Human Cord Blood-derived Mast Cells Research Articles

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.

Identification of Nash1, a Novel Protein Containing a Nuclear Localization Signal, a Sterile α Motif, and an SH3 Domain Preferentially Expressed in Mast Cells

By using a serial analysis of gene expression (SAGE), we have identified a novel full-length cDNA that is preferentially expressed in human cord blood-derived mast cells. The predicted protein showed unique primary structure with a nuclear localization signal (NLS), a sterile α motif (SAM), and a Src homology 3 domain (SH3) (termed Nash1). Nash1 was mapped to human chromosome 21q11.1 and highly expressed in spleen, liver, peripheral blood, and mast cell lines. In consistent with the presence of NLS, Nash1 was localized in the nucleus. Interestingly, screening gene databases for Nash1-related sequences revealed the existence of a Nash1-related gene termed SLY that was preferentially detected in lymphoid cells. We also found at least two additional candidates for this gene family in the database. These findings suggested that Nash1 and Nash1-related proteins consisted of a novel family of signaling/adaptor proteins, and Nash1 might function as a signaling component of mast cells, possibly in the nucleus.

Autocrine regulation of cord blood–derived human mast cell activation by IL-10

Background: Ligation of the high-affinity receptor for IgE on human mast cells (MCs) induces the release of proinflammatory mediators, including vasoactive amines and cytokines (TNF-α, IL-5, and IL-8). Moreover, we have recently shown that IL-10 inhibits the release of proinflammatory mediators by activated MCs. Objective: We investigated whether human cord blood–derived MCs (CBMCs) could produce IL-10 and whether this production could inhibit their activation in an autocrine fashion. Methods: IL-10 synthesis by resting or activated human MCs derived from cord blood progenitors was investigated in cell supernatants or by using immunostaining and RT-PCR methods. In addition, the effect of IL-4 on such synthesis was also studied. Anti-IL-10–neutralizing antibodies were used to investigate the validity of the hypothesis of an autocrine regulation of MCs by IL-10. Finally, the presence of specific receptors for IL-10 was searched on human CBMCs by using flow cytometric analysis. Results: Human CBMCs spontaneously synthesize and release IL-10, and this synthesis is increased after IgE/anti-IgE stimulation. In addition, the presence of IL-10 in resting or in activated MCs was proved by immunostaining. Interestingly, the release of IL-10 was also increased after incubation of the cells with IL-4. Besides, the use of neutralizing antibodies against IL-10 confirmed that IL-10 released inhibited MC activation in an autocrine fashion. Finally, the presence of specific receptors for this cytokine was observed on the membranes of our population of human CBMCs. Conclusion: Taken together, our data are in favor of an autocrine regulation pathway through synthesis and release of IL-10 by human MCs. Such an autoregulatory mechanism is, to our knowledge, the first described for these elements. (J Allergy Clin Immunol 2001;108:80-6.)

Identification of novel mast cell genes by serial analysis of gene expression in cord blood-derived mast cells

The gene expression profile of human cord blood-derived mast cells (MCs) was investigated using serial analysis of gene expression (SAGE). A total of 22 914 tags, representing 9181 unique transcripts, were sequenced. By selecting tags that were detected more frequently in MCs than in other tissues, genes characteristic of MCs were enriched. Reverse transcription-PCR and the high-density oligonucleotide array hybridization confirmed the validity of our SAGE result. About 70% of the selected genes were previously uncharacterized. Northern blot analysis showed the MC-specific expression of selected genes. This inventory will be useful to identify novel genes with important functions in MCs.

GM-CSF downmodulates c-kit, Fc(epsilon)RI(alpha) and GM-CSF receptor expression as well as histamine and tryptase levels in cultured human mast cells.

