Umbilical Cord Blood-derived Mast Cells Research Articles

Characterization of human umbilical cord blood-derived mast cells using high-throughput expression profiling and next-generation knowledge discovery platforms

Mast cells (MCs) are tissue-resident innate immune cells that express the high-affinity receptor for immunoglobulin E and are responsible for host defense and an array of diseases related to immune system. We aimed in this study to characterize the pathways and gene signatures of human cord blood-derived MCs (hCBMCs) in comparison to cells originating from CD34− progenitors using next-generation knowledge discovery methods. CD34+ cells were isolated from human umbilical cord blood using magnetic activated cell sorting and differentiated into MCs with rhIL-6 and rhSCF supplementation for 6–8 weeks. The purity of hCBMCs was analyzed by flow cytometry exhibiting the surface markers CD117+CD34−CD45−CD23−FcεR1αdim. Total RNA from hCBMCs and CD34− cells were isolated and hybridized using microarray. Differentially expressed genes were analyzed using iPathway Guide and Pre-Ranked Gene Set Enrichment Analysis. Next-generation knowledge discovery platforms revealed MC-specific gene signatures and molecular pathways enriched in hCBMCs and pertain the immunological response repertoire.

Novel MRGPRX2 antagonists inhibit IgE-independent activation of human umbilical cord blood-derived mast cells.

Human MCs are primary effectors implicated in immune surveillance and defense by secreting histamine and various inflammatory mediators, a mechanism termed as degranulation. MCs can be activated by two pathways: IgE-dependent classical pathway and the IgE-independent pathway that utilizes various cationic molecules including substance P (SP) and pituitary adenylate cyclase-activating polypeptides, which are host defense peptides collectively known as basic secretagogues. Our pharmacological study investigated whether or not IgE-independent MC activation is mediated via MRGPRX2. We identified two novel MRGPRX2 antagonists, which completely inhibited the degranulation of human cord blood-derived MCs (hCMCs) induced by basic secretagogues and pseudoallergic drug, icatibant, but IgE- or A23187-challenged hCMCs were resistant to MRGPRX2 antagonists. The MRGPRX2 antagonists markedly inhibited the de novo synthesis of SP-induced prostaglandin D2 in hCMCs. Moreover, the antagonists were able to inhibit p42/44 mitogen-activated protein kinase signal in hCMCs activated by SP. This study strongly suggests that MRGPRX2 antagonists may be a promising drug to prevent the IgE-independent allergic reactions, and thus, MRGPRX2 antagonist development may lead to a promising therapeutic medication for the IgE-independent allergic reactions.

Evaluation of the Stabilization of Human Umbilical Cord Blood-Derived Mast Cells in Accordance with Ketotifen and Olopatadine Concentration

Evaluation of the Stabilization of Human Umbilical Cord Blood-Derived Mast Cells in Accordance with Ketotifen and Olopatadine Concentration

Proteasome inhibition upregulates Bim and induces caspase-3-dependent apoptosis in human mast cells expressing the Kit D816V mutation

The majority of patients with systemic mastocytosis exhibit a D816V mutation in the activating loop of the Kit receptor expressed on mast cells. The Kit ligand regulates mast cell survival by transcriptional repression of the proapoptotic BH3-only protein Bim and by promoting Bim phosphorylation that makes it vulnerable for proteasomal-dependent degradation. We investigated here whether prevention of Bim degradation by a proteasomal inhibitor, MG132, would induce apoptosis in mast cells with the D816V mutation. Human umbilical cord blood-derived mast cells (CBMCs) with wild-type (wt) Kit and two different subclones of the human mast cell line-1 (HMC-1) were used for the study: HMC-1.1 with the V560G mutation in the juxtamembrane domain and HMC-1.2 carrying the V560G mutation together with the D816V mutation. MG132 at 1 μM induced apoptosis in all cell types, an effect accompanied by increased BH3-only proapoptotic protein Bim. The raise of Bim was accompanied by caspase-3 activation, and a caspase-3 inhibitor reduced MG132-induced apoptosis. Further, MG132 caused a reduction of activated Erk, a negative regulator of Bim expression, and thus Bim upregulation. We conclude that decreased phosphorylation and increased levels of Bim overcome the prosurvival effect of the D816V mutation and that the results warrant further investigations of the clinical effects of proteasomal inhibition in systemic mastocytosis.

