Sebastian Drube

The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells

Members of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R-induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH(2)-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33-induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.

In various tumour cell lines the peptide bradykinin B2 receptor antagonist, Hoe 140 (Icatibant), may act as mitogenic agonist

This study examined the mitogenic effects of bradykinin (BK, Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg), the peptide bradykinin B2 receptor antagonist Hoe 140 (D‐Arg0[Hyp3‐Thi6‐D‐Tic7‐Oic8]BK, and the orally active, nonpeptide B2 receptor antagonist FR 173657 ((E)‐3‐(6‐acetamido‐3‐pyridyl)‐N‐[N‐2‐4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl) oxymethyl]phenyl]‐N‐methylaminocarbonyl‐methyl]acrylamide) in three different human tumour cell lines: the small cell lung carcinoma (SCLC) cell line H‐69, the breast carcinoma cell line EFM‐192A, and the colon carcinoma cell line SW‐480. In these cell lines activation of mitogen‐activated protein kinase (MAPK) is involved in BK‐induced stimulation of cell proliferation and may be mediated by both Gq proteins (SW‐480) and Gi proteins (EFM‐192A; H‐69). In these cells BK as well as Hoe 140 increased the rate of DNA synthesis measured with the [3H]‐thymidine uptake assay. Hoe 140 did neither antagonize nor potentiate the effect of BK. FR 173657 did not stimulate [3H]‐thymidine incorporation but clearly antagonized the mitogenic effects of BK as well as Hoe 140. In H‐69 cells, FR 173657 induced a decrease in the basal rate of DNA synthesis. In all three cell lines BK and Hoe 140 stimulated the activity of MAPK. Their effect on MAPK activity was completely abolished by FR 173657 which itself did not increase the activity of MAPK. In H‐69 cells, the basal activity of MAPK was slightly inhibited by FR 173657. In the cell lines SW‐480 and H‐69 both BK and Hoe 140 but not FR 173657 stimulated phosphatidylinositol hydrolysis. In H‐69 cells, FR 173657 decreased basal inositol phosphate formation. Our results show that in certain tumour cell lines the classical peptide B2 receptor antagonist, Hoe 140, may act as mitogenic B2 receptor agonist whereas the nonpeptide B2 receptor antagonist, FR 173657, does not. In H‐69 cells FR 173657 was found to exhibit properties of an inverse agonist.

Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit

Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in cells of adult mice, including hematopoietic precursors, caused severe mastocytosis with 100% penetrance at young age frequently associated with additional hematopoietic (mostly B lineage-derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain-of-function mutation.

PI3Kγ deficiency delays the onset of experimental autoimmune encephalomyelitis and ameliorates its clinical outcome.

PI3Ks control signal transduction triggered by growth factors and G‐protein‐coupled receptors and regulate an array of biological processes, including cellular proliferation, differentiation, survival and migration. Herein, we investigated the role of PI3Kγ in the pathogenesis of EAE. We show that, in the absence of PI3Kγ expression, clinical signs of EAE were delayed and mitigated. PI3Kγ‐deficient myelin oligodendrocyte glycoprotein (MOG)35–55‐specific CD4+ T cells appeared later in the secondary lymphoid organs and in the CNS than their WT counterparts. Transfer of WT CD4+ cells into PI3Kγ−/− mice prior to MOG35–55 immunisation restored EAE severity to WT levels, supporting the relevance of PI3Kγ expression in Th cells for the pathogenesis of EAE; however, PI3Kγ was dispensable for Th1 and Th17 differentiation, thus excluding an altered expression of these pathogenetically relevant cytokines as the cause for ameliorated EAE in PI3Kγ−/− mice. These findings demonstrate that PI3Kγ contributes to the development of autoimmune CNS inflammation.

C-Kit controls IL-1β-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-1β-induced signalling and effector functions in HMC mast cell lines. HMC-cells were stimulated with IL-1β 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-1β-induced signalling and cytokine responses. Treatment of HMC-cells with the c-Kit inhibitor STI571 blocked the IL-1β-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-1β-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.

Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion

&NA; Gastro‐intestinal helminth infections trigger the release of interleukin‐33 (IL‐33), which induces type‐2 helper T cells (Th2 cells) at the site of infection to produce IL‐13, thereby contributing to host resistance in a T cell receptor (TCR)‐independent manner. Here, we show that, as a prerequisite for IL‐33‐induced IL‐13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL‐33R) and EGFR. This shared signaling complex allowed IL‐33 to induce the EGFR‐mediated activation of the MAP‐kinase signaling pathway and consequently the expression of IL‐13. Lack of EGFR expression on T cells abrogated IL‐13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR‐signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation. Graphical Abstract Figure. No caption available. HighlightsMice lacking EGFR expression on T cells are more susceptible to worm infectionsEGFR forms a complex with T1/ST2, allowing for IL‐33 induced IL‐13 expressionAmphiregulin‐mediated EGFR activation is essential for complex formation with T1/ST2EGFR expression is induced by TCR engagement and sustained by cytokines, such as TSLP &NA; At the site of infection, Th2 cells secrete IL‐13 upon exposure to IL‐33. Minutti et al. now show that TCR‐induced expression of the EGFR and its ligand amphiregulin was essential for IL‐33‐induced IL‐13 secretion, revealing a mechanism whereby antigen‐specific activation controls the innate effector function of Th2 cells.

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.

A complicated liaison: IL-33 and IL-33R in arthritis pathogenesis

Interruption of cytokine signaling, by targeting either the cytokine itself or its cellular receptor, is a mainstay in the therapy for patients with rheumatic diseases. Interleukin (IL)-33, a member of the IL-1 cytokine family, has emerged as an important mediator of inflammatory responses. In a side-by-side examination of IL-33-deficient and IL-33 receptor (IL-33R)-deficient mice in the K/BxN serum transfer model, arthritis was ameliorated in the IL-33R knockout (KO) mice but not in the IL-33 KO mice. These findings complement previous knowledge on IL-33R signaling, demonstrating that the IL-33R cross-activates other signaling pathways in addition to IL-33-mediated signals. The results reported by Martin and colleagues in a previous issue of Arthritis Research & Therapy underline the clinical relevance of IL-33R cross-signaling and further illustrate that targeting a cytokine receptor (IL-33R) can have completely different clinical outcomes than targeting the respective cytokine.

Membrane-bound stem cell factor is the major but not only driver of fibroblast-induced murine skin mast cell differentiation

The maintenance and modulation of cutaneous mast cell (MC) numbers is held to be important for skin immune responses to allergens and pathogens. The increase in MC numbers in the skin is achieved by proliferation and the differentiation of precursor to mature MCs. Fibroblast‐derived SCF is thought to be the major skin MC growth factor and it potently induces MC proliferation. The mechanisms of fibroblast‐induced skin MC differentiation, including the role of SCF, however, remain insufficiently characterized and understood. Using cocultures of immature murine MCs and fibroblasts, we found that the adhesion of immature MCs to fibroblasts via VCAM‐1 and α4β7 integrin is very important for subsequent differentiation, which is driven by fibroblast membrane‐bound SCF and additional fibroblast‐derived membrane‐bound signals. Thus, our results show that fibroblast‐induced MC differentiation is induced by direct cell–cell contact and involves both Kit‐dependent and Kit‐independent pathways. Our findings add to the understanding of how immature mast cells mature in murine skin and encourage further analyses of the underlying mechanisms, which may result in novel targets for the modulation of skin mast cell driven diseases.

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.