Mast Cell Differentiation Research Articles

Long-term treatment with imatinib results in profound mast cell deficiency in Ph+ chronic myeloid leukemia.

Although mast cells (MC) play an important role in allergic reactions, their physiologic role remains unknown. In mice, several models of MC-deficiency have been developed. However, no comparable human model is available. We examined the in vitro- and in vivo effects of the KIT-targeting drug imatinib on growth and development of human MC. Imatinib was found to inhibit stem cell factor (SCF)-induced differentiation of MC in long-term suspension cultures (IC50: 0.01 µM). Correspondingly, long-term treatment of chronic myeloid leukemia (CML) patients with imatinib (400 mg/day) resulted in a marked decrease in MC. In patients with continuous complete molecular response during therapy, bone marrow MC decreased to less than 5% of pre-treatment values, and also serum tryptase concentrations decreased significantly (pre-treatment: 32.0 ± 11.1 ng/ml; post-therapy: 3.4 ± 1.8, p<0.01). Other myeloid lineages, known to develop independently of KIT, were not affected by imatinib-therapy. Imatinib also produced a substantial decrease in MC-development in mice. However, no clinical syndrome attributable to drug-induced MC-deficiency was recorded in our CML patients. Together, imatinib suppresses MC production in vitro and in vivo. However, drug-induced MC depletion is not accompanied by adverse clinical events, suggesting that MC are less relevant to homeostasis in healthy tissues than we assumed so far.

Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation.

Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (FcεRI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on FcεRI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (∼50-fold) of cells to FcεRI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLCγ1. In contrast, degranulation and calcium responses elicited by the G protein-coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed FcεRI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate FcεRI-mediated degranulation independently of mTORC2.

C/EBPα controls mast cell function

CCAAT/enhancer binding protein alpha (C/EBPα) is a transcription factor that influences immune cell fate and differentiation. However, the effect of C/EBPα on mast cells is not fully understood. In this study, we showed that C/EBPα suppressed granule formation in mast cells and increased macrophage inflammatory protein (MIP)-2 production from mast cells upon bacterial stimulation. These results indicate that C/EBPα regulates the balance between the allergic response and the innate immune response of mast cells. Furthermore, we showed that stimulation of mast cells with the Lactobacillus casei JCM1134T strain during late differentiation up-regulated C/EBPα expression in differentiated mast cells. This suggests that intestinal commensal bacteria modulate C/EBPα expression and thereby regulate mast cell function.

TSLP Induces Mast Cell Development and Aggravates Allergic Reactions through the Activation of MDM2 and STAT6

Thymic stromal lymphopoietin (TSLP) is known to promote T helper type 2 cell-associated inflammation. Mast cells are major effector cells in allergic inflammatory responses. We noted that the population and maturation of mast cells were reduced in TSLP-deficient mice (TSLP-/-). Thus, we hypothesized that TSLP might affect mast cell development. We found that TSLP induced the proliferation and differentiation of mast cells from bone marrow progenitors. TSLP-induced mast cell proliferation was abolished by depletion of mouse double minute 2 (MDM2) and signal transducers and activators of transcription 6 (STAT6), as an upstream activator of MDM2. TSLP-/-, in particular, had a considerable deficit in the expression of MDM2 and STAT6. Also, the TSLP deficiency attenuated mast cell-mediated allergic reactions through the downregulation of STAT6 and MDM2. In an antibody microarray chip analysis, MDM2 expression was increased in atopic dermatitis patients. These observations indicate that TSLP is a factor for mast cell development, and that it aggravates mast cell-mediated immune responses.

Editorial introductions

Editorial introductions

Tenascin-C is required for normal Wnt/β-catenin signaling in the whisker follicle stem cell niche

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.

Mechanisms controlling mast cell and basophil lineage decisions.

