have no financial conflicts of interest.