Perhaps the most important factor for mast cell development, survival and proliferation is Stem-Cell Factor (SCF or c-KIT ligand) 4, although several lines of evidence also indicate IL-3 as a crucial factor for mast cell development, survival and proliferation 4. Human mast cells are very difficult to isolate in numbers that would allow for wide-genome expression profiling studies: for that reason we isolated CD34+ progenitor hematopoietic cells from umbilical cord blood and differentiated them to mast cells by culturing with the cytokines SCF and IL-3. After culturing CD34⁺ progenitor cells with cytokines for 5–6 weeks, cells were characterized as Mast Cells by flow cytometry as positive for the specific mast cell marker intracellular chymase; positive c-Kit/CD117⁺ and FcεRI⁺, and by Toluidine blue staining (Figure 1). Purity was estimated at >97%.

Our aim is to study the global gene expression pattern induced by IgE sensitization and FcεRI aggregation on human mast cells. Gene expression analysis using cDNA or oligo-DNA microarrays has proven to be a sensitive method to develop and refine the molecular determinants of several human disorders, including cancer and autoimmune diseases. We analyzed the expression pattern of 8,793 transcripts from the stimulated mast cells, and compared the expression patterns with control/unstimulated samples. The complete gene expression data of our experiments, representing 8,793 probeID is available at the NCBIs Gene Expression Omnibus 25, and is accessible through GEO Series accession number GSE1933 (25). The microarray analysis revealed that 760 genes (~8.6%) were differentially expressed between resting and stimulated mast cells with statistical significance (P ≤ 0.05), which were hierarchically clustered (Figure 2). Because of the relatively large number of genes that were differentially regulated, we focused on genes that were upregulated by at least a 2-fold in any time point of mast cell stimulation. Of the 760 genes, 58 genes were initially upregulated (at least 2-fold) by IgE-sensitization alone (Table. 1), and a total of 115 genes were overexpressed (by 2-fold or more in at least one time point), during the time course of mast cell activation by IgE-alone or after crosslinking FcεRI (Table. 1). In order to examine the global characteristics of these genes, we used the Gene Ontology Consortium database for Biological processes 26. Using this database we analyzed the genes that were upregulated by at least 2-fold, thus, allowing us to separate the 115 genes, into the following functional families: (a) cytokines and cytokine receptors; (b) chemokines and chemokine receptors; (c) other immunoregulatory genes; (d) cell proliferation and anti-apoptosis; (e) adhesion and cytoskeleton remodeling; (f) transcription factors and regulators of transcription; (g) signal transduction; (h) genes involved in other cellular functions "others" (Table 1 & Figure 3)

Our study revealed that substantial changes in gene expression in response to monomeric-IgE sensitization alone. In order to ensure that the human IgE (IgE, Cat: 30-AI05, Lot number A01071004, Fitzgerald, Concord, MA), used in this study was indeed monomeric IgE, prior to each experiment; we run a sample of the IgE through non-denaturing polyachrylamide-gel-electrophosys (non-reducing-PAGE). No aggregates were observed at any time (data not shown).

We focused the analysis of our data on genes that were upregulated by at least 2-fold, over basal levels, and identified 58 genes that were increased by IgE sensitization alone (Table. 1). We then separated them into different categories, based on their biological function (determined by public databases). Among the most prominent findings was the upregulation of genes coding for the cytokines IL-1β (3.3 fold), IL-6 (2.7 fold), and CSF1 (1.6 fold); genes coding for the chemokines IL-8 (CXCL8) (2.1 fold), MIP1β (CCL4) (3.5 fold), MCP3 (CCL7) (2.1 fold), GROα (CXCL1) (2.3 fold) and GROγ (CXCL3) (1.6 fold), were also upregulated. Other than these, several genes coding for other receptors involved in immune-responses; immunoregulatory genes; adhesion and/or cytoskeleton remodeling; regulators of apoptosis; signal transduction; transcription factors; were also upregulated by monomeric IgE (Table. 1). Thus, these results suggest that "passive" sensitization of mast cells, with monomeric IgE, may not only prime mast cells to be ready for the challenge to come, but that mast cells may also have the potential to trigger the priming and/or recruitment of other immune-effector cells.

An interesting finding in our study was upregulation of genes coding for the pyrogenic and proinflammatory cytokines IL-1β, IL-6 (Table. 1a). One of the key roles for IL-1β is to trigger the upregulation of key proinflammatory proteins 27. We also observed the upregulation of PTX3 (pentaxin-related gene) (Table. 1c): PTX3 is a protein involved in inflammation that is rapidly upregulated by IL-1β. Other cytokine genes that were upregulated are CSF1 (a growth promoting cytokine), and the cytokine receptors IL1R1, IL27RA, TNFRSF9, TNFRSF12 and IL1RN (Table. 1a). We also found that the level of expression of genes coding for the chemokines IL-8 (CXCL8), CCL7 (MCP3), CCL4 (MIP1β) and CXCL1 (GROα) was increased (Table 1b). IL-8 plays a major role in inflammatory responses mainly due to its ability to recruit and activate neutrophils. CCL7, CCL4 and CXCL1 are known to recruit monocytes, NK, basophils, dendritic cells and TH2 cells. Moreover, our data also show that several other chemokine genes were upregulated during mast cell stimulation: these include CCL5 (RANTES), CXCL3 (GROγ), and the chemokine receptor CCRL2 (Table. 1b). These data suggest a key role for FcεRI in triggering an a wide range of inflammatory responses, as the over-expression of cytokines and chemokines is a prerequisite to triggering inflammation, including vascular permeability, and leukocyte and lymphocyte recruitment, differentiation and activation.