Basophils and mast cells have long been known to play critical roles in allergic disease and host defense against parasitic infections. Recent recognition of these effector cells in immune regulations, host defense against bacteria and virus, and autoimmune diseases entices increased interest in studying these cells. However, origin and molecular regulation of basophil and mast cell differentiation remain incompletely understood. In this review, we focus on recent advances of the understanding the origin and molecular regulation of mouse basophil and mast cell development. We also summarize progress in the understanding of the origin and molecular regulation of human basophil and mast cell development. A more complete understanding of molecular regulation of basophils and mast cells will lead to the development of interventions that are more effective in achieving long-term success.

Identification of microRNAs regulating the developmental pathways of bone marrow derived mast cells.

BackgroundMicroRNAs (miRNAs) play important roles in leukocyte differentiation, although those utilised for specific programs and key functions remain incompletely characterised. As a global approach to gain insights into the potential regulatory role of miRNA in mast cell differentiation we characterised expression in BM cultures from the initiation of differentiation. In cultures enriched in differentiating mast cells we characterised miRNA expression and identified miRNA targeting the mRNA of putative factors involved in differentiation pathways and cellular identity. Detailed pathway analysis identified a unique miRNA network that is intimately linked to the mast cell differentiation program.Methodology/Principal FindingsWe identified 86 unique miRNAs with expression patterns that were up- or down- regulated at 5-fold or more during bone marrow derived mast cells (BMMC) development. By employing TargetScan and MeSH databases, we identified 524 transcripts involved in 30 canonical pathways as potentially regulated by these specific 86 miRNAs. Furthermore, by applying miRanda and IPA analyses, we predict that 7 specific miRNAs of this group are directly associated with the expression of c-Kit and FcεRIα and likewise, that 18 miRNAs promote expression of Mitf, GATA1 and c/EBPα three core transcription factors that direct mast cell differentiation. Furthermore, we have identified 11 miRNAs that may regulate the expression of STATs-3, -5a/b, GATA2 and GATA3 during differentiation, along with 13 miRNAs that target transcripts encoding Ndst2, mMCP4 and mMCP6 and thus may regulate biosynthesis of mast cell secretory mediators.Conclusions/SignificanceThis investigation characterises changes in miRNA expression in whole BM cultures during the differentiation of mast cells and predicts functional links between miRNAs and their target mRNAs for the regulation of development. This information provides an important resource for further investigations of the contributions of miRNAs to mast cell differentiation and function.

Transcription Factor GATA1 Is Dispensable for Mast Cell Differentiation in Adult Mice

Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1(-/y)), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1(-/y) bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5' end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit(+) FcεRIα(+) mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.

Foreign body response to subcutaneous biomaterial implants in a mast cell-deficient Kitw-Sh murine model

Mast cells (MCs)_are recognized for their functional role in wound-healing and allergic and inflammatory responses – host responses that are frequently detrimental to implanted biomaterials if extended beyond acute reactivity. These tissue reactions impact especially on the performance of sensing implants such as continuous glucose monitoring (CGM) devices. Our hypothesis that effective blockade of MC activity around implants could alter the host foreign body response (FBR) and enhance the in vivo lifetime of these implantable devices motivated this study. Stem cell factor and its ligand c-KIT receptor are critically important for MC survival, differentiation and degranulation. Therefore, an MC-deficient sash mouse model was used to assess MC relationships to the in vivo performance of CGM implants. Additionally, local delivery of a tyrosine kinase inhibitor (TKI) that inhibits c-KIT activity was also used to evaluate the role of MCs in modulating the FBR. Model sensor implants comprising polyester fibers coated with a rapidly dissolving polymer coating containing drug-releasing degradable microspheres were implanted subcutaneously in sash mice for various time points, and the FBR was evaluated for chronic inflammation and fibrous capsule formation around the implants. No significant differences were observed in the foreign body capsule formation between control and drug-releasing implant groups in MC-deficient mice. However, fibrous encapsulation was significantly greater around the drug-releasing implants in sash mice compared to drug-releasing implants in wild-type (e.g. MC-competent) mice. These results provide insights into the role of MCs in the FBR, suggesting that MC deficiency provides alternative pathways for host inflammatory responses to implanted biomaterials.