Mitochondria Distinguish Granule-Stored from de novo Synthesized Tumor Necrosis Factor Secretion in Human Mast Cells

Background: Mast cells are immune cells derived from hematopoietic precursors that mature in the tissue microenvironment. Mast cells are critical for allergic, immune and inflammatory processes, many of which involve tumor necrosis factor (TNF). These cells uniquely store TNF in their secretory granules. Upon stimulation, mast cells rapidly (30 min) secrete β-hexosaminidase and granule-stored TNF through degranulation, but also increase TNF mRNA and release de novo synthesized TNF 24 h later. The regulation of these two distinct pathways is poorly understood. Methods: Human LAD2 leukemic mast cells are stimulated by substance P. TNF secretion and gene expression were measured by ELISA and real-time PCR, and mitochondrial dynamics was observed in live cells under confocal microscopy. Cell energy consumption was measured in terms of oxygen consumption rate. Results: Here, we show that granule-stored TNF is preformed, and its secretion from LAD2 mast cells stimulated by substance P (1) exhibits higher energy consumption and is inhibited by the mitochondrial ATP pump blocker oligomycin, (2) shows rapid increase in intracellular calcium levels, and (3) exhibits reversible mitochondrial translocation, from a perinuclear distribution to the cell surface, as compared to de novo synthesized TNF release induced by lipopolysaccharide. This mitochondrial translocation is confirmed using primary human umbilical cord blood-derived mast cells stimulated by an allergic trigger (IgE/streptavidin). Conclusion: Our findings indicate that unique mitochondrial functions distinguish granule-stored from newly synthesized TNF release from human mast cells, thus permitting the versatile involvement of mast cells in different biological processes.

Mast cells and histamine enhance the proliferation of non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer with an extremely low survival rate. It is characterized by a chronic inflammatory process with intense mast cell infiltrate that is associated with reduced survival. The aim of this study was to test the hypothesis that mast cells have an enhancing effect on NSCLC proliferation. To assess the tumor-promoting potential of mast cells, we used the human alveolar basal adenocarcinoma (A549) and the mouse Lewis lung carcinoma (LLC) cell lines, umbilical cord blood-derived mast cells (CBMC) and the mast cell-deficient mouse Sash model. The proliferation rate of A549/LLC cells was markedly increased by mast cells and histamine. Histamine proliferating activity was mediated via H1, H2 and H4 receptors and caused ERK phosphorylation. LLC induced in Sash mice or in wild-type mice treated with the mast cell stabilizer nedocromil sodium displayed an accelerated growth (number of metastic colonies in the lungs, total lung area and lung/total mice weight ratio). In summary, we have shown a significant effect of mast cells and histamine in enhancing NSCLC/LLCX growth in vitro, while in a mouse LLC model in vivo we have found that mast cells are important negative regulators of cancer development. Therefore our results would indicate a pro-tumorogenic effect of the mast cells in vitro on established lung tumor cell lines, and anti-tumorogenic effect in mice at lung cancer induction. In conclusion, mast cell/anti-histamine targeted therapies should carefully consider this dual effect.

Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis

Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

Characterization of three human sec14p-like proteins: α-Tocopherol transport activity and expression pattern in tissues

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled α-tocopherol to mitochondria in the same order of magnitude as the human α-tocopherol transfer protein (α-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

IL-33 can promote survival, adhesion and cytokine production in human mast cells

IL-33 is a recently identified member of the IL-1 family of molecules, which also includes IL-1 and IL-18. IL-33 binds to the receptor, T1/ST2/IL-1R4, and can promote cytokine secretion by Th2 cells and NF-κB phosphorylation in mouse mast cells. However, the effects of these molecules, especially IL-33, in human mast cells are poorly understood. Expression of the receptors for IL-1 family molecules, specifically, IL-1R1, IL-18R and T1/ST2, was detectable intracellularly in human umbilical cord blood-derived mast cells (HUCBMCs) by flow cytometry, but was scarcely detectable on the cells' surface. However, IL-1β, IL-18 or IL-33 induced phosphorylation of Erk, p38 and JNK in naïve HUCBMCs, and IL-33 or IL-1β, but not IL-18, enhanced the survival of naive HUCBMCs and promoted their adhesion to fibronectin. IL-33 or IL-1β also induced IL-8 and IL-13 production in naïve HUCBMCs, and enhanced production of these cytokines in IgE/anti-IgE-stimulated HUCBMCs, without enhancing secretion of either PGD2 or histamine. Moreover, IL-33-mediated IL-8 production by HUCBMCs was markedly reduced by the p38 MAPK inhibitor, SB203580. In contrast to findings with mouse mast cells, IL-18 neither induced nor enhanced secretion of the mediators PGD2 or histamine by HUCBMCs. Our findings identify previously unknown functions of IL-33 in human mast cells. One of these is that IL-33, like IL-1β, can induce cytokine production in human mast cells even in the absence of stimuli of FcɛRI aggregation. Our findings thus support the hypothesis that IL-33 may enhance mast cell function in allergic disorders and other settings, either in the presence or absence of co-stimulation of mast cells via IgE/antigen–FcɛRI signals.

Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison

Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE(2)) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE(2) and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE(2) induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE(2) receptors EP2 and EP4 eliminated the PGE(2)-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE(2)-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE(2) response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE(2) stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE(2) plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter.

Human umbilical cord blood-derived mast cells

Findings obtained using animal models have often failed to reflect the processes involved in human disease. Moreover, human cultured cells do not necessarily function as their actual tissue counterparts. Therefore, there is great demand for sources of human progenitor cells that may be directed to acquire specific tissue characteristics and be available in sufficient quantities to carry out functional and pharmacological studies. Acase in point is the mast cell, well known for its involvement in allergic reactions, but also implicated in inflammatory diseases. Mast cells can be activated by allergens, anaphylatoxins, immunoglobulin-free light chains, superantigens, neuropeptides, and cytokines, leading to selective release of mediators. These could be involved in many inflammatory diseases, such as asthma and atopic dermatitis, which worsen by stress, through activation by local release of corticotropin-releasing hormone or related peptides. Umbilical cord blood and cord matrix-derived mast cell progenitors can be separated magnetically and grown in the presence of stem cell factor, interleukin-6, interleukin-4, and other cytokines to yield distinct mast cell populations. The recent use of live cell array, with its ability to study such interactions rapidly at the single-cell level, provides unique new opportunities for fast output screening of mast cell triggers and inhibitors.

Human Umbilical Cord Blood-Derived Mast Cells: A Unique Model for the Study of Neuro-Immuno-Endocrine Interactions

Human Umbilical Cord Blood-Derived Mast Cells: A Unique Model for the Study of Neuro-Immuno-Endocrine Interactions

Corticotropin-Releasing Hormone Induces Vascular Endothelial Growth Factor Release from Human Mast Cells via the cAMP/Protein Kinase A/p38 Mitogen-Activated Protein Kinase Pathway

Mast cells are involved in allergic reactions but also in innate immunity and inflammation. Corticotropin-releasing hormone (CRH), the key regulator of the hypothalamic-pituitary-adrenal axis, also has proinflammatory effects, apparently through mast cells. We showed recently that CRH selectively stimulates human leukemic mast cells and human umbilical cord blood-derived mast cells to release newly synthesized vascular endothelial growth factor (VEGF) without release of either preformed mediators or cytokines. This effect was mediated through the activation of CRH receptor-1 and adenylate cyclase with increased intracellular cAMP. However, the precise mechanism by which CRH induces VEGF secretion has not yet been defined. Here, we show that CRH-induced VEGF release was dose-dependently inhibited by the specific protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) or the p38 mitogen-activated protein kinase (MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) but not by the specific inhibitor 2'-amino-3'-methoxyflavone (PD98059) of mitogen-activated protein kinase kinase, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) or the c-Jun N-terminal kinase (JNK) inhibitor 1,9-pyrazoloanthrone anthra-(1,9-cd)pyrazol-6(2H)-one (SP600125). Furthermore, CRH significantly increased protein kinase A activity, which could be mimicked by the cell-permeable cAMP analog 8-bromo-cAMP, and was blocked by H89 or the adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CRH also induced rapid phosphorylation of p38 MAPK, which was mimicked by 8-bromo-cAMP and was inhibited by H89 or SB203580. CRH did not stimulate ERK or JNK phosphorylation and did not increase intracellular calcium levels. These results indicate that CRH induces VEGF release in human mast cells via selective activation of the cAMP/protein kinase A/p38 MAPK signaling pathway, thereby providing further insight into the molecular mechanism of how CRH affects the release of a key proinflammatory mediator.

