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Codeine stimulates skin mast cells and is therefore used in skin tests and as an inducer of experimental itch. MRGPRX2 responds to various drugs, including opioids, to elicit pseudoallergic reactions, but whether it represents the main opiate receptor of skin mast cells remains unknown. By combining a number of approaches, including the silencing of MRGPRX2, we now report that MRGPRX2 is indeed the dominant codeine receptor of dermal mast cells. Activation by codeine displayed profound subject variability and correlated with secretion elicited by compound 48/80 or substance P but not by FcεRI aggregation. Degranulation by codeine was attenuated by stem cell factor, whereas the opposite was found for FcεRI. Compound 48/80 or codeine alone was able to achieve maximum MRGPRX2 activation. MRGPRX2 was rapidly internalized on codeine binding in a β-arrestin-1‒dependent manner. Codeine-triggered β-arrestin activation was also established by the Tango assay. Prestimulation with MRGPRX2 agonists (but not C3a or FcεRI aggregation) resulted in refractoriness to further stimulation by the same or another MRGPRX2 ligand (cross desensitization). This was duplicated in a cell line (RBL-MRGPRX2). Collectively, codeine degranulates skin mast cells through MRGPRX2, at which it acts as a balanced ligand. It has yet to be determined whether codeine-induced refractoriness could be exploited to desensitize MRGPRX2 to prevent severe pseudoallergic reactions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The organotypic co-culture skin model has been providing an advanced approach to in vitro investigations of the skin. Mast cells, containing various mediators such as tryptase and chymase, are thought to contribute to many physiological and pathological events of the skin interactively with other cells. Here, we introduce an organotypic co-culture skin model which successfully integrates human dermal mast cells for further study of mast cell interactions with fibroblasts and keratinocytes.

Mast cells (MCs) are the principal effector cells of IgE-mediated allergy. IL-33 is released by resident skin cells as alarmin upon tissue damage or allergen contact. Owing to their pronounced receptor expression, MCs are important targets of IL-33 action, but consequences for skin MCs are ill-defined, especially upon chronic exposure to IL-33. Mimicking the inflammatory milieu of skin disorders, we found that persistent exposure to IL-33 (over a 5-week period) strengthened skin MC numbers through accelerated cell-cycle progression and restriction of apoptosis. Conversely, IL-33 attenuated degranulation and FcεRI expression, potentially as a feedback to chronic "alarmin" exposure. Interestingly, the negative impact on histamine release was counterbalanced by amplified histamine production. Considering the clinical significance of histamine and scarce information on its regulation, we explored the molecular underpinnings. IL-33 induced swift phosphorylation of p38 and JNK (but not of ERK1/2 or AKT), and stimulated histidine decarboxylase expression. Combining pharmacological inhibition and kinase elimination by Accell-facilitated RNA interference in skin MCs revealed a p38-dependent, but JNK-independent mechanism. Collectively, IL-33 exerts multifaceted effects on cutaneous MCs at a post-maturation stage. The IL-33-skin MC axis may contribute to and balance inflammation in chronic skin disorders., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Clinically relevant exocytosis of mast cell (MC) mediators can be triggered by high-affinity IgE receptor (FcεRI)-aggregation (allergic route) or by the so-called pseudo-allergic pathway elicited via MAS-related G protein-coupled receptor-X2 (MRGPRX2). The latter is activated by drugs and endogenous neuropeptides. We recently reported that FcεRI-triggered degranulation is attenuated when human skin mast cells are chronically exposed to IL-33. Here, we were interested in the regulation of the MRGPRX2-route. Chronic exposure of skin MCs to IL-33 basically eliminated the pseudo-allergic/neurogenic route as a result of massive MRGPRX2 reduction. This downregulation seemed to partially require c-Jun N-terminal Kinase (JNK), but not p38, the two kinases activated by IL-33 in skin MCs. Surprisingly, however, JNK had a positive effect on MRGPRX2 expression in the absence of IL-33. This was evidenced by Accell ® -mediated JNK knockdown and JNK inhibition. In stark contrast to the dampening effect upon prolonged exposure, IL-33 was able to prime for increased degranulation by MRGPRX2 ligands when administered directly before stimulation. This supportive effect depended on p38, but not on JNK activity. Our data reinforce the concept that exposure length dictates whether IL-33 will enhance or attenuate secretion. IL-33 is, thus, the first factor to acutely enhance MRGPRX2-triggered degranulation. Finally, we reveal that p38, rarely associated with MC degranulation, can positively affect exocytosis in a context-dependent manner.

