Human Mast Cell Progenitors Research Articles

CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation

The differentiation of hematopoietic stem and progenitor cells into the various cell lineages is regulated by cell-intrinsic factors and the progenitors' microenvironment. Experiments in mice have allowed the identification of transcriptional modulators that control mast cell differentiation. However, a comprehensive approach that allows efficient disruption of individual transcriptional modulators in primary human hematopoietic progenitors coupled with a mast cell formation readout has not been described. Here, we report a simple electroporation- and ribonucleoprotein-based knockout system that allows the identification of genes that are required and dispensable for human mast cell differentiation. We show that the transcription factor MITF is upregulated in human mast cell progenitors and reveal that the loss of MITF results in the suppressed formation of mast cells. By contrast, CITED2, another transcriptional modulator that is upregulated along the mast cell differentiation trajectory, was dispensable for human mast cell differentiation. Taken together, we report a CRISPR/Cas9-based framework that serves to identify genes involved in regulating the formation of human mast cells, and the results uncover the role of two transcriptional modulators in controlling human mast cell differentiation.

Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors

AbstractMast cell accumulation is a hallmark of a number of diseases, including allergic asthma and systemic mastocytosis. Immunoglobulin E–mediated crosslinking of the FcεRI receptors causes mast cell activation and contributes to disease pathogenesis. The mast cell lineage is one of the least studied among the hematopoietic cell lineages, and controversies remain about whether FcεRI expression appears during the mast cell progenitor stage or during terminal mast cell maturation. Here, we used single-cell transcriptomics analysis to reveal a temporal association between the appearance of FcεRI and the mast cell gene signature in CD34+ hematopoietic progenitors in adult peripheral blood. In agreement with these data, the FcεRI+ hematopoietic progenitors formed morphologically, phenotypically, and functionally mature mast cells in long-term culture assays. Single-cell transcriptomics analysis further revealed the expression patterns of prospective cytokine receptors regulating development of mast cell progenitors. Culture assays showed that interleukin-3 (IL-3) and IL-5 promoted disparate effects on progenitor cell proliferation and survival, respectively, whereas IL-33 caused robust FcεRI downregulation. Taken together, we showed that FcεRI expression appears at the progenitor stage of mast cell differentiation in peripheral blood. We also showed that external stimuli regulate FcεRI expression of mast cell progenitors, providing a possible explanation for the variable FcεRI expression levels during mast cell development.

IgE cross-linking induces activation of human and mouse mast cell progenitors

The concept of innate and adaptive effector cells that are repleted by maturing inert progenitor cell populations is changing. Mast cells develop from rare mast cell progenitors populating peripheral tissues at homeostatic conditions, or as a result of induced recruitment during inflammatory conditions. Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation invivo, we hypothesized that they are activated by IgE cross-linking. Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen, or stimulated with anti-IgE, and the mast cell progenitor population analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. Sensitized mouse peritoneal mast cell progenitors demonstrate increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors, and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produce IL-13. Mast cell progenitors become activated by IgE cross-linking and may contribute to the pathology associated with acute allergic airway inflammation.

Localization-Specific Expression of CCR1 and CCR5 by Mast Cell Progenitors.

Mast cells are powerful immune cells found predominately in barrier tissues. They play an important role in immune surveillance and act as effector cells in allergic reactions. Mast cells develop from mast cell progenitors (MCp), which migrate to the peripheral tissues via the blood circulation. Presumably, the homing of MCp to the peripheral sites and localization is regulated by chemotactic signals. Due to the scarce abundance of these cells, chemotactic receptors have not been previously characterized on primary MCp. Here, mRNA transcripts for CCR1 and CX3CR1 were identified in mouse bone marrow and lung MCp in a gene expression screen of chemotactic receptors. However, surface expression of CCR1 was only found in the bone marrow MCp. Flow cytometry-based screening identified distinct surface expression of CCR5 by mouse peritoneal mast cells and MCp, while surface expression of CXCR2-5, CX3CR1, CCR1-3, CCR6-7, and CCR9 was not detected. Low surface expression of CCR5 was detected in mouse MCp in the bone marrow, spleen, and lung. To translate the findings to human, blood and bone marrow MCp from healthy donors were analyzed for possible CCR1 and CCR5 expression. Human MCp showed distinct surface expression of both CCR1 and CCR5. The expression levels of these chemokine receptors were higher in human bone marrow MCp than in the peripheral blood, suggesting that CCR1 and CCR5 may mediate retention in the bone marrow. In conclusion, mouse and human MCp show differential expression of CCR1 and CCR5 depending on their localization.