GM-CSF is known primarily as a hematopoietic growth factor, but it has also been shown to inhibit mast cell differentiation in vitro. In order elucidate the mechanisms involved, we investigated the effects of GM-CSF in vitro on the differentiation of human leukemic mast cells (HMC-1 cells) and normal cord blood-derived mast cells (CBMC) under the influence of SCF, NGF, and fibroblast supernatant (FS). Under all culture conditions, GM-CSF induced a dose- and time-dependent reduction in intracellular histamine levels, tryptase activity, and numbers of cells immunoreactive for c-Kit and FcepsilonRIalpha. This effect leveled off between 10-100 ng/ml and after 4 days of culture. There was an associated decrease in mRNA expression for c-kit, FcepsilonRIalpha and tryptase. In contrast, no significant changes in the expression of the NGF receptor TrkA were noted under the same conditions. The GM-CSF receptor was found in HMC-1 cells and CBMC at both the mRNA and protein levels, but its expression decreased during culture with FS, and even more markedly during culture with GM-CSF. GM-CSF thus selectively inhibits in vitro induction and/or upregulation of all major mast cell characteristics in HMC-1 cells and CBMC irrespective of the growth factors present, and a concomitant downregulation of GM-CSF receptors can counteract these effects. GM-CSF may therefore function as a regulatory factor in mast cell growth and differentiation under normal and pathological conditions.

SDF-1 Induces IL-8 Production and Transendothelial Migration of Human Cord Blood-Derived Mast Cells

Background: Mast cell numbers and expression of chemokines are known to increase in the context of angiogenesis and inflammation, but the mechanisms by which this occurs are not understood. Stromal-derived factor-1 (SDF-1) is an important chemokine in angiogenesis and cell migration. The effects of SDF-1 on human mast cells were examined. Methods: Expression of the SDF-1 receptor CXC chemokine receptor 4 (CXCR4) on mast cells was examined by RT-PCR and flow cytometry. The ability of labeled cord blood-derived mast cells to migrate across HUVEC monolayers in response to SDF-1 was determined. The cytokine and chemokine responses of cord blood-derived mast cells to SDF-1 treatment over 24 h were examined by ELISA. Results: Cord blood-derived human mast cells expressed the CXCR4 receptor for SDF-1 and migrated across HUVEC monolayers in response to this chemokine. Treatment of cord blood-derived mast cells with SDF-1 did not induce degranulation or the production of several cytokines but did induce a highly selective IL-8 response. Conclusion: Human mast cells can both migrate across vascular endothelium and produce the pro-angiogenic chemokine IL-8 in response to SDF-1. These responses may be important in angiogenic processes.

T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4.

Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE-stimulated hMCs elaborated minimal cys-LT (0.1 +/- 0.1 ng/10(6) hMCs) and abundant prostaglandin (PG)D(2) (16.2 +/- 10.3 ng/10(6) hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE-dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD(2) (by 2. 5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE-mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD(2) generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C(4) synthase (LTC(4)S) mRNA within 6 h, and increased the expression of LTC(4)S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC(4)S biosynthetic pathway (induction of LTC(4)S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.

IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production.

Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMC in vitro. Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcvarepsilonRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-alpha, IL-5, IL-13, macrophage inflammatory protein 1alpha, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcvarepsilonRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcvarepsilonRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.

Stem cell factor and IL-6 do not promote complete maturation of human cultured mast cells from umbilical cord blood cells: an ultrastructural study

Human mast cells are classified into two phenotypes based on their neutral protease compositions. One type is a tryptase-positive and chymase-positive MCTC cell that is predominant in the skin, another is a tryptase-positive and chymase-negative MCT cell that is predominant in the lung. Cord blood-derived human mast cells cultured in the presence of stem cell factor and interleukin-6 are a mixture of MCTC and MCT at various ratios, as revealed by immunocytochemical staining. We performed an electron microscopic analysis of cord blood-derived human cultured mast cells and found that they were so immature that we could not distinguish MCT and MCTC from their ultrastructural morphology. The response to secretagogues was not the response of MCTC but rather of MCT. Although human cultured mast cells are the most useful cells for use in in vitro experiments, the present culture condition supplemented with stem cell factor and interleukin-6 does not develop fully mature mast cells in vitro.