MRGPRX2 was recently uncovered as the "missing link" in clinically relevant mast cell (MC) activation explaining previously puzzling phenomena. It is the receptor for various endogenous ligands and exogenous compounds alike, whose binding evokes rapid degranulation much like allergen-mediated exocytosis. While the perceivable outcomes are similar, the two activation routes differ regarding mechanism and regulation. We recently reported that acute SCF administration curbs responses evoked by MRGPRX2 in human skin MCs. Maintenance of MCs in culture requires the presence of MC supportive factors and renders the cells functionally and molecularly unequal to ex vivo counterparts. Here, we asked whether expansion in culture impacts the pseudo-allergic route, and if so, what contribution SCF and IL-4 play in this scenario. We report that the in vitro micromilieu dampens (but does not erase) pseudo-allergic responses and that this is accompanied by strongly reduced MRGPRX2 expression. Withdrawal of SCF or IL-4 individually, but most potently of both collectively, partially reinstates the MRGPRX2 pathway, revealing that SCF and IL-4 make negative adjustments to the pseudo-allergic pathway. Under all conditions, the FcεRI-triggered route showed the inverse pattern of regulation, substantiating that allergic and pseudo-allergic MC activation can obey opposite rules, hinting at possible competition between them., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

The Vitamin-A-metabolite retinoic acid (RA) acts as a master regulator of cellular programs. Mast cells (MCs) are primary effector cells of type-I-allergic reactions. We recently uncovered that human cutaneous MCs are enriched with RA network components over other skin cells. Yet, direct experimental evidence on the significance of the RA-MC axis is limited. Here, skin-derived cultured MCs were exposed to RA for seven days and investigated by flow-cytometry (BrdU incorporation, Annexin/PI, FcεRI), microscopy, RT-qPCR, histamine quantitation, protease activity, and degranulation assays. We found that while MC size and granularity remained unchanged, RA potently interfered with MC proliferation. Conversely, a modest survival-promoting effect from RA was noted. The granule constituents, histamine and tryptase, remained unaffected, while RA had a striking impact on MC chymase, whose expression dropped by gene and by peptidase activity. The newly uncovered MRGPRX2 performed similarly to chymase. Intriguingly, RA fostered allergic MC degranulation, in a way completely uncoupled from FcεRI expression, but it simultaneously restricted MRGPRX2-triggered histamine release in agreement with the reduced receptor expression. Vitamin-A-derived hormones thus re-shape skin-derived MCs numerically, phenotypically, and functionally. A general theme emerges, implying RA to skew MCs towards processes associated with (allergic) inflammation, while driving them away from the skin-imprinted MC TC ("MCs containing tryptase and chymase") signature (chymase, MRGPRX2). Collectively, MCs are substantial targets of the skin retinoid network.

Mast cells (MCs), unique cellular elements of the body, are commonly associated with IgE-mediated reactions and manifestations of Th2-type immunity. A key characteristic of the lineage is its heterogeneity, with subsets displaying significant variation depending on maturation stage, species, tissue, microenvironment and other. Heterogeneity also affects MC responses to extracellular cues. Indeed, IL-4, the signature cytokine of Th2-immunity, can affect MCs in opposing ways ranging from the induction of apoptosis to positive regulation of lineage characteristics. It is unknown, however, whether IL-4 alters the phenotype of terminally differentiated human cutaneous MCs. Using our well-established technique for homogeneous purification of human skin MCs, we now report that prolonged contact with IL-4 not only increases MC expansion, but also phenotypically and functionally re-shapes the cells. FcεRI cell surface expression, FcεRIα-specific mRNA and FcεRI-mediated histamine release are all augmented by IL-4, while histamine release elicited by the non-immunological stimulus, substance P, remains unaffected. IL-4's potential to mold MCs is broad and similarly detectable across donors. Intriguingly, IL-4 impacts granule-associated mediators, especially histamine whose synthesis is boosted in the presence of IL-4. To our knowledge, an increase in histamine production by IL-4 has not been described yet for any type of MCs, but may well contribute to its pro-allergic effect given the significance of this biogenic amine to allergic symptoms. Collectively, IL-4 alters human skin MCs after long-term exposure mimicking chronic disorders by strengthening MC numbers and intensifying processes associated with allergic inflammation.