Mast Cells and Their Progenitors in Allergic Asthma.

Mast cells and their mediators have been implicated in the pathogenesis of asthma and allergy for decades. Allergic asthma is a complex chronic lung disease in which several different immune cells, genetic factors and environmental exposures influence the pathology. Mast cells are key players in the asthmatic response through secretion of a multitude of mediators with pro-inflammatory and airway-constrictive effects. Well-known mast cell mediators, such as histamine and bioactive lipids are responsible for many of the physiological effects observed in the acute phase of allergic reactions. The accumulation of mast cells at particular sites of the allergic lung is likely relevant to the asthma phenotype, severity and progression. Mast cells located in different compartments in the lung and airways have different characteristics and express different mediators. According to in vivo experiments in mice, lung mast cells develop from mast cell progenitors induced by inflammatory stimuli to migrate to the airways. Human mast cell progenitors have been identified in the blood circulation. A high frequency of circulating human mast cell progenitors may reflect ongoing pathological changes in the allergic lung. In allergic asthma, mast cells become activated mainly via IgE-mediated crosslinking of the high affinity receptor for IgE (FcεRI) with allergens. However, mast cells can also be activated by numerous other stimuli e.g. toll-like receptors and MAS-related G protein-coupled receptor X2. In this review, we summarize research with implications on the role and development of mast cells and their progenitors in allergic asthma and cover selected activation pathways and mast cell mediators that have been implicated in the pathogenesis. The review places an emphasis on describing mechanisms identified using in vivo mouse models and data obtained by analysis of clinical samples.

Murine and human mast cell progenitors

The development of mature mast cells (MCs) from hematopoietic progenitor cells as well as the identification and characterization of committed progenitor cells are a current focus of mast cell research. Most published reports in this area are on the origin and differentiation of MCs in mice. Evidence for the human system, i.e. derived from primary human MCs, is widely lacking. Based on the published data, MCs develop either from a committed progenitor or from a common basophil/mast cell precursor. This review summarizes the current knowledge on MC development and MC differentiation.

Mast Cells as Reservoirs for HIV Latency

Mast Cells as Reservoirs for HIV Latency More than a decade ago, Bannert and colleagues first reported that human mast cell (MC) progenitors are susceptible to infection with HIV-1. This finding was confirmed and extended by other groups in the following years providing in vivo evidence that tissue MCs, developing from infected human MC progenitors, remain productively infected in infected people. Moreover, evidence was shown that Toll-like receptor (TLR) 2, TLR4, or TLR9 stimulation of latently infected mast cells induced re-initiation of HIV-1 replication.

Differential expression of leukocyte immunoglobulin-like receptors on cord blood-derived human mast cell progenitors and mature mast cells

The leukocyte Ig-like receptors (LILRs) comprise a family of cell-surface immunoregulatory receptors with activating and inhibitory members. The inhibitory LILRs possess cytoplasmic ITIMs that down-regulate signaling by nonreceptor tyrosine kinase cascades. The activating members have a truncated cytoplasmic domain and signal through the FcR gamma chain. We examined the expression of LILRs on human mast cells during their development in vitro. Progenitor mast cells expressed cell surface inhibitory LILRB1, -B2, -B3, and -B4 and activating LILRA1. However, although mature cord blood-derived mast cells (hMCs) had detectable mRNA encoding multiple LILRs, none were expressed on the cell surface. Culture of progenitor mast cells or hMCs with various cytokine combinations failed to retain or induce cell surface expression of the LILRs. It is interesting that hMCs expressed LILRB5 in cytoplasmic granules and upon cross-linking of the high-affinity IgE receptor, released LILRB5 into the culture medium. Our results demonstrate that LILRs are developmentally regulated in human mast cells and that LILRB5 is expressed in mast cell granules and the release of soluble LILRB5 following IgE FcR-dependent stimulation, which has potential for amplification of mast cell-dependent, inflammatory responses.