Establishment of a mast cell line, NCL-2, without Kit mutation, derived from NC mouse bone marrow

Immortal mast cell lines, such as RBL-2H3 and HMC-1 cells, are commonly utilized to investigate the function of mast cells. However, they are tumor cells carrying a gain-of-function mutation of Kit. We established an immortal mast cell line without Kit mutation, NCL-2, derived from NC mouse bone marrow. NCL-2 cells could be maintained without additional growth factors and thus could respond to exogenous growth signals. Moreover, NCL-2 cells expressed FcεRI and KIT, and release histamine and LTB4 in response to antigen stimulation. This cell line could be a useful tool to analyze proliferation, differentiation, and function of normal mast cells.

Imatinib Inhibits SCF-Induced Development Of Human Mast Cells In Vitro and Induces Profound and Selective Mast Cell Deficiency In Patients With Ph+ CML

Although mast cells (MC) play an important role as effector cells of allergic reactions and may contribute to vascular repair processes after a thromboembolic event, their physiological role in healthy tissues is currently unknown. In mice, several ´MC-deficiency models´ have been developed. In these models, MC-deficiency is usually triggered by a lack of KIT or lack of a functional KIT-ligand, stem cell factor (SCF). Currently, no comparable human model is available. We examined the in vitro and in vivo effects of the KIT-targeting drug imatinib on growth and differentiation of MC. Imatinib was found to inhibit SCF-induced development of human MC from cord blood-derived progenitors in an in vitro long-term culture assay (100 ng/ml SCF, day 28). The effects of imatinib on MC differentiation in this assay were dose-dependent (IC50: 0.01 µM). Correspondingly, efficacious long-term treatment of CML patients with imatinib (400 mg daily) resulted in a profound reduction of MC numbers. Whereas no substantial decrease in the numbers of MC was seen within the first year of treatment, the numbers of tryptase+ and KIT+ MC in the bone marrow (BM) decreased to less than 5% of pre-treatment values in 23 patients who were in continuous complete molecular response (CMR) for at least 2 years during imatinib therapy (numbers of tryptase+ BM cells: pre-treatment values: 21.1±13.7% of nucleated BM cells vs CMR-values in the same patients: 0.5±0.6% vs normal BM: 3.8±1.6%; p<0.001). Tryptase mRNA levels and KIT mRNA levels in the BM also decreased significantly during treatment with imatinib in these patients (p<0.01). Moreover, during treatment with imatinib, serum tryptase concentrations decreased to low or even undetectable levels (before therapy: 32.0±11.1 ng/ml vs CMR: 3.4±1.8 ng/ml, p<0.01). Other myeloid lineages in the BM, known to develop independently of KIT, were not affected by imatinib therapy. Remarkably, imatinib-induced MC deficiency was not accompanied by any specific symptoms or side effects (no increased rate of bacterial infections, thromboembolic events or secondary neoplastic lesions). In a next step, we examined MC numbers in C57BL/6J mice and cellular histamine levels in BALB/c mice treated with imatinib. In these experiments, we found that treatment with imatinib (60 mg/kg/day vs control saline by intraperitoneal=i.p. injection over 42 days; 4 mice per group) leads to a significant decrease in the numbers of MC (skin MC numbers per high power field in control mice: 36.7±10.1 versus imatinib-treated mice: 15.7±4.6, p<0.001). MC numbers and cellular histamine levels in peritoneal samples of BALB/c mice treated with imatinib (50 mg/kg/day i.p. vs control saline; 3 mice each) also decreased substantially during treatment with imatinib (histamine levels on day 24: imatinib: 9.2±1.4 ng/106 cells vs control: 75.7±2.1 ng/106cells, p<0.001). In conclusion, our data show that imatinib produces a profound MC deficiency in mice and men, without causing a particular clinical condition or syndrome. Based on these observations, we hypothesize that MC may be less relevant in physiologic processes and tissue homeostasis in healthy organs than has so far been assumed. Part of the work, including data on mast cells in BM sections, has been presented at EHA 2013. However, in our current ASH abstract, we have included additional data on mast cell numbers and histamine levels in 2 different mouse models. These mouse data have not been presented at EHA or any other congress before. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Cardiac Failure In Juvenile Rats Caused By Continuous Long-Term Exposure To The Tyrosine Kinase Inhibitor Dasatinib Can Be Circumvented By An Intermittent Application Schedule