Mast cells (MCs) are unique constituents of the human body. While inter-individual differences may influence the ways by which MCs operate in their skin habitat, they have not been surveyed in a comprehensive manner so far. We therefore set out to quantify skin MC variability in a large cohort of subjects. Pathophysiologically relevant key features were quantified and correlated: transcripts of c-kit, FcεRIα, FcεRIβ, FcεRIγ, histidine decarboxylase, tryptase, and chymase; surface expression of c-Kit, FcεRIα; activity of tryptase, and chymase; histamine content and release triggered by FcεRI and Ca(2+) ionophore. While there was substantial variability among subjects, it strongly depended on the feature under study (coefficient of variation 33-386%). Surface expression of FcεRI was positively associated with FcεRIα mRNA content, histamine content with HDC mRNA, and chymase activity with chymase mRNA. Also, MC signature genes were co-regulated in distinct patterns. Intriguingly, histamine levels were positively linked to tryptase and chymase activity, whereas tryptase and chymase activity appeared to be uncorrelated. FcεRI triggered histamine release was highly variable and was unrelated to FcεRI expression but unexpectedly tightly correlated with histamine release elicited by Ca(2+) ionophore. This most comprehensive and systematic work of its kind provides not only detailed insights into inter-individual variability in MCs, but also uncovers unexpected patterns of co-regulation among signature attributes of the lineage. Differences in MCs among humans may well underlie clinical responses in settings of allergic reactions and complex skin disorders alike., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Retinoic acid (RA), the active vitamin-A-metabolite, has well-established functions in skin homeostasis and in the immune system. Skin mast cells (MCs) combine traits of both structures, being of hematopoietic origin, but functional in the skin environment. It remains largely unknown whether mature MCs are targeted by the retinoid network. Here, we demonstrate that human skin MCs display substantial susceptibility to RA by which they are instructed to increase pro-inflammatory mediators (IL-1β, IL-8, TNF-α) but not histamine release. The effects are observed at physiological RA levels, in different microenvironments, and are largely donor-independent. RA susceptibility is owed to the cells' abundant expression of RARA, the receptor mediating MC cytokine responses. Unexpectedly, bioinformatics calculations on the FANTOM5 expression atlas revealed general enrichment of retinoid network components in MCs against other skin cells, and MCs rapidly upregulated RA responsive genes. In conclusion, MCs are important yet hitherto overlooked retinoid targets in the skin., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Mast cells (MCs) mature exclusively in peripheral tissues, hampering research into their developmental and functional programs. Here, we employed deep cap analysis of gene expression on skin-derived MCs to generate the most comprehensive view of the human MC transcriptome ever reported. An advantage is that MCs were embedded in the FANTOM5 project, giving the opportunity to contrast their molecular signature against a multitude of human samples. We demonstrate that MCs possess a unique and surprising transcriptional landscape, combining hematopoietic genes with those exclusively active in MCs and genes not previously reported as expressed by MCs (several of them markers of unrelated tissues). We also found functional bone morphogenetic protein receptors transducing activatory signals in MCs. Conversely, several immune-related genes frequently studied in MCs were not expressed or were weakly expressed. Comparing MCs ex vivo with cultured counterparts revealed profound changes in the MC transcriptome in in vitro surroundings. We also determined the promoter usage of MC-expressed genes and identified associated motifs active in the lineage. Befitting their uniqueness, MCs had no close relative in the hematopoietic network (also only distantly related with basophils). This rich data set reveals that our knowledge of human MCs is still limited, but with this resource, novel functional programs of MCs may soon be discovered.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

The organotypic co-culture skin model has been providing an advanced approach to the in vitro investigation of the skin. Mast cells, containing various mediators such as tryptase and chymase, are thought to contribute to many physiological and pathological events of the skin interactively with other cells. Here, we introduce an organotypic co-culture skin model which successfully integrates human dermal mast cells for further study of mast cell interactions with fibroblasts and keratinocytes.