Human mast cell progenitors use α4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions

AbstractMast cells (MCs) are central to asthma and other allergic diseases, and for responses to infection and tissue injuries. MCs arise from committed progenitors (PrMCs) that migrate from the circulation to tissues by incompletely characterized mechanisms, and differentiate in situ in perivascular connective tissues of multiple organs. PrMCs derived in vitro from human cord blood were examined for adhesion molecule expression and their ability to adhere to human umbilical vein endothelial cells (HUVECs) under conditions that mimic physiologic shear flow. The PrMCs expressed α4β1, low levels of β7, and the β2-integrins αLβ2 and αMβ2. The PrMCs also expressed PSGL-1, but not L-selectin. At low (0.5 dynes/cm2-1.0 dynes/cm2) shear stress, PrMCs attached and rolled on recombinant E-selectin and P-selectin and VCAM-1. An anti–PSGL-1 monoclonal antibody (mAb) blocked essentially all adhesion to P-selectin but reduced adhesion to E-selectin by only 40%, suggesting PrMCs express other ligands for E-selectin. PrMCs adhered strongly to tumor necrosis factor-α (TNF-α)–activated HUVECs, whereas adhesion to interleukin 4 (IL-4)–activated HUVECs was lower. PrMC adhesion to IL-4–activated HUVECs was totally α4-integrin– and VCAM-1–dependent. Adhesion to TNF-α–activated HUVECs was blocked by 50% by mAbs against α4-integrin, vascular cell adhesion molecule–1 (VCAM-1), E-selectin, or PSGL-1, whereas combinations of mAbs to α4-integrin plus PSGL-1, or VCAM-1 plus E-selectin, blocked adhesion by greater than 70%. Thus, PrMCs derived in vitro predominantly use α4-integrin, VCAM-1, PSGL-1, and other ligands that bind E-selectin for adhesion to cytokine-activated HUVEC monolayers. These observations may explain the abundance of MCs at sites of mucosal inflammation, where VCAM-1 and E-selectin are important inducible receptors.

Human Mast cell progenitors can be infected by macrophagetropic human immunodeficiency virus type 1 and retain virus with maturation in vitro.

Mast cells are critical components of innate and adaptive immunity that differentiate in tissues in situ from circulating committed progenitor cells. We now demonstrate that human cord blood-derived mast cell progenitors are susceptible to infection with macrophagetropic (M-tropic) and dualtropic human immunodeficiency virus type 1 (HIV-1) isolates but not with T-cell-tropic (T-tropic) strains. Mast cell progenitors (c-kit(+) CD13(+) cells with chloroacetate esterase activity) were purified from 4-week-old cultures of cord blood mononuclear cells maintained in stem cell factor, interleukin-6 (IL-6), and IL-10 using a CD14 depletion column. These progenitors expressed CCR3, CCR5, and CXCR4, as well as low levels of CD4. When infected in vitro with viruses pseudotyped with different HIV and simian immunodeficiency virus envelope glycoproteins, only M-tropic and dualtropic, but not T-tropic, viruses were able to enter mast cell progenitors. Both the CCR5-specific monoclonal antibody 2D7 and TAK-779, a nonpeptide inhibitor of CCR5-mediated viral entry, blocked HIV-1 strain ADA infection by >80%. Cultures infected with replication-competent virus produced progressively increasing amounts of virus for 21 days as indicated by p24 antigen detection. Mast cell progenitors that were exposed to an M-tropic, green fluorescent protein-expressing HIV-1 strain exhibited fluorescence indicative of viral entry and replication on a single-cell level and retained virus production during differentiation. The trafficking of mast cell progenitors to multiple tissues, combined with the long life span of mature mast cells, suggests that they could provide a widespread and persistent HIV reservoir in AIDS.

The chemokine receptor CXCR4 is expressed within the mast cell lineage and its ligand stromal cell-derived factor-1alpha acts as a mast cell chemotaxin.

