Mast Cell Survival Research Articles

In vitro effects of histamine receptor 1 antagonists on proliferation and histamine release in canine neoplastic mast cells.

Canine mastocytomas (MCTs) are characterized by rapid proliferation of neoplastic mast cells (MCs) and clinical signs caused by MC‐derived mediators. In dogs suffering from MCT, histamine receptor 1 (HR1) antagonists are frequently used to control mediator‐related clinical symptoms. Previous studies have shown that the HR1 antagonists loratadine and terfenadine exert some growth‐inhibitory effects on neoplastic MCs. We examined whether other HR1 antagonists used in clinical practice (desloratadine, rupatadine, cyproheptadine, dimetindene, diphenhydramine) affect proliferation and survival of neoplastic MCs. Furthermore, we analysed whether these HR1 antagonists counteract IgE‐dependent histamine release from a MC line harbouring a functional IgE‐receptor. HR1 antagonists were applied on two canine MC lines, C2 and NI‐1, and on primary MCs obtained from three MCT samples. The HR1 antagonists desloratadine, rupatadine and cyproheptadine were found to be more potent in decreasing proliferation of C2 and NI‐1 cells when compared with dimetindene and diphenhydramine. Similar effects were seen in primary neoplastic MCs, except for diphenhydramine, which exerted more potent growth‐inhibitory effects than the other HR1 antagonists. Drug‐induced growth‐inhibition in C2 and NI‐1 cells was accompanied by apoptosis. Loratadine, desloratadine and rupatadine also suppressed IgE‐dependent histamine release in NI‐1 cells. However, drug concentrations required to elicit substantial effects on growth or histamine release were relatively high (>10 µM). Therefore, it remains unknown whether these drugs or similar, more potent, HR1‐targeting drugs can suppress growth or activation of canine neoplastic MCs in vivo.

Inhibition of mast cells: a novel mechanism by which nintedanib may elicit anti-fibrotic effects

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone (unclear mechanism of...

Computational Design for the Production of Secretory Recombinant Codon-Optimized Human Stem Cell Factor (SCF) in Chinese Hamster Ovary (CHO) Cells With an Appropriate Signal Peptide: An Intensive In Silico Study

Background: Production of the recombinant proteins in mammalian cells is an important issue with a bio-therapeutic purpose. Numerous efforts have been focused on the improvement of the yields of recombinant proteins, which include optimization of conventional biological processes, selection of appropriate signal peptides, codon optimization, and re-engineering of cells to produce more proteins. Stem cell factor (SCF) is a blood cytokine which activates the c-Kit receptor. This factor is crucial not only for the differentiation of hematopoietic progenitor cells but also for the survival, proliferation, and differentiation of mast cells. Recently, its therapeutic role in several diseases such as Alzheimer’s and myocardial infarction has been investigated. Therefore, the aim of this study was to design a secretory recombinant human SCF with the maximal yield in an appropriate mammalian host cell as Chinese hamster ovary (CHO) cells using the computational studies. Methods: As the first step, computational simulation studies were carried out to design the appropriate signal peptide for the human SCF protein. Codon optimized coding sequence of hSCF was transferred into a eukaryotic expression vector (pBudCE4.1). Recombinant vector (pBudCE4.1/SCF) was transfected into CHO cells and the stably transformed cells were screened and isolated. Subsequently, the expression of SCF was defined by reverse transcription polymerase chain reaction (RT-qPCR) in stably transformed cells. Results: Our bioinformatics studies indicated that Azurocidin signal peptide could be a suitable signal peptide for the production of SCF proteins in the CHO cells. Accordingly, computational studies revealed that the presence of 6×His-tag did not have a significant impact on the three-dimensional structure of the protein. Furthermore, the expression of hSCF was significant in the stable CHO cells. Conclusion: The use of this approach may, therefore, lead to the production of highly efficient recombinant hSCF, which would be feasible for the mass production of this factor for therapeutic purposes.

Fluvastatin Induces Apoptosis in Primary and Transformed Mast Cells.

Statin drugs are widely employed in the clinic to reduce serum cholesterol. Because of their hydroxymethylglutaryl coenzyme A reductase antagonism, statins also reduce isoprenyl lipids necessary for the membrane anchorage and signaling of small G-proteins in the Ras superfamily. We previously found that statins suppress immunoglobulin E (IgE)-mediated mast cell activation, suggesting these drugs might be useful in treating allergic disease. Although IgE-induced function is critical to allergic inflammation, mast cell proliferation and survival also impact atopic disease and mast cell neoplasia. In this study, we describe fluvastatin-mediated apoptosis in primary and transformed mast cells. An IC50 was achieved between 0.8 and 3.5 μM in both cell types, concentrations similar to the reported fluvastatin serum Cmax value. Apoptosis was correlated with reduced stem cell factor (SCF)-mediated signal transduction, mitochondrial dysfunction, and caspase activation. Complementing these data, we found that p53 deficiency or Bcl-2 overexpression reduced fluvastatin-induced apoptosis. We also noted evidence of cytoprotective autophagy in primary mast cells treated with fluvastatin. Finally, we found that intraperitoneal fluvastatin treatment reduced peritoneal mast cell numbers in vivo These findings offer insight into the mechanisms of mast cell survival and support the possible utility of statins in mast cell-associated allergic and neoplastic diseases. SIGNIFICANCE STATEMENT: Fluvastatin, a statin drug used to lower cholesterol, induces apoptosis in primary and transformed mast cells by antagonizing protein isoprenylation, effectively inhibiting stem cell factor (SCF)-induced survival signals. This drug may be an effective means of suppressing mast cell survival.

Systemic Mastocytosis: Following the Tyrosine Kinase Inhibition Roadmap.

Systemic mastocytosis is a rare and heterogeneous disease characterized by mast cell proliferation and activation. KIT is a transmembrane tyrosine kinase which plays a key role in mast cell growth, differentiation and survival. After interaction with its ligand, the stem cell factor, KIT dimerizes activating downstream pathways involving multiple tyrosine kinases (PI3K, JAK/STAT, RAS/ERK). Activating mutations in KIT are detected in most cases of systemic mastocytosis, being the most common KIT D816V. Therefore, since the emergence of tyrosine kinase inhibitors, KIT inhibition has been an attractive approach when facing mastocytosis treatment. Initial reports showed that only the rare KIT D816V negative cases were responsive to tyrosine kinase inhibitors. However, the development of new tyrosine kinase inhibitors such as midostaurin or avapritinib with activity against mast cells carrying the D816V KIT mutation, has changed the landscape of this disease.

Low Stromal Mast Cell Density in Canine Mammary Gland Tumours Predicts a Poor Prognosis

Tumour histological classification and grade are frequently used to predict the prognosis of canine mammary gland tumours. While these techniques provide some information about tumour behaviour, it is currently difficult to predict which tumours will metastasize. Mast cell density has been shown to predict metastasis in human breast cancer. The present study investigated whether the average mast cell density in 10 high-power (×400) microscopical fields (10 HPFs), evaluated by toluidine blue staining, similarly predicted the behaviour of canine mammary gland tumours. Mast cell density was evaluated in 53 canine mammary neoplasms for which the clinical outcome was known. Stromal mast cell density in malignant tumours that had subsequently developed radiographical evidence of metastasis (n=21) was significantly lower (P<0.001) than in malignant tumours that did not show evidence of metastases (n=20) or in benign tumours (n=12). The density of stromal mast cells that best predicted the disease outcome was ≤10/10 HPFs. Eighty-one percent of malignant tumours with ≤10 stromal mast cells/10 HPFs subsequently metastasized, while only 9.5% of malignant tumours with >10 stromal mast cells/10 HPFs developed metastases. There was a positive correlation between stromal mast cell density and survival time (rs=0.50, P<0.001). These findings suggest that assessing stromal mast cell density using toluidine blue staining may represent an easy to perform and cost-effective histopathological measure that, in conjunction with classification and grading, could better predict the behaviour of canine mammary neoplasms.

D100 CDX-0159, AN ANTI-KIT MONOCLONAL ANTIBODY, AS A MODULATOR OF MAST CELL-RELATED DISEASES

CDX-0159 is a humanized anti-KIT IgG1 monoclonal antibody being evaluated as a modulator of mast cell (MC)-related diseases including chronic idiopathic urticaria. The receptor KIT regulates MC growth, survival, chemotaxis and degranulation, suggesting KIT inhibition as a unique approach to modulate MC activity. CDX-0159 is a re-engineered variant of CDX-0158, a potent KIT inhibitor that decreased MC numbers in several preclinical models and decreased plasma tryptase levels in cancer patients, indicating a reduction in MC numbers/activity. However, CDX-0158 was associated with infusion-related reactions. CDX-0159 was designed to improve its safety profile and increase its serum half-life.