BackgroundTreatment of chronic myeloid leukemia (CML) by tyrosine kinase inhibitors (TKIs, e.g. imatinib, dasatinib) can result in cardiac failure as these drugs exert off-target effects via so far ill-defined mechanisms. Especially in pediatric patients long-term drug exposure is of great concern as this cohort may require life-long treatment for CML. Dasatinib-induced side effects require treatment interruption or dose modification in particular due to hematologic toxicity or cardiac and pleural effusions. Here we investigated the impact of prolonged dasatinib exposure on the growing heart in juvenile rodents. In analogy to clinical data on older adults (La Rosee P, et al, Ann Hematol 2013; in press) we also questioned whether intermittent treatment would result in regression of dasatinib-dependent off-target cardiac toxicity. MethodsMale 4-week old Wistar rats were exposed via the drinking water to dasatinib at a standard-dose (cohort S: 50 µM), at a high-dose (cohort H: 100 µM), and also at the high dose intermittently (cohort I; Mon.-Wed. “on”; Thu.-Sun. “off”), respectively, over 10 weeks continuously. Each cohort comprised 10 animals; controls (cohort K) received water only. Thus, the animals were challenged with drug exposure from shortly after weaning (3 weeks old) over puberty (6-8 weeks old) until young adolescence (14 weeks old). Animals' behaviour, development, and body weight gain was monitored three times weekly. Using a dedicated small animal device echocardiography to determine left ventricular ejection fraction (EF) as well as analysis of blood serums markers indicating cardiac impairment (BNP, IL-6, TNF-alpha, Troponin I) by Luminex assay (Millipore, USA) were performed every two weeks. Necropsy was performed in all rats after spontaneous death while all survivors were sacrificed after 10 weeks of exposure, respectively. Total heart weights and microscopic histopathological changes were examined. ResultsDose-dependently animals died spontaneously: 8/10 animals from cohort H (high dose continuously), 6/10 animals from cohort S (standard-dose), and 1/10 animals from cohort I (high dose intermittently) , respectively. Two 2/10 controls died from infections. For ethical reason the experiment was terminated in cohort H after 5 weeks of drug exposure. Details on survival time for each cohort are shown in the Kaplan-Meier-Plots below. In concordance with survival data animals also exhibited dose-dependently impaired EF and elevated serum BNP. At necropsy median relative heart weights (% of total body weight) were found increased when compared to controls (0.35 %) for cohort H (0.45 %, p= 0.026), but not significantly higher for cohort S (0.38 %) and cohort I (0.39%), respectively. Findings of heart structural changes comprised a non-significant trend to an increased mast cell (MC) number (median MC counts per visual microscopic field: cohort K= 15 MC; cohort H= 23.5 MC; cohort S= 22.5 MC; cohort I= 17.5 MC). ConclusionContinuous long-term exposure to dasatinib at a high dosage is life-threatening to juvenile rats. However, intermittent exposure (treatment and interruption intervals of comparable lengths) will avoid cardiac failure. MCs have been identified to play an important role for cardiac adverse remodelling and increased numbers of MCs have been reported in explanted human hearts with dilated cardiomyopathy and in animal models of experimentally induced hypertension, myocardial infarction, and secondary chronic volume overload (Levick SP, et al, 2011; Cardiovas Res 89:12). Treatment with imatinib has been shown to result in vivo and in vitro in severe bone marrow mast cell depletion via c-kit induced blockade of MC differentiation (Cerny-Reiterer S, et al, 2013; Hematology, EHA abstract P 699). However, it is still unknown whether this also holds true for dasatinib. We conclude that it might be prudent to carefully monitor cardiac function in still growing individuals with CML if treated with dasatinib continuously over long periods. [Display omitted] JT Tauer and N Steinbronn contributed equally to the work. Disclosures:No relevant conflicts of interest to declare.