Human Mast Cells Express Corticotropin-Releasing Hormone (CRH) Receptors and CRH Leads to Selective Secretion of Vascular Endothelial Growth Factor

Mast cells are critical for allergic reactions, but also for innate or acquired immunity and inflammatory conditions that worsen by stress. Corticotropin-releasing hormone (CRH), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. We investigated the expression of CRH receptors and the effects of CRH in the human leukemic mast cell (HMC-1) line and human umbilical cord blood-derived mast cells. We detected mRNA for CRH-R1alpha, 1beta, 1c, 1e, 1f isoforms, as well as CRH-R1 protein in both cell types. CRH-R2alpha (but not R2beta or R2gamma) mRNA and protein were present only in human cord blood-derived mast cells. CRH increased cAMP and induced secretion of vascular endothelial growth factor (VEGF) without tryptase, histamine, IL-6, IL-8, or TNF-alpha release. The effects were blocked by the CRH-R1 antagonist antalarmin, but not the CRH-R2 antagonist astressin 2B. CRH-stimulated VEGF production was mediated through activation of adenylate cyclase and increased cAMP, as evidenced by the fact that the effect of CRH was mimicked by the direct adenylate cyclase activator forskolin and the cell-permeable cAMP analog 8-bromo-cAMP, whereas it was abolished by the adenylate cyclase inhibitor SQ22536. This is the first evidence that mast cells express functional CRH receptors and that CRH can induce VEGF secretion selectively. CRH-induced mast cell-derived VEGF could, therefore, be involved in chronic inflammatory conditions associated with increased VEGF, such as arthritis or psoriasis, both of which worsen by stress.

Molecular regulation of interleukin-13 and monocyte chemoattractant protein-1 expression in human mast cells by interleukin-1beta.

Mast cells play pivotal roles in immunoglobulin (Ig) E-mediated airway inflammation, expressing interleukin (IL)-13 and monocyte chemoattractant protein-1 (MCP-1), which in turn regulate IgE synthesis and/or inflammatory cell recruitment. The molecular effects of IL-1beta on cytokine expression by human mast cells (HMC) have not been studied well. In this report, we provide evidence that human umbilical cord blood-derived mast cells (CBDMC) and HMC-1 cells express the type 1 receptor for IL-1. We also demonstrate that IL-1beta and tumor necrosis factor-alpha are able to induce, individually or additively, dose-dependent expression of IL-13 and MCP-1 in these cells. The induction of IL-13 and MCP-1 gene expression by IL-1beta was accompanied by the activation of IL-1 receptor-associated kinase and translocation of the transcription factor, nuclear factor (NF) kappaB into the nucleus. Accordingly, Bay-11 7082, an inhibitor of NF-kappaB activation, inhibited IL-1beta-induced IL-13 and MCP-1 expression. IL-1beta also induced IL-13 promoter activity while enhancing the stability of IL-13 messenger RNA transcripts. Dexamethasone, a glucocorticoid, inhibited IL-1beta-induced nuclear translocation of NF-kappaB and also the secretion of IL-13 from mast cells. Our data suggest that IL-1beta can serve as a pivotal costimulus of inflammatory cytokine synthesis in human mast cells, and this may be partly mediated by IL-1 receptor-binding and subsequent signaling via nuclear translocation of NF-kappaB. Because IL-1beta is a ubiquitously expressed cytokine, these findings have important implications for non-IgE-mediated signaling in airway mast cells as well as for innate immunity and airway inflammatory responses, such as observed in extrinsic and intrinsic asthma.

IL-1 induces vesicular secretion of IL-6 without degranulation from human mast cells.

Fc epsilon RI cross-linkage in mast cells results in release of granule-associated mediators, such as histamine and proteases, as well as the production of numerous cytokines, including IL-6. Mast cells have been increasingly implicated in inflammatory processes where explosive degranulation is not commonly observed. Here, we show that IL-1 stimulates secretion of IL-6 without release of the granule-associated protease tryptase in normal human umbilical cord blood-derived mast cells (hCBMCs). IL-6 secretion stimulated by IL-1 in hCBMCs is potentiated by priming with IL-4 and reflects the higher levels of IL-6 secreted from human leukemic mast cell line (HMC-1). Stimulating HMC-1 cells by both IL-1 and TNF-alpha results in synergistic secretion of IL-6. IL-6 is de novo synthesized, as its secretion is blocked by inhibitors of transcription or protein synthesis. IL-1 does not increase intracellular calcium ion levels in either hCBMCs or HMC-1 cells, and IL-6 stimulation proceeds in the absence of extracellular calcium ions. Ultrastructural Immunogold localization shows that IL-6 is excluded from the secretory granules and is compartmentalized in 40- to 80-nm vesicular structures. Selective secretion of IL-6 from mast cells appears distinct from degranulation and may contribute to the development of inflammation, where the importance of IL-6 has been recognized.