Expression of suppressor of cytokine signaling 3 (SOCS3) and interleukin-6 (-174-G/C) polymorphism in atopic conditions.

Hypersensitivity of the immune system is caused by elevated immunoglobulin E (IgE) levels in the serum, in response to a discrete allergen leading to allergic reactions. IgE-mediated inflammation is regulated by the cascade of defense related signaling molecules including interleukin-6 (IL-6) that plays pivotal role in the survival and maturation of mast cells during an allergic reaction. IL-6 mediated defense responses are tightly regulated by Suppressor of Cytokine Signaling 3 (SOCS3), an inhibitory molecules of Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling, in a negative feedback mechanism. The given study focuses on the assessment of crosstalk between SOCS3 and IL-6 to unravel the molecular significance of SOCS3 and IL-6 in the diagnosis and prognosis of allergy. The expression study of SOCS3 through real-time PCR analysis revealed, a 5.9 mean fold increase in SOCS3 expression in atopic cases in comparison to control cases. Moreover, IL-6 has, also, been found significantly enhanced in the serum level of atopic cases (26.4 pg/ml) as compared to control cases (3.686 pg/ml). Female population was found to be at a higher risk to develop atopic condition than male population as females exhibited higher expression of both SOCS3 and IL-6 than males. Furthermore, the polymorphic study of IL-6 promoter region (IL-6 174-G/C) in atopic population has reasserted the importance of SOCS3 and IL-6 in the diagnosis and prognosis of allergy. Expression of SOCS3 and IL-6 serum levels were found to be highly correlated. Therefore establishing the role of IL-6 (-174-G/C) polymorphism on the expression of SOCS3 and IL-6 in atopic cases. Notably, the study established SOCS3 and IL-6 as potential targets for the diagnosis/prognosis of allergy and for the development of reliable therapeutic strategies to control atopic conditions in the near future.

Human Skin-Derived Mast Cells Spontaneously Secrete Several Angiogenesis-Related Factors.

Mast cells are classically recognized as cells that cause IgE-mediated allergic reactions. However, their ability to store and secrete vascular endothelial growth factor (VEGF) suggests a role in vascular development and tumorigenesis. The current study sought to determine if other angiogenesis-related factors, in addition to VEGF, were also secreted by human tissue-derived mast cells. Using proteome array analysis and ELISA, we found that human skin-derived mast cells spontaneously secrete CXCL16, DPPIV, Endothelin-1, GM-CSF, IL-8, MCP-1, Pentraxin 3, Serpin E1, Serpin F1, TIMP-1, Thrombospondin-1, and uPA. We identified three groups based on their dependency for stem cell factor (SCF), which is required for mast cell survival: Endothelin-1, GM-CSF, IL-8, MCP-1, and VEGF (dependent); Pentraxin 3, Serpin E1, Serpin F1, TIMP-1, and Thrombospondin-1 (partly dependent); and CXCL16, DPPIV, and uPA (independent). Crosslinking of FcεRI with multivalent antigen enhanced the secretion of GM-CSF, Serpin E1, IL-8, and VEGF, and induced Amphiregulin and MMP-8 expression. Interestingly, FcεRI signals inhibited the spontaneous secretion of CXCL16, Endothelin-1, Serpin F1, Thrombospondin-1, MCP-1 and Pentraxin-3. Furthermore, IL-6, which we previously showed could induce VEGF, significantly enhanced MCP-1 secretion. Overall, this study identified several angiogenesis-related proteins that, in addition to VEGF, are spontaneously secreted at high concentrations from human skin-derived mast cells. These findings provide further evidence supporting an intrinsic role for mast cells in blood vessel formation.

