The p38-MK2/3 Module Is Critical for IL-33-Induced Signaling and Cytokine Production in Dendritic Cells.

IL-33 is an IL-1 cytokine superfamily member. Binding of IL-33 to the IL-33R induces activation of the canonical NF-κB signaling and activation of MAPKs. In bone marrow-derived dendritic cells, IL-33 induces the production of IL-6, IL-13, and TNF-α. However, the signaling pathways resulting in IL-33-induced effector functions of dendritic cells are unknown. In this article, we show that the IL-33-induced cytokine production is only partly dependent on p65. Thereby, p65 mediates the production of IL-6, but not of IL-13, whereas the p38-Mapk-activated protein kinases 2/3 (MK2/3) signaling module mediates the IL-13, but not the IL-6, production. In addition, GM-CSF, which is critical for the differentiation and proliferation of bone marrow-derived dendritic cells, potentiates the p65-dependent IL-6 and the p38-MK2/3-dependent IL-13 production. Furthermore, we found that effective TNF-α production is only induced in the presence of GM-CSF and IL-33 via the p38-MK2/3 signaling module. Taken together, we found that the p38-MK2/3 signaling module is essential to mediate IL-33-induced cytokine production in dendritic cells.

The Neurobeachin-like 2 Protein Regulates Mast Cell Homeostasis.

The neurobeachin-like 2 protein (Nbeal2) belongs to the family of beige and Chediak-Higashi (BEACH) domain proteins. Loss-of-function mutations in the human NBEAL2 gene or Nbeal2 deficiency in mice cause gray platelet syndrome, a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of α-granules in megakaryocytes and platelets. We found that in mast cells, Nbeal2 regulates the activation of the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome. Furthermore, Nbeal2 mediates granule formation and restricts the expression of the transcription factors, IRF8, GATA2, and MITF as well as of the cell-cycle inhibitor p27, which are essential for mast cell differentiation, proliferation, and cytokine production. These data demonstrate the relevance of Nbeal2 in mast cells above and beyond granule biosynthesis.

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

The IL-1R family member IL-33R mediates Fcε-receptor-I (FcεRI)-independent activation of mast cells leading to NF-κB activation and consequently the production of cytokines. IL-33 also induces the activation of MAPKs, such as p38. We aimed to define the relevance of the p38-targets, the MAPK-activated protein kinases 2 and 3 (MK2 and MK3) in IL-33-induced signaling and the resulting mast cell effector functions in vitro and in vivo. We demonstrate that the IL-33-induced IL-6 and IL-13 production strongly depends on the MK2/3-mediated activation of ERK1/2 and PI3K signaling. Furthermore, in the presence of the stem cell factors, IL-33 did induce an MK2/3-, ERK1/2- and PI3K-dependent production of TNF-α. In vivo, the loss of MK2/3 in mast cells decreased the IL-33-induced leukocyte recruitment and the resulting skin inflammation. Therefore, the MK2/3-dependent signaling in mast cells is essential to mediate IL-33-induced inflammatory responses. Thus, MK2/3 are potential therapeutic targets for suppression of IL-33-induced inflammation skin diseases such as psoriasis.

TAK1 and IKK2, novel mediators of SCF-induced signaling and potential targets for c-Kit-driven diseases.

NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases.

Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells.

Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca²âº-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation".This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33.We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo.Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.

C-Kit controls IL-1ß-induced effector functions in HMC-cells.

The receptor tyrosine kinase c-Kit is important for mast cell differentiation, proliferation, and cytokine release. Recently, we reported that c-Kit acts as an intermediate signalling molecule regulating IL-33-induced signalling and effector functions in mast cells. Here, we investigated the influence of c-Kit on the IL-1ß-induced signalling and effector functions in HMC mast cell lines. HMC-cells were stimulated with IL-1ß and the resulting signalling and cytokine responses were analysed. Furthermore, we used pharmacological inhibitors to investigate the relevance of several signalling molecules for the IL-1ß-induced signalling and cytokine responses. Treatment of HMC-cells with the c-Kit inhibitor STI571 blocked the IL-1ß-induced activation of Erk1/2 and JNK1/2 but not p38 and NFκB. Furthermore, inhibition of these signalling pathways blocked the IL-6 production in HMC-cells. These findings indicate that IL-1ß-induced signalling in mast cells branches into c-Kit- dependent and -independent pathways, both relevant for IL-6 release. Therefore, c-Kit is an important regulator of IL-1 receptor 1-induced signalling and effector functions in HMC-cells.

The fate of osteochondral grafts after autologous osteochondral transplantation: a one-year follow-up study in a minipig model.

INTRODUCTION: Because articular cartilage shows little intrinsic capacity of spontaneous regeneration, a variety of treatment options are currently at use to repair cartilage damage. One of these is the autologous osteochondral transplantation (AOT). The aim of the present work was to study the histological changes during the progress of 1 year after AOT in the knee joint. MATERIALS AND METHODS: Twelve Minipigs underwent an AOT on the medial femoral condyles of both knees using cooled diamond studded trephines with a diameter of the grafts of 4.6 mm. Three animals were sacrificed at each 2, 8, 26 and 52 weeks after the operation. The condyles were analyzed histologically and immunohistologically for collagen types I and II. RESULTS: A successful bony incorporation was observed in all specimens. The transplant demonstrated an increasingly stable integration of the chondral matrix into the cartilage of the surrounding femoral condyle. At 52 weeks after the operations 5 of 6 condyles showed a chondral integration at least at one side of the graft. Immunohistologically all specimens showed physiological staining characteristics up to 52 weeks after operation. The quality of the chondral part of the graft showed a wide range of variations, ranging from vital tissue resembling native cartilage after 52 weeks, to severe degenerative signs beginning 2 weeks after operation and ending at 52 weeks with deep fissures fragmenting the cartilage and the complete loss of vital cells. CONCLUSION: The press-fit technique allows a stable bony incorporation. A chondral integration of the graft seems to occur, provided that a close contact between the interfaces can be achieved. Present results demonstrate a vital cartilagenous transplant for up to 52 weeks. However, some specimens showed in part severe degenerative signs. A possible explanation is an insufficient cooling of the trephines in relation to the small diameter of the grafts used in the minipig model. The collagen network seems not to be affected for up to 52 weeks.