In this study we provide evidence that the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) acts as a mast cell chemoattractant through interactions with its receptor CXCR4 expressed on mast cell progenitors in the blood as well as on in vitro-developed and leukemic mast cells. We found expression of CXCR4 on cord blood-derived mast cells (CBMC) and on the human mast cell line HMC-1, analyzed by RNAse protection assay and flow cytometry. SDF-1alpha induced intracellular calcium mobilization in HMC-1 cells and was chemotactic for both HMC-1 cells and CBMC. The activity of SDF-1alpha was completely blocked by treating the cells with pertussis toxin, indicating the involvement of Gi-proteins in the signaling. By applying a transwell assay we could show that SDF-1alpha induces migration of a cell population in peripheral blood that is enriched for cells with the capacity to differentiate into mast cells. These findings thus suggest a mechanism by which human mast cell progenitors may be recruited from circulation into the tissue.

Regulation of chymase production in human mast cell progenitors

Background: Although mature tryptase-positive mast cells (MCs) and tryptase and chymase double-positive MCs are recognized using in situ staining and are preferentially distributed in different tissues, recent findings suggest that tryptase-positive MCs can give rise to tryptase and chymase double-positive MCs. Objective: We investigated the regulation of chymase production in developing MCs. Methods: Human cord blood or peripheral blood cells were cultured in the presence of stem cell factor and IL-6 with or without IL-4 in methylcellulose or liquid medium. Intracellular chymase and tryptase were determined with immunocytochemistry, flow cytometry, and ELISA. Chymase messenger RNA expression was examined with 3 different methods, such as Northern blotting. Results: Flow cytometric analysis always showed a unimodal histogram of chymase-positive, as well as tryptase-positive, cells in the presence of various cytokines, even when chymase was not detected in some MCs with immunocytochemistry. The chymase protein expression increased by culture duration and was enhanced by cytokines, such as a high concentration of stem cell factor or IL-4. Chymase messenger RNA was expressed higher in immature MCs than mature chymase protein–rich MCs. We generated macroscopic MC colonies in methylcellulose by culturing CD34+ cells for 10 weeks and measured cellular chymase, tryptase, and histamine. The chymase/histamine ratio widely varied (0.07-1.01) depending on MC colony, even under the same culture conditions, including IL-4, whereas the tryptase/histamine ratio was relatively constant (1.02-1.89). Conclusion: All human MCs in culture are capable of producing chymase, and the production is clonally regulated at their progenitors by cytokine-independent mechanisms, as well as being totally controlled by cytokine-dependent mechanisms accompanied by maturation. (J Allergy Clin Immunol 2000;106:321-8.)

Retinoic acid is a negative regulator for the differentiation of cord blood-derived human mast cell progenitors

We examined the effects of retinoids on the human mast cell development using a serum-deprived culture system. When 10-week cultured mast cells derived from CD34(+) cord blood cells were used as target cells, both all-trans retinoic acid (ATRA) and 9-cis RA inhibited the progeny generation under stimulation with stem cell factor (SCF) in a dose-dependent manner (the number of progeny grown by SCF plus RA at 10(-7) mol/L was one tenth of the value obtained by SCF alone). The early steps in mast cell development appear to be less sensitive to RA according to the single CD34(+)c-kit(+) cord blood cell culture study. The optimal concentration of RAs also reduced the histamine concentration in the cultured mast cells (3.00 +/- 0.47 pg per cell in SCF alone, 1.44 +/- 0.18 pg per cell in SCF+ATRA, and 1.41 +/- 0.10 pg per cell in SCF+9-cis RA). RT-PCR analyses showed the expression of RARalpha, RARbeta, RXRalpha, and RXRbeta messenger ribonucleic acid (mRNA) in 10-week cultured mast cells. The addition of an RAR-selective agonist at 10(-10) mol/L to 10(-7) mol/L decreased the number of mast cells grown in SCF, whereas an RXR-selective agonist at up to 10(-8) mol/L was inactive. Among RAR subtype selective retinoids used at 10(-9) mol/L to 10(-7) mol/L, only the RARalpha agonist was equivalent to ATRA at 10(-7) mol/L in its ability to inhibit mast cell growth. Conversely, the addition of excess concentrations of a RARalpha antagonist profoundly counteracted the retinoid-mediated suppressive effects. These results suggest that RA inhibits SCF-dependent differentiation of human mast cell progenitors through a specific receptor. (Blood. 2000;95:2821-2828)

The potential of human mast cell progenitors to differentiate into mature mast cells remains after prolonged culture with flt3 ligand, interleukin-3 or granulocyte-macrophage colony stimulating factor.