Transcriptional response of human mast cells stimulated via the FcεRI and identification of mast cells as a source of IL-11

BackgroundIn asthma and other allergic disorders, the activation of mast cells by IgE and antigen induces the cells to release histamine and other mediators of inflammation, as well as to produce certain cytokines and chemokines. To search for new mast cell products, we used complementary DNA microarrays to analyze gene expression in human umbilical cord blood-derived mast cells stimulated via the high-affinity IgE receptor (FcεRI).ResultsOne to two hours after FcεRI-dependent stimulation, more than 2,400 genes (about half of which are of unknown function) exhibited 2–200 fold changes in expression. The transcriptional program included changes in the expression of IL-11 and at least 30 other cytokines and chemokines. Human mast cells secreted 130–529 pg of IL-11/106 cells by 6 h after stimulation with anti-IgE.ConclusionOur initial analysis of the transcriptional program induced in in vitro-derived human mast cells stimulated via the FcεRI has identified many products that heretofore have not been associated with this cell type, but which may significantly influence mast cell function in IgE-associated host responses. We also have demonstrated that mast cells stimulated via the FcεRI can secrete IL-11. Based on the previously reported biological effects of IL-11, our results suggest that production of IL-11 may represent one link between IgE-dependent mast cell activation in subjects with allergic asthma and the development of a spectrum of structural changes in the airways of these individuals; such changes, collectively termed "airway remodeling," can constitute an important long term consequence of asthma.

IgE Enhances Fcε Receptor I Expression and IgE-Dependent Release of Histamine and Lipid Mediators from Human Umbilical Cord Blood-Derived Mast Cells: Synergistic Effect of IL-4 and IgE on Human Mast Cell Fcε Receptor I Expression and Mediator Release

AbstractWe investigated the effects of IgE versus IL-4 on FcεRI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 μg/ml) much more strikingly enhanced surface expression of FcεRI than did IL-4 (at 0.1–100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance FcεRI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4−/− or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of FcεRI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on FcεRI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.

Expression of functional major histocompatibility complex class II molecules on HMC-1 human mast cells.

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC-1 human mast cell line, umbilical cord blood-derived mast cells express HLA-DR, -DP and -DQ molecules on their surface. MHC class II molecules expressed on HMC-1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural changes, suggesting their degranulation. Recognition of SEA-bound MHC class II molecules on HMC-1 mast cells by the T cell receptor of K25 cells, an SEA-specific murine T cell hybridoma, triggers significant IL-2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity.

Expression of Functional TrkA Receptor Tyrosine Kinase in the HMC-1 Human Mast Cell Line and in Human Mast Cells

AbstractNerve growth factor (NGF ) can influence mast cell development and function in murine rodents by interacting with its receptors on mast cells. We now report the identification of mRNA transcripts of full-length tyrosine kinase-containing trkA, trkB, and trkC neurotrophin receptor genes in HMC-1 human mast cell leukemia cells. Although HMC-1 cells lacked p75 mRNA, they expressed transcripts for the exon-lacking splice variant of trkA (trkAI), truncated trkB (trkB.T1), and truncated trkC. By flow cytometry, HMC-1 cells exhibited expression of TrkA, TrkB, and TrkC receptor proteins containing full-length tyrosine kinase domains. NGF stimulation of HMC-1 cells induced tyrosine phosphorylation of TrkA protein, increased expression of the early response genes c-fos and NGF1-A, and activation of ERK-mitogen–activated protein (MAP) kinase, results which indicate that TrkA receptors in HMC-1 cells are fully functional. Highly purified populations of human lung mast cells expressed mRNAs for trkA, trkB and trkC, whereas preparations of human umbilical cord blood-derived mast cells expressed mRNAs for trkA and trkC, but not trkB. Moreover, preparations of human umbilical cord blood-derived immature mast cells not only expressed mRNA transcript and protein for TrkA, but exhibited significantly higher numbers of chymase-positive cells after the addition of NGF to their culture medium for 3 weeks. In addition, HMC-1 cells expressed mRNAs for NGF, brain-derived neurotrophic factor (BDNF ), and neurotrophin-3 (NT-3), the cognate ligands for TrkA, TrkB, and TrkC, whereas NGF and BDNF transcripts were detectable in human umbilical cord blood mast cell preparations. Taken together, our findings show that human mast cells express a functional TrkA receptor tyrosine kinase and indicate that NGF may be able to promote certain aspects of mast cell development and/or maturation in humans. Our studies also raise the possibility that human mast cells may represent a potential source for neurotrophins.