Sven Guhl

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.

Evidence for a restricted rather than generalized stimulatory response of skin-derived human mast cells to substance P

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.

Comparative cytokine profile of human skin mast cells from two compartments—strong resemblance with monocytes at baseline but induction of IL‐5 by IL‐4 priming

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 twohuman skin compartments produce cytokines that are primarily associated with inflammation and innate immunity [interleukin (IL)‐1β, IL‐6, IL‐8, tumor necrosis factor α (TNF‐α)]. Although these are detectable even without stimulation, immunoglobulin (Ig)E receptor cross‐linking is able to enhance only TNF‐α production, but phorbol 12‐myristate 13‐acetate additionally promotes IL‐1β and IL‐8. With the exception of TNF‐α, 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‐1β 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.

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.

The transcription factor profile of human mast cells in comparison with monocytes and granulocytes

Abstract.Expression profiles of mRNAs and proteins for various transcription factors were determined for human skin mast cells (sMCs), leukemic HMC-1 MCs, monocytes and granulocytes. By quantitative RT-PCR, sMCs expressed lower levels of c-fos, PU.1, C/EBPα, and C/EBPɛ than monocytes and granulocytes, but higher levels of MITF, SCL, GATA-1 and GATA-2. At the protein level, MITF, SCL, GATA-2, Elf-1 and c-fos were clearly detectable in sMCs. With the exception of c-fos, these proteins were absent or expressed only slightly in monocytes and granulocytes. The expression of NF-E2p45, GATA-1, PU.1, Ets-1, C/EBPα and C/EBPɛ was below the detection limit in sMCs, but detectable in other myelocytes. The high expression of SCL and GATA-2 in sMCs is reminiscent of stem cells. The absence of C/EBPɛ in sMCs, but strong expression in HMC-1, suggests it may impair MC maturation. In summary, mature human MCs can be characterized as C/EBPαlow, C/EBPɛ−, PU.1low, GATA-1low, GATA-2+, SCL+, MITFhigh.

Infection of in vivo differentiated human mast cells with hantaviruses.

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.

Baseline and stimulated turnover of cell surface c-Kit expression in different types of human mast cells

Abstract:  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.

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, 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 Ca2+ 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 Ca2+ 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.

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.

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.