Ikaros limits basophil development by suppressing C/EBP-α expression

AbstractThe Ikaros gene (Ikzf1) encodes a family of zinc-finger transcription factors implicated in hematopoietic cell differentiation. Here we show that Ikaros suppresses the development of basophils, which are proinflammatory cells of the myeloid lineage. In the absence of extrinsic basophil-inducing signals, Ikaros−/− (Ik−/−) mice exhibit increases in basophil numbers in blood and bone marrow and in their direct precursors in bone marrow and the spleen, as well as decreased numbers of intestinal mast cells. In vitro culture of Ik−/− bone marrow under mast cell differentiation conditions also results in predominance of basophils. Basophil expansion is associated with an increase in basophil progenitors, increased expression of Cebpa and decreased expression of mast cell-specifying genes Hes1 and microphthalmia-associated transcription factor (Mitf). Ikaros directly associates with regulatory sites within Cebpa and Hes1 and regulates the acquisition of permissive H3K4 tri-methylation marks at the Cebpa locus and reduces H3K4 tri-methylation at the Hes1 promoter. Ikaros blockade in cultured cells or transfer of Ik−/− bone marrow into irradiated Ik+/+ recipients also results in increased basophils confirming a cell-intrinsic effect of Ikaros on basophil development. We conclude that Ikaros is a suppressor of basophil differentiation under steady-state conditions and that it acts by regulating permissive chromatin modifications of Cebpa.

Editorial introductions

Editorial introductions

Non-tumor mast cells cultured in vitro on a honeycomb-like structured film proliferate with multinucleated formation

Mast cells are released from bone marrow into the circulatory system as immature precursors and differentiate upon their arrival at diverse organs and tissues. Because mast cell functions can be altered in these tissues, we propose that mast cells are sensitive to their surrounding microenvironment. To examine the morphological responses of mast cells, we cultured a proliferative mouse non-tumor cell line of mast cells (NCL-2 cells) on a honeycomb-like structured polystyrene film (HCF) representing a microenvironmental scaffold. In this study, the NCL-2 cells cultured on the HCF proliferated without apoptosis. Furthermore, NCL-2 cells cultured on 3- and 5-μm HCFs exhibited multinuclear formation. These observations of different NCL-2 cell morphologies and proliferation rates on HCF scaffolds with different hole sizes suggest that mast cells undertake specific proliferative shapes depending on the surrounding microenviroment. Moreover, HCFs may lead to the regulation of mast cell differentiation. From the Clinical EditorThis team reports on the development of a honeycomb-like structured film to study mast cell differentiation of non-cancerous origin, demonstrating that different microenvironments provided by different honeycomb hole sizes determine the morphology of the differentiated cells.

Assessment of relief from pruritus due to Kampo medicines by using murine models of atopic dermatitis

Aim: Atopic dermatitis is a representative intractable chronic eczematous skin disease. Because the symptoms vary between individuals, several therapies are required for atopic dermatitis. Since kampo medicines should be used according to the particular physical and mental conditions, the use of distinctive animal models is necessary for the experimental assessment of the effi cacy of kampo medicines. In this study, 7 kampo medicines were assessed for their anti-itching eff ect in 2 types of atopic dermatitis-like pruritus models for characterizing. Methods: Long-lasting scratching behavior was induced by multiple treatments with 2,4,6-trinitrochlorobenzene (TNCB) for 4 weeks in NC mice. Kampo medicines were administered orally for 11 d from the following day of the last TNCB-treatment to assess curing pruritus. In the other model, TNCB was applied to ears of BALB/c mice for 10 d every other day to induce scratching behavior. Kampo medicines were administered orally everyday for the same term of the induction of scratching behavior to assess the prevention of dermatitis. Results: Shofusan (SFS) had an instant eff ect, even on using a single dose, on scratching behavior in the model of NC mice; it ameliorated chronic itching on oral administration for 11 d, by preventing mast cell diff erentiation and degranulation. In the model of BALB/c mice, another kampo medicine, hochuekkito (HET) inhibited the induction of scratching behavior. Conclusion: Kampo medicines such as SFS and HET may improve chronic itching sensation in atopic dermatitis to the same extent as or better than existing antiallergic medicines.

