Mast cells are known to have a detrimental impact on a variety of pathological conditions. There is therefore an urgent need of developing strategies that limit their harmful effects. The aim of this study was to accomplish this by developing a means of inducing mast cell apoptosis. The strategy was to identify novel compounds that induce mast cell apoptosis by permeabilization of their secretory lysosomes (granules). As a candidate, we assessed mefloquine, an anti-malarial drug that has been proposed to have lysosome-permeabilizing activity. Mefloquine was added to mast cells and administered in vivo, followed by assessment of the extent and mechanisms of mast cell death. Mefloquine was cytotoxic to murine and human mast cells. Mefloquine induced apoptotic cell death of wild-type mast cells whereas cells lacking the granule compounds serglycin proteoglycan or tryptase were shown to undergo necrotic cell death, the latter finding indicating a role of the mast cell granules in mefloquine-induced cell death. In support of this, mefloquine was shown to cause compromised granule integrity and to induce leakage of granule components into the cytosol. Mefloquine-induced cell death was refractory to caspase inhibitors but was completely abrogated by reactive oxygen species inhibition. These findings identify mefloquine as a novel anti-mast cell agent, which induces mast cell death through a granule-mediated pathway. Mefloquine may thus become useful in therapy aiming at limiting harmful effects of mast cells.

Mast cell secretory granules are densely packed with various bioactive mediators including proteases of chymase, tryptase and CPA3 type. Previous studies have indicated that mast cells can affect the outcome of melanoma but the contribution of the mast cell granule proteases to such effects has not been clear. Here we addressed this issue by assessing mice lacking either the chymase Mcpt4, the tryptase Mcpt6 or carboxypeptidase A3 (Cpa3), as well as mice simultaneously lacking all three proteases, in a model of melanoma dissemination from blood to the lung. Although mice with individual deficiency in the respective proteases did not differ significantly from wildtype mice in the extent of melanoma colonization, mice with multiple protease deficiency (Mcpt4/Mcpt6/Cpa3-deficient) exhibited a higher extent of melanoma colonization in lungs as compared to wildtype animals. This was supported by higher expression of melanoma-specific genes in lungs of Mcpt4/Mcpt6/CPA3-deficient vs. wildtype mice. Cytokine profiling showed that the levels of CXCL16, a chemokine with effects on T cell populations and NKT cells, were significantly lower in lungs of Mcpt4/Mcpt6/Cpa3-deficient animals vs. controls, suggesting that multiple mast cell protease deficiency might affect T cell or NKT cell populations. In line with this, we found that the Mcpt4/Mcpt6/Cpa3-deficiency was associated with a reduction in cells expressing CD1d, a MHC class 1-like molecule that is crucial for presenting antigen to invariant NKT (iNKT) cells. Together, these findings indicate a protective role of mast cell-specific proteases in melanoma dissemination, and suggest that this effect involves a CXCL16/CD1d/NKT cell axis.

Mast cells (MCs) have a profound impact on allergic asthma. Under such conditions, MCs undergo degranulation, resulting in the release of exceptionally large amounts of MC-restricted proteases. However, the role of these proteases in asthma is only partially understood.

Mast cells contribute to the pathology of allergic and other disorders. Strategies to interfere with harmful mast cell-related activities are therefore warranted. Previously we established a principle for inducing selective apoptosis of mast cells, by the use of lysosomotropic agents that cause secretory granule permeabilization, leading to production of reactive oxygen species (ROS). However, the mechanism of ROS production has not been known. Here we addressed this issue. Live microscopy analysis showed that the secretory granules comprise major subcellular compartments for ROS production in response to mefloquine. As further signs for the primary involvement of secretory granules, both ROS production and cell death was blunted in mast cells lacking serglycin, a secretory granule-restricted proteoglycan. Inhibition of granule acidification caused an essentially complete blockade of granule permeabilization, ROS production and cell death in response to mefloquine. ROS production was also attenuated in the presence of an iron chelator, and after inhibition of either granzyme B or the ERK1/2 MAP kinase signaling pathway. Together, our findings reveal that the mast cell secretory granules constitute major sites for ROS production in mast cells subjected to lysosomotropic challenge. Moreover, this study reveals a central role for granule acidification in ROS generation and the pro-apoptotic response triggered downstream of secretory granule permeabilization.

Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs.

It has been recognized for a long time that the secretory granules of mast cells are acidic, but the functional importance of maintaining an acidic pH in the mast cell granules is not fully understood. Here we addressed this issue by examining the effects of raising the pH of the mast cell secretory granules. Mast cells were incubated with bafilomycin A1, an inhibitor of the vacuolar-type ATPase proton pump. Supporting a role of vacuolar-type ATPase in mast cell granule acidification, bafilomycin A1 treatment caused a robust increase in granule pH. This was accompanied by marked effects on mast cell granules, including swelling and acquisition of vacuole-like morphology. Moreover, bafilomycin A1 caused extensive, yet selective effects on the granule content. These included aberrant processing of pro-carboxypeptidase A3 and a reduction in the level of intracellular histamine, the latter being accompanied by an increase in extracellular histamine. In contrast, the storage of β-hexosaminidase, a prototype lysosomal hydrolase known to be stored in mast cell granules, was not affected by abrogation of granule acidification. Moreover, bafilomycin A1 caused a reduction of tryptase enzymatic activity and appearance of tryptase degradation products. Tryptase inhibition prevented the formation of such degradation products, suggesting that the pH elevation causes tryptase to undergo autoproteolysis. Taken together, our findings reveal that mast cell secretory granule homeostasis is critically dependent on an acidic milieu.

Mast cells (MCs) are characterized by an abundance of lysosome-like secretory granules filled with immunomodulatory compounds including histamine, cytokines, lysosomal hydrolases, MC-restricted proteases, and serglycin proteoglycans. The latter are essential for promoting the storage of other granule compounds and are built up of the serglycin core protein to which highly sulfated and thereby negatively charged glycosaminoglycan (GAG) side chains of heparin or chondroitin sulfate type are attached. In the search for mechanisms operating in regulating MC granule homeostasis, we here investigated the role of mitogen-activated protein kinase (MAPK) signaling. We show that inhibition of MEK1/2 (a MAPK kinase) leads to increased metachromatic staining of MC granules, indicative of increased proteoglycan content. Indeed, MEK1/2 inhibition caused a profound increase in the expression of the gene coding for the serglycin core protein and of genes coding for various enzymes involved in the biosynthesis/sulfation of the GAGs attached to the serglycin core protein. This was accompanied by corresponding increases in the levels of the respective GAGs. Deletion of the serglycin core protein abrogated the induction of enzymes operative in proteoglycan synthesis, indicating that availability of the serglycin proteoglycan core protein has a regulatory function impacting on the expression of the various serglycin-modifying enzymes. MEK1/2 inhibition also caused a substantial increase in the expression of granule-localized, proteoglycan-binding proteases. Altogether, this study identifies a novel role for MAPK signaling in regulating the content of secretory granules in MCs.

Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 μM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

To obtain a more detailed picture of macrophage (MΦ) biology, in the current study, we analyzed the transcriptome of mouse peritoneal MΦs by RNA-seq and PCR-based transcriptomics. The results show that peritoneal MΦs, based on mRNA content, under non-inflammatory conditions produce large amounts of a number of antimicrobial proteins such as lysozyme and several complement components. They were also found to be potent producers of several chemokines, including platelet factor 4 (PF4), Ccl6, Ccl9, Cxcl13, and Ccl24, and to express high levels of both TGF-β1 and TGF-β2. The liver is considered to be the main producer of most complement and coagulation components. However, we can now show that MΦs are also important sources of such compounds including C1qA, C1qB, C1qC, properdin, C4a, factor H, ficolin, and coagulation factor FV. In addition, FX, FVII, and complement factor B were expressed by the MΦs, altogether indicating that MΦs are important local players in both the complement and coagulation systems. For comparison, we analyzed human peripheral blood monocytes. We show that the human monocytes shared many characteristics with the mouse peritoneal MΦs but that there were also many major differences. Similar to the mouse peritoneal MΦs, the most highly expressed transcript in the monocytes was lysozyme, and high levels of both properdin and ficolin were observed. However, with regard to connective tissue components, such as fibronectin, lubricin, syndecan 3, and extracellular matrix protein 1, which were highly expressed by the peritoneal MΦs, the monocytes almost totally lacked transcripts. In contrast, monocytes expressed high levels of MHC Class II, whereas the peritoneal MΦs showed very low levels of these antigen-presenting molecules. Altogether, the present study provides a novel view of the phenotype of the major MΦ subpopulation in the mouse peritoneum and the large peritoneal MΦs and places the transcriptome profile of the peritoneal MΦs in a broader context, including a comparison of the peritoneal MΦ transcriptome with that of human peripheral blood monocytes and the liver.