Human skin mast cells proliferated in the presence of interleukin (IL)-4+SCF (expanding 18-fold in 8 weeks) and acquired profound responsiveness towards high affinity IgE receptor (FcεRI) cross-linking, liberating about 75% of their histamine. In a proof-of-concept, we found that these cells are useful for pharmacological testing. Even a subtle inhibition of degranulation can be visualized. This model might prove valuable in tests of novel anti-allergic drugs.

Increased vascular permeability is a key feature of the pathological symptoms caused by hantaviruses. Here, we analysed the interaction between hantaviruses and mast cells, which regulate vascular homeostasis. In highly purified human skin mast cells increasing amounts of Hantaan (HTNV) and, to a lower extent, Prospect Hill (PHV) virions were produced. Replication was confirmed by the production of viral plus-strand RNA as determined by a virus strand-specific RT-PCR. PHV but not HTNV elicited early expression of beta interferon, MxA, ISG15 and CCL5 consistent to studies with other cell types. The data demonstrate that mature mast cells are permissive to infection with hantaviruses. This interaction might contribute to the development of vascular leakage syndrome.

The receptor tyrosine kinase c-Kit is fundamental to mast cell (MC) development and maintenance. Its regulation can occur at various levels, but nothing is known about how this is accomplished in normal human tissue MC. Likewise, the baseline turnover of c-Kit has not been addressed yet. We used mature MC from human skin, along with the MC lines LAD-2 and HMC-1 and treated them with stem cell factor (SCF), cycloheximide, actinomycin D (AD) and combinations thereof, and determined expression levels of c-Kit and other surface receptors by flow cytometry. Ligand-induced internalization of c-Kit was found to be a universal mechanism and detectable in all MC subtypes. By Western blot analysis of LAD-2 cells, c-Kit was found to nearly disappear 3 h after the addition of SCF to slowly recover thereafter. Investigations into the baseline turnover of c-Kit expression revealed that c-Kit is strongly affected by the inhibition of de novo translation in all MC subsets, while a suppression of transcription had a weaker effect and displayed greater cell-to-cell variation. Only a minor impact on other cell surface receptors (CD29, CD50 and CD54) was noted. On combined treatment, cycloheximide, AD and SCF displayed additive effects, resulting in a complete disappearance of c-Kit from the cell surface. In conclusion, c-Kit represents a rapidly cycling cell surface receptor. It is not only immediately internalized upon binding of its ligand, but it is also heavily affected by the inhibition of translation or transcription when viewed against an average background. Interestingly, c-Kit regulation seems largely independent of the MC subtype.

To resolve the controversy regarding substance P (SP) mediated stimulation of mast cells (MC), we demonstrate that SP triggers histamine release from purified human skin MC (sMC), but contrast to stimulation via FcepsilonRI, does not effect the production of TNF-alpha or IL-8. Conversely, both anti-IgE and SP are suppressive in terms of IL-6. By quantitative RT-PCR, the amount of templates at baseline (per 25 ng total RNA) is 2178 (IL-6), 2,665 (IL-8) and 94 (TNF-alpha), and remains unaltered by SP. Contrast to sMC, LAD2 MC respond to SP with stronger histamine release and robust TNF-alpha production in an only partially neurokinin-1R mediated manner, while histamine release of sMC is chiefly mediated by this receptor. We conclude that human sMC are responsive to SP in a selective manner by eliciting degranulation without the induction of cytokines and that SP-triggered cytokine production varies among MC subtypes, likely through differences in signaling mechanisms.

Ultraviolet (UV) irradiation is an established treatment for inflammatory skin diseases, although the precise mode of action is still unclear. Activating and suppressive effects on mast cell (MC) mediator release have been described. The aim of this study was to investigate systematically the effects of UVB, UVA-1, and psoralen plus UVA-1 at therapeutic doses on skin-derived human MC. Baseline and stimulated release of histamine, tryptase, and of interleukin (IL)-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) were examined. In resting MC, UV light induced a slight, yet significant histamine release corresponding to enhanced surface levels of lysosome-associated membrane proteins (LAMP). In contrast, UV pre-treatment caused a marked suppression of the anti-IgE-induced histamine release, accompanied by a diminished, anti-IgE-mediated increase in LAMP expression. The secretion of IL-6, IL-8, and TNF-alpha was inhibited in resting and activated MC, suggesting a different mode of action. Regarding the importance of MC in a variety of allergic and inflammatory processes, our data show a high susceptibility of this cell type towards UV light, which seems to partially depend on the state of cellular activation. Immunosuppressive effects predominate in activated MC, thus corresponding with the beneficial effects in inflammatory diseases, whereas in resting MC, both stimulatory and inhibitory effects are observed.