The upregulation of Pim kinases is essential in coordinating the survival, proliferation, and migration of KIT D816V-mutated neoplastic mast cells

About ˜80% of mast cell neoplasm patients harbor the c-Kit activating mutation D816 V, which is associated with c-Kit inhibitor resistance and poor prognosis. However, the molecular basis for these effects is not fully known. To address this issue, in this study we screened molecules whose expression is altered by KIT D816 V mutation and found that Pim kinases were overexpressed in D816V-mutant neoplastic mast cells. This was accompanied by upregulation of signal transducer and activator of transcription (STAT) and mammalian target of rapamycin (mTOR) and downregulation of Akt and extracellular signal-regulated kinase (ERK1/2). Activated Pim kinases promoted the survival of D816 V cells by maintaining mTOR and p70S6K activation even under nutrient starvation. Conversely, cell proliferation was suppressed by inhibiting Pim kinases. The mRNA level of C-X-C chemokine receptor type 4 (CXCR4) was about 2-fold higher in D816 V cells; this was associated with a 2-fold increase in migratory capacity, which was modulated by Pim kinases. We also confirmed that upregulation of Pim kinases is a feature specific to cells with the D816 V mutation and is not observed in cells with the c-Kit activating N822 K mutation. These data suggest Pim kinases as a promising therapeutic target for the treatment of mast cell neoplasms with KIT D816 V mutation.

IL-9high GATA3low ILC2s are potent inducers of allergic airway inflammation

Abstract IL-9 is key driver of chronic and allergic inflammation at mucosal surfaces, with important roles in the activation and survival of mast cells and type 2 innate lymphoid cells (ILC2s). IL-9-reporter mice have established T cells (Th9) and ILC2s as major sources of IL-9 in vivo. The discovery that TGF-β and IL-4 cooperate for induction of Th9 cells has been an important breakthrough. However, the signals that induce IL-9high ILC2s remain incompletely characterized. Here, we show that IL-33, a critical activator of ILC2s, with important roles in type-2 innate immunity and allergic inflammation (Cayrol, Duval et al., Nature Immunology 2018) synergizes with TNF-family cytokine TL1A to induce prodigious amounts of IL-9 secretion by ILC2s. Unbiased global proteomic analyses revealed that IL-9 was the most-induced protein in ILC2s treated with IL-33 and TL1A. More than 99% of ILC2s expressed IL-9 intracellularly after co-stimulation with IL-33 and TL1A. ILC2s are known to produce very large amounts of IL-5 and IL-13, but ILC2s producing such high levels of IL-9 have not been described previously. Interestingly, IL-9 production by ILC2s was transient and associated with a phenotypic shift characterized by upregulation of IRF4, pSTAT5 and downregulation of ICOS, KLRG1 as well as master transcription factor GATA-3 and its target genes (IL-9R, ST2). Lastly, we detected an expansion of IL-9high ILC2s in the lungs of mice after IL-33/TL1A inhalation and adoptive transfer experiments revealed that IL-9high ILC2s are potent inducers of allergic airway inflammation in vivo. Thus, our study supports the rising plasticity of ILC2s and their critical role in the induction of type-2 inflammatory responses in the lungs.

The HMGCR Inhibitor Fluvastatin Induces Apoptosis and Autophagy in Primary and Neoplastic Mast Cells

Abstract Mast cell hyperplasia is noted in many inflammatory and allergic diseases. Mast cells can also generate a subtype of acute myelogenous leukemia. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and mastocytoma cells. Apoptosis was suppressed in p53 KO and Bcl-2 Tg BMMC, consistent with detectable mitochondrial damage and Caspase-9 activation. Because apoptosis induction could be mimicked by suppressing geranylgeranyl transferase (GGT), and fluvastatin effects were reversed by adding the product of the GGT reaction, loss of GGT-mediated protein lipidation is the likely mechanism of cell death. In addition to apoptosis, fluvastatin also induced autophagy. Chemical blockade of autophagy enhanced fluvastatin-mediated apoptosis in primary mast cells, but reduced it in mastocytomas. These data suggest that the FDA-approved statin drugs may be useful in suppressing mast cell hyperplasia and leukemia.

Frontline Science: Mast cells regulate neutrophil homeostasis by influencing macrophage clearance activity.