The human copper (Cu) chaperone Atox1 delivers Cu to P1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC) motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.

Mast cells are key effector cells in allergic reactions. When activated to degranulate, they release a plethora of bioactive compounds from their secretory granules, including mast cell-restricted proteases such as tryptase. In a previous study, we showed that tryptase, in addition to its intragranular location, can be found within the nuclei of mast cells where it truncates core histones at their N-terminal ends.

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre- R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre- R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre- R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

Mast cells are implicated as detrimental players in inflammatory lung diseases, particularly asthma. Mast cells respond to activating stimuli by releasing a wide panel of pro-inflammatory compounds that can contribute profoundly to the pathology, and there is currently an unmet need for strategies that efficiently ameliorate harmful effects of mast cells under such conditions. Here, we sought to evaluate a novel concept for targeting human lung mast cells, by assessing the possibility of selectively depleting the lung mast cells by induction of apoptosis. For this purpose, we used lysosomotropic agents, i.e., compounds that are known to permeabilize the secretory granules of mast cells, thereby releasing the contents of the granules into the cytosol. Either intact human lung tissue, purified human lung mast cells or mixed populations of human lung cells were incubated with the lysosomotropic agents mefloquine or siramesine, followed by measurement of apoptosis, reactive oxygen species (ROS) production, and release of cytokines. We show that human lung mast cells were highly susceptible to apoptosis induced by this strategy, whereas other cell populations of the lung were largely refractory. Moreover, we demonstrate that apoptosis induced by this mode is dependent on the production of ROS and that the treatment of lung tissue with lysosomotropic agents causes a decrease in the release of pathogenic cytokines. We conclude that selective apoptosis of human lung mast cells can be accomplished by administration of lysosomotropic agents, thus introducing the possibility of using such drugs as novel therapeutics in the treatment of inflammatory lung disorders such as asthma.

Mast cells (MCs) are primarily resident hematopoietic tissue cells that are localized at external and internal surfaces of the body where they act in the first line of defense. MCs are found in all studied vertebrates and have also been identified in tunicates, an early chordate. To obtain a detailed insight into the biology of MCs, here we analyzed the transcriptome of MCs from different mouse organs by RNA-seq and PCR-based transcriptomics. We show that MCs at different tissue locations differ substantially in their levels of transcripts coding for the most abundant MC granule proteins, even within the connective tissue type, or mucosal MC niches. We also demonstrate that transcript levels for the major granule proteins, including the various MC-restricted proteases and the heparin core protein, can be several orders of magnitude higher than those coding for various surface receptors and enzymes involved in protease activation, as well as enzymes involved in the synthesis of heparin, histamine, leukotrienes, and prostaglandins. Interestingly, our analyses revealed an almost complete absence in MCs of transcripts coding for cytokines at baseline conditions, indicating that cytokines are primarily produced by activated MCs. Bone marrow-derived MCs (BMMCs) are often used as equivalents of tissue MCs. Here, we show that these cells differ substantially from tissue MCs with regard to their transcriptome. Notably, they showed a transcriptome indicative of relatively immature cells, both with respect to the expression of granule proteases and of various enzymes involved in the processing/synthesis of granule compounds, indicating that care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs. Furthermore, the latter finding indicates that the development of fully mature tissue-resident MCs requires a cytokine milieu beyond what is needed for in vitro differentiation of BMMCs. Altogether, this study provides a comprehensive quantitative view of the transcriptome profile of MCs resident at different tissue locations that builds nicely on previous studies of both the mouse and human transcriptome, and form a solid base for future evolutionary studies of the role of MCs in vertebrate immunity.

Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells.