MRGPRX2 Is the Codeine Receptor of Human Skin Mast Cells: Desensitization through ß-Arrestin and Lack of Correlation with the FcεRI Pathway.

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 ß-arrestin-1‒dependent manner. Codeine-triggered ß-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.

Integration of the Human Dermal Mast Cell into the Organotypic Co-culture Skin Model.

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.

IL-33 and MRGPRX2-Triggered Activation of Human Skin Mast Cells-Elimination of Receptor Expression on Chronic Exposure, but Reinforced Degranulation on Acute Priming.

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.

Yin-Yang of IL-33 in Human Skin Mast Cells: Reduced Degranulation, but Augmented Histamine Synthesis through p38 Activation.

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.

MRGPRX2 is negatively targeted by SCF and IL-4 to diminish pseudo-allergic stimulation of skin mast cells in culture.

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.

An integrated expression atlas of miRNAs and their promoters in human and mouse.

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.

An efficient method for gene knock-down by RNA interference in human skin mast cells.

Mast cells (MCs) from human skin have been notoriously resistant to gene manipulation, and a method to knock-down gene expression in in situ differentiated MCs is highly desired. The Dharmacon Accell® transfection system proved successful on several "difficult-to-transfect" cells. In the present work, we therefore tested this method on skin-derived MCs using different siRNA entities. The siRNA was readily taken up, followed by pronounced, specific reduction of gene and protein expression. Hence, we present the first efficient technique for the manipulation of gene expression in primary skin MCs ex vivo, which combines high transfection rates with retained cell viability.

Retinoic Acid Negatively Impacts Proliferation and MCTC Specific Attributes of Human Skin Derived Mast Cells, but Reinforces Allergic Stimulability.

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 MCTC ("MCs containing tryptase and chymase") signature (chymase, MRGPRX2). Collectively, MCs are substantial targets of the skin retinoid network.

IL-4 and human skin mast cells revisited: reinforcement of a pro-allergic phenotype upon prolonged exposure.

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.

Phenotypic variability in human skin mast cells.

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, FcεRIß, 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.

Retinoic acid potentiates inflammatory cytokines in human mast cells: identification of mast cells as prominent constituents of the skin retinoid network.

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-1ß, 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.

Skin mast cells develop non-synchronized changes in typical lineage characteristics upon culture.

Despite their hematopoietic origin, mast cells (MCs) develop exclusively in tissues, hampering their ample use in research. To circumvent this problem, tissue-derived MCs are typically first expanded in culture, but the changes MCs may undergo in the novel micromilieu are poorly defined. Here, we monitor skin MCs from a number of donors over time, revealing profound yet non-synchronized modulations in culture. While tryptase and chymase, the most specific markers, strongly decline, FcεRI surface expression, and FcεRI-mediated histamine release steeply increase (from ≈15.5% to ≈60%), replicated by similar increments in TNF-α secretion. Interestingly, the modulations are independent of cell cycle progression, as they are comparable in the growth and postgrowth phase, implying they primarily result from microenvironmental conditioning. The data highlight a high degree of MC versatility, but also advise that results based on cultured MCs should be viewed with some caution, as they may not accurately reflect their counterparts in situ.

Integration of the human dermal mast cell into the organotypic co-culture skin model.

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.

Redefinition of the human mast cell transcriptome by deep-CAGE sequencing.

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.

Testosterone exerts selective anti-inflammatory effects on human skin mast cells in a cell subset dependent manner.

Androgens are known to exert anti-inflammatory effects but their impact on mast cells (MCs) remains to be determined. Here, we show that MCs isolated from human foreskin samples (male) and those from breast skin (female) express the androgen receptor, albeit with a 10-fold difference between the subsets. While fundamental MC properties (FcεRI, c-Kit, tryptase; histamine release upon FcεRI cross-linking) were unaffected or slightly reduced (chymase) by testosterone, the hormone had a more profound impact on the production of cytokines, with IL-6 being a target (reduction by 53%). Interestingly, this effect was limited to breast skin MCs (15 of 16 donors displayed this phenomenon), but was not reproduced by foreskin MCs. Collectively, effector functions of human skin MCs are modulated by androgens in a gene-selective and MC subset-specific fashion. Possibly, MCs from women are more susceptible to testosterone. We also demonstrate that MC IL-6 production is highly variable among individuals.

Mast cell-derived TNF-α and histamine modify IL-6 and IL-8 expression and release from cutaneous tumor cells.

The coincidence of skin tumors and elevated mast cell (MC) numbers has been known for many years. However, it has remained controversial whether, in this context, MCs promote or inhibit tumor growth. Addressing this problem, different melanoma and squamous cell carcinoma cell lines were co-cultivated with primary, dermal MC for 24 h and gene or protein expression of cytokines tumor necrosis factor (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) estimated. Co-culture with MCs led to an increase in IL-8 gene expression and IL-8 protein release from melanoma cells and IL-6 and IL-8 gene expression and protein release from squamous cell carcinoma cells, respectively. Moreover induction of IL-6 and IL-8 was primarily regulated by MC-derived TNF-α. Our data suggest an interplay between MCs and tumor cells, which results in altered cytokine release and may, thus, have an impact on tumor growth, invasion and neovascularisation.

Long-term cultured human skin mast cells are suitable for pharmacological studies of anti-allergic drugs due to high responsiveness to FcεRI cross-linking.

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.

Mast cell lines HMC-1 and LAD2 in comparison with mature human skin mast cells--drastically reduced levels of tryptase and chymase in mast cell lines.

To circumvent the costly isolation procedure associated with tissue mast cells (MC), two human MC lines, i.e. HMC-1 and LAD2, are frequently employed, but their relation to mature MC is unknown. Here, we quantitatively assessed their expression of MC markers in direct comparison to skin MC (sMC). sMC expressed all lineage markers at highest and HMC-1 cells at lowest levels. LAD2 cells expressed comparable high-affinity IgE receptor alpha (FcepsilonRIalpha) and FcepsilonRIgamma but less FcepsilonRIbeta than sMC and displayed slightly reduced, but robust FcepsilonRI-mediated histamine release. Only minor differences were found for total histamine content and c-Kit expression. Huge, and to this level unexpected, differences were found for MC tryptase and chymase, with sMC >>> LAD2 > HMC-1. Taken together, HMC-1 cells represent very immature malignantly transformed MC, whereas LAD2 cells can be considered intermediately differentiated. Because of the minute levels of MC proteases, MC lines can serve as surrogates of tissue MC to a limited degree only.