The receptor tyrosine kinase cKit and its ligand stem cell factor are essential for mast cells (MC) development and survival. Strains with mutations affecting the Kit gene display a profound MC deficiency in all tissues and have been extensively used to investigate the role of MC in both physiologic and pathologic conditions. However, these mice present a variety of abnormalities in other immune cell populations that can affect the interpretation of MC-related responses. C57BL/6 KitW-sh are characterized by an aberrant extramedullary myelopoiesis and systemic neutrophilia. MC deficiency in KitW-sh mice can be selectively repaired by engraftment with in vitro-differentiated MC to validate MC-specific functions. Nevertheless, the impact of MC reconstitution on other immune populations has never been evaluated in detail. Here, we specifically investigated the neutrophil compartment in primary and secondary lymphoid organs of C57BL/6 KitW-sh mice before and after MC reconstitution. We found that, albeit not apparently affecting neutrophils phenotype or maturation, MC reconstitution of KitW-sh mice restored the number of neutrophils at a level similar to that of wild-type C57BL/6 mice. In vitro and ex vivo experiments indicated that MC can influence neutrophil clearance by increasing macrophages' phagocytic activity. Furthermore, the G-CSF/IL-17 axis was also influenced by the presence or absence of MC in KitW-sh mice. These data suggest that MC play a role in the control of neutrophil homeostasis and that this aspect should be taken into account in the interpretation of results obtained using KitW-sh mice.

The CDK4/6 Inhibitor Palbociclib Exerts Growth-Inhibitory Effects on Neoplastic Mast Cells and Synergizes with Midostaurin in Producing Growth Arrest

Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are rare, malignant diseases with an unfavorable prognosis. In a majority of patients, the transforming KIT mutation D816V is detectable. Currently, several drugs are available for the treatment of ASM/MCL, including midostaurin, a KIT D816V-targeting drug that has recently been approved for the treatment of advanced SM in the US and in Europe. However, when applied as single drug, midostaurin usually fails to induce durable remissions in patients with ASM/MCL, and the same holds true for all other drugs tested in the ASM/MCL context so far. Therefore, drug combinations, including established drugs and novel targeted drugs are currently being examined for their anti-neoplastic effects in ASM/MCL. CDK4 and CDK6 are kinases that play an essential role in cell cycle-initiation in normal and neoplastic cells. However, the role of CDK4/6 as potential therapeutic targets in neoplastic mast cells (MC) has not been analyzed so far. Recently, three CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib, have been translated into clinical application. The aim of the current study was to evaluate the effects of these CDK4/6 inhibitors on cell cycle progression, proliferation and survival of neoplastic MC. In initial experiments, we employed the MCL-related cell lines HMC-1.1 (lacking KIT D816V), HMC-1.2 (KIT D816V+), ROSAKIT WT, ROSAKIT D816V and MCPV-1 (expressing RAS G12V, Large T and hTert). In 3H-thymidine incorporation experiments, all three CDK4/6-inhibitors were found to block proliferation in both HMC-1 sub-clones and both ROSA sub-clones, with comparable IC50 values (<0.5 µM). In MCPV-1 cells, similar results were obtained, but higher concentrations of palbociclib, ribociclib and abemaciclib were required to block proliferation (IC50 1-5 µM). These data suggest that CDK4/6-inhibitors exert anti-proliferative effects in neoplastic MC independent of the presence of KIT D816V. In a next step, we examined drug effects on primary bone marrow cells obtained from patients with KIT D816V+ indolent SM (n=3), ASM (n=1), SM with an associated hematologic neoplasm, ASM-AHN (n=5) and MCL (n=2). As determined by 3H-thymidine uptake, palbociclib was found to inhibit cell proliferation at pharmacologically meaningful concentrations in all donors tested, with IC50 values ranging between 5 nM and 250 nM. Similar effects were obtained when applying ribociclib (25-500 nM) and abemaciclib (5-500 nM). To learn more about the mechanisms underlying the effects of the CDK4/6 inhibitors on neoplastic MC, cell cycle progression and apoptosis were examined in HMC-1.1 and HMC-1.2 cells after drug exposure. In both cell lines, the palbociclib-induced growth inhibition was found to be accompanied by cell cycle arrest in the G1-phase. Moreover, all three CDK4/6 inhibitors were found to produce time- and dose-dependent apoptosis in HMC-1.1 and HMC-1.2 cells during 72 hours of incubation. In a next step, Western blot experiments were performed using antibodies against the main downstream target of CDK6, retinoblastoma protein-1 (Rb-1). The Rb-1 antigen was found to be expressed in phosphorylated form (p-Rb-1) in HMC-1.1 and HMC-1.2 cells. As expected, all 3 CDK4/6 inhibitors were found to suppress p-Rb-1 expression in both HMC-1 cell lines, suggesting specific drug effects. In a final step, we examined potential cooperative drug effects using palbociclib and the KIT D816V-targeting drug midostaurin. In these experiments, palbociclib was found to synergize with midostaurin in inducing growth inhibition in HMC-1 cells. In conclusion our data suggest that inhibition of CDK4/6 may be a new promising approach for the treatment of patients with advanced SM. In addition, our data suggest that CDK4/6 inhibitors may represent promising combination partners for midostaurin in the treatment of ASM/MCL. Whether treatment with CDK4/6 inhibitors alone or in combination with KIT inhibition, is indeed sufficient to control proliferation of neoplastic MC in vivo in patients with advanced SM remains to be determined in forthcoming studies. DisclosuresHoermann:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Reiter:Incyte: Consultancy, Honoraria. Valent:Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria.