Increased circulating levels of neurotrophins and elevated expression of their high-affinity receptors on skin and gut mast cells in mastocytosis

AbstractMastocytosis is a rare heterogeneous disease characterized by increase of mast cells (MCs) in different organs. Neurotrophins (NTs) have been shown to promote differentiation and survival of MCs, which in turn represent a major source of NTs. Thus, a contribution of NTs to mastocytosis seems highly conceivable but has not yet been investigated. We could demonstrate expression of high-affinity NT receptors tropomyosin-related kinase A (TrkA) for nerve growth factor (NGF)-β, TrkB for brain-derived neurotrophic factor, and NT-4 and TrkC for NT-3 on skin MCs; and of TrkA and TrkC on intestinal MCs of patients with mastocytosis. Moreover, increased expression of NGF-β; NT-3; TrkA, TrkB, and TrkC; and isoforms truncated TrkB-T1 and truncated TrkC were observed on skin MCs. Patients with mastocytosis featured elevated serum levels of NGF, NT-3, and NT-4. Levels of NGF-β and NT-4 correlated with tryptase levels, suggesting a link between MC load and blood levels of NGF and NT-4. Migration of CD117+ progenitor cells from the blood was enhanced toward NGF-β gradient in both mastocytosis and controls. Together with enhanced NT levels, the elevated expression of modified Trk receptors on skin and gut MCs might contribute to the pathophysiology of mastocytosis in autocrine and paracrine loops.

Active Caspase-3 Is Stored within Secretory Compartments of Viable Mast Cells

Caspase-3 is a main executioner of apoptotic cell death. The general notion is that, in viable cells, caspase-3 is found as a cytosolic inactive proenzyme and that caspase-3 activation is largely confined to processes associated with cell death. In this study, we challenge this notion by showing that enzymatically active caspase-3 is stored in viable mast cells. The enzymatically active caspase-3 was undetectable in the cytosol of viable cells, but was recovered in subcellular fractions containing secretory granule-localized proteases. Moreover, active caspase-3 was rapidly released into the cytosolic compartment after permeabilization of the secretory granules. Using a cell-permeable substrate for caspase-3, the presence of active caspase-3-like activity in granule-like compartments close to the plasma membrane was demonstrated. Moreover, it was shown that mast cell activation caused release of the caspase-3 to the cell exterior. During the course of mast cell differentiation from bone marrow cells, procaspase-3 was present in cells of all stages of maturation. In contrast, active caspase-3 was undetectable in bone marrow precursor cells, but increased progressively during the process of mast cell maturation, its accumulation coinciding with that of a mast cell-specific secretory granule marker, mouse mast cell protease 6. Together, the current study suggests that active caspase-3 can be stored within secretory compartments of viable mast cells.

Microfilaments make mast cells migrate (rather than degranulate)

Expression of the high-affinity receptor for IgE (FcεRI) provides mast cells with the ability to react in a proinflammatory manner to antigens (Ags). In particular, the immediate secretion of preformed mediators from secretory lysosomes (degranulation) is typical for FcεRI-mediated mast cell activation. In addition to the FcεRI, the stem cell factor receptor, KIT, is expressed at high levels on the surface of mast cells. KIT activation controls mast cell differentiation and survival in vivo and potently stimulates the chemotaxis of these cells. Although FcεRI and KIT initiate many of the same early signaling events in mast cells, FcεRI activation results in potent degranulation and a poor chemotactic response while KIT activation triggers very little degranulation and a strong chemotactic response. Novel data published in this issue of the European Journal of Immunology [Smrž et al. Eur. J. Immunol. 2013. 43: 1873-1882] demonstrate that actin de- and repolymerization, involved in both degranulation and chemotaxis, make all the difference: Pharmacological suppression of F-actin formation converts activated KIT into a strong degranulator. The possible implications for mast cell physiology and pathophysiology are discussed in this Commentary.