Mast cells generate and release histamine during anaphylactic reactions, and there is pharmacological evidence that histamine regulates this process via specific receptors. Therefore, we examined human leukemic (HMC-1) and normal skin mast cells for the expression of all four currently known histamine receptors. Both cell types expressed H2 and H4 receptors at mRNA and protein levels, whereas H3 receptor specific mRNA and receptor protein was undetectable. Similarly, immunohistochemistry of cutaneous tissue showed an absence of H3 receptor in these cells. Despite transcription of mRNA, H1 receptor protein was only moderately expressed in HMC-1 cells and was virtually absent in skin mast cells. Furthermore, only H1, H2, and H4 receptors were detectable by Western blot analysis of HMC-1 cells. Radiolabeled histamine binding was strongly inhibited only by H2 (ranitidine)- and H3/H4 (FUB 108)-specific antagonists. Histamine-induced increase of cAMP was inhibited by the H2 receptor antagonist famotidine, whereas induction of IP3 was not observed, making signaling via the H1 receptor unlikely. These data show that human mast cells constitutively express primarily H2 and H4 receptors and that H2 receptors are functionally linked to cellular processes. They provide new insights into the mechanisms that govern auto- and paracrine histamine-induced mast cell functions.

Although known as heterogenous, mast cells (MC) are believed to induce allergic inflammation, partially by secretion of T helper cell type 2 (Th2) cytokines. We show here that MC purified from two human skin compartments produce cytokines that are primarily associated with inflammation and innate immunity [interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha)]. Although these are detectable even without stimulation, immunoglobulin (Ig)E receptor cross-linking is able to enhance only TNF-alpha production, but phorbol 12-myristate 13-acetate additionally promotes IL-1beta and IL-8. With the exception of TNF-alpha, the presence of serum has a positive impact on cytokine production. Although IL-13 transcripts (but not those for IL-4 and -5) are produced by skin MC, all Th2 cytokines remain undetectable in the supernatants or lysates of MC from foreskin and breast skin by all treatments. Therefore, rather than sharing similarity with Th2 cells, the cytokine profile of skin MC at baseline resembles that of monocytes. Of note, MC precultured in the presence of IL-4 [alone or plus stem cell factor (SCF)] before anti-IgE stimulation, acquired the ability to produce IL-5, and IL-1beta was concomitantly suppressed. Additionally, strong up-regulation of IL-6 by SCF was observed, which was inhibited by IL-4. In summary, we present a detailed analysis of the cytokine array of human skin MC immediately upon isolation; demonstrate that MC from different skin compartments, although producing the same pattern of cytokines, display quantitative differences in several aspects; and provide further evidence that MC possess a proinflammatory capacity, which can, however, be altered by microenvironmental stimuli, substantiating the marked plasticity of the cells.

As diverse pruritic cutaneous diseases respond to ultraviolet treatment, we have examined whether ultraviolet light is capable of inducing apoptosis in mast cells. Human mast cell line 1 (HMC1) derived from a patient with malignant mastocytosis and purified skin mast cells were irradiated with single doses of ultraviolet B or ultraviolet A1, or pretreated with 8-methoxypsoralen prior to ultraviolet A1 exposure. After 0 to 48 h of incubation, the percentage of apoptotic and dead cells was assessed. In HMC1 cells, morphologic features of apoptosis were further evaluated by electron microscopy. All ultraviolet treatment induced apoptosis of HMC1 cells in a time- and dose-dependent manner. Apoptosis was associated with activation of caspase-3, release of cytochrome C, cleavage of poly(ADP-ribose)-polymerase, and nuclear accumulation of p53. In contrast, resting skin mast cells were resistant to ultraviolet light induced apoptosis. After incubation with stem cell factor and interleukin-4 for 2 wk, however, slowly proliferating skin mast cells also underwent apoptosis in response to ultraviolet light. In conclusion, these data demonstrate that ultraviolet light directly affects mast cells, but mainly aims at the proliferating mast cells as found in mastocytosis and mast cell dependent pruritic diseases, where increased numbers are observed due to the recruitment mast cell precursors from the blood.