Human mast cells transmigrate through human umbilical vein endothelial monolayers and selectively produce IL-8 in response to stromal cell-derived factor-1 alpha.

Mature mast cells are generally considered to be less mobile cells residing within tissue sites. However, mast cell numbers are known to increase in the context of inflammation, and mast cells are recognized to be important in regulating local neutrophil infiltration. CXC chemokines may play a critical role in this process. In this study two human mast cell-like lines, HMC-1 and KU812, and human cord blood-derived primary cultured mast cells were employed to examine role of stromal cell-derived factor-1 (SDF-1) in regulating mast cell migration and mediator production. It was demonstrated that human mast cells constitutively express mRNA and protein for CXCR4. Stimulation of human mast cells with SDF-1, the only known ligand for CXCR4, induced a significant increase in intracellular calcium levels. In vitro, SDF-1 alpha mediated dose-dependent migration of human cord blood-derived mast cells and HMC-1 cells across HUVEC monolayers. Although SDF-1 alpha did not induce mast cell degranulation, it selectively stimulated production of the neutrophil chemoattractant IL-8 without affecting TNF-alpha, IL-1beta, IL-6, GM-CSF, IFN-gamma, or RANTES production, providing further evidence of the selective modulation of mast cell function by this chemokine. These findings provide a novel, SDF-1-dependent mechanism for mast cell transendothelial migration and functional regulation, which may have important implications for the local regulation of mast cells in disease.

Serial Analysis of Gene Expression in Human Cord Blood-Derived Mast Cells

Serial Analysis of Gene Expression in Human Cord Blood-Derived Mast Cells

Internalization of FimH+ Escherichia coli by the human mast cell line (HMC-1 5C6) involves protein kinase C.

Rodent mast cells (MC) play critical roles in host defense against bacterial infection. However, bacteria-mediated signaling mechanisms in MC have not been studied. In addition, the response of human MC to bacteria is not fully investigated. This study examined the interaction between human MC and type 1 fimbriated Escherichia coli and the mechanisms involved using the human MC line HMC-1 5C6 and human cord blood-derived MC. These MC internalized significant numbers of FimH+ E. coli, but not its isogenic FimH- mutant. In HMC-1 cells, bacterial internalization was stimulated by protein kinase C (PKC) activation [short-term phorbol myristate acetate (PMA) treatment] and dramatically decreased by PKC inhibitors or PKC depletion (long-term PMA treatment). Moreover, bacterial internalization was accompanied by significant expression of PKCbeta1 and delta. Fluorescence microscopy demonstrated accumulation of PKCbeta1 on internalized bacteria. These data indicate that human MC has the capacity to internalize bacteria and PKC may be a critical intracellular mediator of this function.

Mast Cell Migratory Response to Interleukin-8 Is Mediated Through Interaction With Chemokine Receptor CXCR2/Interleukin-8RB

To explore the role of chemokines in mast cell chemotaxis and accumulation at sites of inflammation, we first investigated the response of human mast cells to 18 different chemokines by induction of intracellular calcium mobilization in the human mast cell line, HMC-1. Only a subgroup of CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR) tripeptide motif, which included interleukin-8 (IL-8), growth-regulated oncogene  (GRO), neutrophil-activating peptide-2 (NAP-2), and epithelial cell–derived neutrophil activating peptide-78 (ENA-78), induced calcium flux in the cells. These observations suggested that the receptor CXCR2 (IL-8RB) should be expressed on the surface of these cells. Using the RNAse protection assay, CXCR2 mRNA, but not CXCR1 (IL-8RA) mRNA expression was detected in HMC-1 cells. Flow cytometry analysis documented the surface expression of CXCR2. A binding analysis performed with125I-IL-8 determined that there were approximately 3,600 high affinity IL-8 binding sites per HMC-1 cell, with a calculated kd of 1.2 to 2 nmol/L. The activity of this receptor was further explored using IL-8, which was found to induce dose-dependent chemotactic and haptotactic responses in both HMC-1 cells and in vitro cultured human cord blood–derived mast cells. These results show the expression of functional CXCR2 receptors on the surface of human mast cells, which may play an important role in mast cell recruitment during the genesis of an inflammatory response.