The Mast Cell-Aryl Hydrocarbon Receptor Interplay at the Host-Microbe Interface.

Mast cells are increasingly being recognized as crucial cells in the response of the organism to environmental agents. Interestingly, the ability of mast cells to sense and respond to external cues is modulated by the microenvironment that surrounds mast cells and influences their differentiation. The scenario that is emerging unveils a delicate equilibrium that balances the effector functions of mast cells to guarantee host protection without compromising tissue homeostasis. Among the environmental components able to mold mast cells and fine-tune their effector functions, the microorganisms that colonize the human body, collectively known as microbiome, certainly play a key role. Indeed, microorganisms can regulate not only the survival, recruitment, and maturation of mast cells but also their activity by setting the threshold required for the exploitation of their different effector functions. Herein, we summarize the current knowledge about the mechanisms underlying the ability of the microorganisms to regulate mast cell physiology and discuss potential deviations that result in pathological consequences. We will discuss the pivotal role of the aryl hydrocarbon receptor in sensing the environment and shaping mast cell adaptation at the host-microbe interface.

Expression of Stem Cell Factor in Feline Mast Cell Tumour

Stem cell factor (SCF) is a ligand of the molecule Kit, which is expressed in mast cells and is important for mast cell proliferation, migration and survival. Mast cell tumours (MCTs) are associated with mutations of c-kit, a proto-oncogene encoding the Kit protein. In this study, we examined SCF expression in 23 samples of feline MCTs. SCF expression was detected in 10 cutaneous MCTs and a case of splenic mastocytosis. In the cutaneous MCTs, SCF-positive tumour cells were located at the margins. Kit was expressed in eight of the 10 cutaneous cases of SCF-expressing MCTs. In these cases, Kit-positive cells were located near to SCF-positive cells, and SCF/Kit double-positive tumour cells were found. Ki67-positive tumour cells were not found near to SCF-positive cells. These results suggest that SCF autocrine/paracrine mechanisms are involved in the expansion of cutaneous MCTs, but not in tumour proliferation.

Hydroxychloroquine as a novel therapeutic approach in mast cell activation diseases

There is no therapeutic agent approved in cutaneous mastocytosis and mast cell activation syndrome. We report the efficacy of hydroxychloroquine in four patients with cutaneous mastocytosis (n = 2) and mast cell activation syndrome (n = 2).We show that this molecule reduces the long-term survival of primary human mast cells, interferes with lysosome function and leads to the accumulation of non-functional tryptase in the mast cell granules. Furthermore, hydroxychloroquine decreases the production of pro-inflammatory mediators.

IL-3 but not monomeric IgE regulates Fc\u03b5RI levels and cell survival in primary human basophils

Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation.

Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT.

Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.

Correction: The Adaptor 3BP2 Is Required for KIT Receptor Expression and Human Mast Cell Survival.

Ainsua-Enrich, E., E. Serrano-Candelas, D. Alvarez-Errico, C. Picado, J. Sayos, J. Rivera, and M. Martin. 2015. The adaptor 3BP2 is required for KIT receptor expression and human mast cell survival. J. Immunol. 194: [4309–4318][1]. The authors revised the grant footnote to correct a funding