Stem cell factor (SCF) plays a key role in the development of mast cells from haemopoietic progenitor cells. In this study we have investigated the effect of the early acting haemopoietic cytokines flt3 ligand (FL), IL-3 and GM-CSF on the SCF-dependent differentiation of mast cells from cord blood mononuclear cells. By using delayed addition of SCF, we examined the potential of mast cell progenitors to keep their capacity to differentiate into mast cells after exposure to factors signalling differentiation into other lineages. Culture with either cytokine for 3 weeks before transfer to SCF-containing medium resulted in the development of mast cells in all cultures. The appearance of mast cells was attenuated when the cells had been in culture with IL-3 or GM-CSF prior to culture in SCF, compared to cultures exposed to SCF alone for 7 weeks. However, a proportion of the cells had not lost the capacity to develop into mast cells. In contrast, in cultures initiated with FL and transferred to medium containing SCF, the same amount of mast cells developed as in the SCF cultures. Thus, cells committed to the mast cell lineage appear to be resistant to the lineage directives of IL-3 and GM-CSF and keep their potential to differentiate into mature mast cells.

Ultrastructural morphology of human mast cell progenitors in sequential cocultures of cord blood cells and fibroblasts.

The ontogeny of human mast cells (HMCs) is not known, but agranular precursors committed to HMC development are present in cord blood mononuclear cells. Individual cord bloods were cocultured with murine fibroblasts and prepared for light microscopy, immunofluorescent determination of tryptase and electron microscopy after sequential times in culture. Mast cells and their progenitors were identified and quantitated during their evolution from agranular precursors in human cord blood. Each of these methods revealed similar numbers of HMC progenitors. The earliest, visible granule contents were particulate; later, scrolls superimposed on particulate materials were noted. HMC progenitors did not contain crystal granules, but fully mature HMCs present in long-term cocultures did. The developmental sequence for the evolution of the HMC lineage in vitro, made available by this new culture system, is identical to that described for the development of HMCs in vivo in sequentially examined samples of human fetal tissues.

In vitro Development and Functions of Human Mast Cells

A long-term co-culture of mononuclear cells of human umbilical cord blood with mouse embryo-derived 3T3 fibroblasts resulted in the development of human mast cells. These mast cells are morphologically and functionally mature cells, containing 1.4-2.8 micrograms histamine per 10(6) cells and bear approximately 10(5) Fc epsilon RI per cell. The mast cells sensitized with human IgE released histamine upon challenge with anti-IgE. Electron-microscopic analysis of the cells showed that these cells were mature human mast cells, and clearly different from basophilic granulocytes. Most of the mast cells contained some granules with regular crystalline arrays and both tryptase and chymase, resembling human skin mast cells. When mononuclear cells of cord blood were seeded in a millicell insert which was placed on 3T3 fibroblasts monolayer, the number of mast cells developed in the millicell inserts was comparable to those developed in the co-culture of the same cord blood cells with 3T3 fibroblasts. Recent observations that mast cells developed in the presence of concentrated culture supernatants of 3T3 fibroblasts without fibroblasts feeder layers, confirmed that soluble factors released from 3T3 fibroblasts are essential and sufficient for the differentiation of human mast cell progenitors in vitro. Analysis of functional characteristics of cultured mast cells revealed that they respond to anti-IgE, Ca2+ ionophore A23187 and substance P for histamine release, but failed to respond to compound 48/80 and FMLP. Upon anti-IgE challenge, sensitized mast cells generated approximately 80 ng PGD2 per 10(6) cells, and approximately 50 ng of LTC4 per 10(6) cells but no detectable generation of LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)