Mast Cell Migratory Response to Interleukin-8 Is Mediated Through Interaction With Chemokine Receptor CXCR2/Interleukin-8RB

AbstractTo explore the role of chemokines in mast cell chemotaxis and accumulation at sites of inflammation, we first investigated the response of human mast cells to 18 different chemokines by induction of intracellular calcium mobilization in the human mast cell line, HMC-1. Only a subgroup of CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR) tripeptide motif, which included interleukin-8 (IL-8), growth-regulated oncogene α (GROα), neutrophil-activating peptide-2 (NAP-2), and epithelial cell–derived neutrophil activating peptide-78 (ENA-78), induced calcium flux in the cells. These observations suggested that the receptor CXCR2 (IL-8RB) should be expressed on the surface of these cells. Using the RNAse protection assay, CXCR2 mRNA, but not CXCR1 (IL-8RA) mRNA expression was detected in HMC-1 cells. Flow cytometry analysis documented the surface expression of CXCR2. A binding analysis performed with125I-IL-8 determined that there were approximately 3,600 high affinity IL-8 binding sites per HMC-1 cell, with a calculated kd of 1.2 to 2 nmol/L. The activity of this receptor was further explored using IL-8, which was found to induce dose-dependent chemotactic and haptotactic responses in both HMC-1 cells and in vitro cultured human cord blood–derived mast cells. These results show the expression of functional CXCR2 receptors on the surface of human mast cells, which may play an important role in mast cell recruitment during the genesis of an inflammatory response.

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.

Phagocytic and tumor necrosis factor alpha response of human mast cells following exposure to gram-negative and gram-positive bacteria.

Recent studies have implicated rodent mast cells in the innate immune response to infectious bacteria. We report that cord blood-derived human mast cells (CBHMC) obtained from culture of cord blood progenitors phagocytozed and killed various gram-negative and gram-positive bacteria and simultaneously released considerable amounts of tumor necrosis factor alpha. Overall, the extent of the endocytic and exocytic response of CBHMC correlated with the number of adherent bacteria. Thus, human mast cells are intrinsically capable of mediating microbial recognition and of actively contributing to the host defense against bacteria.

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.

C3a and C5a Stimulate Chemotaxis of Human Mast Cells

AbstractThe factors that control migration of mast cells to sites of inflammation and tissue repair remain largely undefined. Whereas several recent studies have described chemotactic factors that induce migration of murine mast cells, only stem cell factor (SCF ) is known to induce migration of human mast cells. We report here that the anaphylatoxins C3a and C5a are chemotactic factors for the human mast cell line HMC-1, human cord blood-derived mast cells (CBMC) and cutaneous mast cells in vitro. The presence of an extracellular matrix protein, laminin, was required for chemotaxis in response to complement peptides. Migration of mast cells towards C3a and C5a was dose-dependent, peaking at 1 μg/mL (100 nmol/L), and was inhibited by specific antibodies. Pretreatment with pertussis toxin inhibited the anaphylatoxin-mediated migration of HMC-1 cells, indicating that Gi proteins are involved in complement-activated signal transduction pathways in human mast cells. Both C3a and C5a also induced a rapid and transient mobilization of intracellular free calcium ([Ca2+]i ) in HMC-1 cells. Besides SCF, other chemotactic factors tested, such as interleukin-3, nerve growth factor, transforming growth factor β, RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β, failed to stimulate migration of human mast cells. In summary, these findings indicate that C3a and C5a serve as chemotaxins for human mast cells. Anaphylatoxin-mediated recruitment of mast cells might play an important role in hypersensitivity and inflammatory processes.