Several genes involved in innate and adaptive immune-response were upregulated at least by 2-fold during mast cell stimulation: these include the Toll Like Receptor 2 (TLR2), as well as several genes of the TNFα signaling pathways, including TNFAIP6 (Table. 1c). These findings support previous reports of the role of mast cells in innate immunity and antibacterial activity 21. Transcripts of genes LIF, CD69, and CD83 were also upregulated, as were the major histocompatibility complex genes (HLA-DQB1, and HKE2) and inhibitory receptor of IgG, FCGR2B (Table. 1c). The generation of transcripts for such genes suggests that mast cells can acquire characteristics typical of cells involved in innate and adaptive immune responses.

Several genes involved in cell proliferation and survival, such as PDGFA (platelet-derived growth factor alpha polypeptide), PDGFB (platelet-derived growth factor beta polypeptide), PBEF1 (pre-B-cell colony-enhancing factor), TIEG (TGFB inducible early growth response), and INSIG1 (insulin induced gene 1) were upregulated, as well as several anti-apoptosis genes including TNFAIP3 (TNF-alpha-induced protein 3), TNFAIP8 (TNF-alpha-induced protein 8), IER3 (immediate early response 3), SERPINB2 (serine or cysteine proteinase inhibitor B2), and IRF2 (interferon regulatory factor 2) (Table. 1d). This supports the fact that, in the initial stages of mast cell activation, several mediators produced are mainly for cell proliferation and survival 28. Thus, FcεRI aggregation may enhance mast cell proliferation and survival, perhaps owing to the autocrine effects of the cytokines, growth factors, and antiapoptotic proteins, triggered by FcεRI aggregation.

Another functional characteristic of immune-cell activation is the coordinated expression of genes involved in cell adhesion and cytoskeleton remodeling (Table. 1e). Of particular importance are the genes coding for proteins involved in cell motility, cytokinesis, endocytosis and exocytosis. We found at least 2-fold upregulation of various genes coding for proteins involved in cell adhesion, such as FLRT2 (fibronectin leucine rich transmembrane protein 2), KAL1 (Kallmann syndrome 1 sequence), CD151 (CD151 antigen), and ALCAM (activated leukocyte cell adhesion molecule); as well as for several gene-transcripts involved in cytoskeleton remodeling, including RASAL1 (RAS protein activator like 1), ARHE (RAS homolog gene family, member E), ARF6 (ADP-ribosylation factor 6), and FLNB (filamin B, β-actin binding protein 278) (Table. 1e). The expression of genes involved in cell adhesion and cytoskeleton remodeling is an essential step in immune-cell activation. Resting immune cells have cytoskeletal structures that sequester antigen, chemokine, and adhesion receptors in accessible regions of the plasma membrane. Upon activation, reorganization of the actin cytoskeleton leads to the formation of supramolecular activation clusters, bringing receptors and costimulatory molecules together, as well as important adaptor proteins that promote the sustained activation of the cell.

Stimulation of immune-effector cells through their antigen receptors initiates cell cycle entry and changes the gene expression pattern, a response generally referred to as "activation". We found that the genes for several transcription factors were upregulated during IgE-sensitization and FcεRI aggregation, including the transcription factors most active during an immune response, such as NFκB and NFAT (Table. 1f). We observed an increase in the transcripts for the nuclear factor of kappa light polypeptide genes 1, alpha, and epsilon (NFκB 1, 1A, and 1E), and the nuclear factor of activated T cells, NFATC1 (Table. 1f). Other transcription factors upregulated were the oncogenes MYC (v-myc myelo-cytomatosis viral oncogene homolog), and MAFF (v-maf musculo-aponeurotic fibrosarcoma oncogene homolog F) (Table. 1f). Interestingly, the activities of NFκB and NFAT together are responsible for the transcription of many proinflammatory genes, including several genes coding for cytokines and chemokines 2930.

During mast cell activation, many signaling molecules are engaged in diverse responses, ranging from calcium release from internal stores, degranulation, the generation of lipid-derived proinflammatory mediators and the production of cytokines and chemokines. In our study we observed that a substantial number of genes coding for intracellular signaling proteins were upregulated, by at least 2-fold (Table. 1g). These include genes for serine protein kinases of the Mitogen Activated Protein Kinase family, such as MAP2K3 and MAP3K14: members of this family of kinases not only play a role in mitogenesis, but can potentially lead to the activation of transcription factors. The gene coding for the tyrosine kinase FYN (FYN oncogene related to SRC, FGR, YES) was also upregulated (Table. 1g): FYN is a member of the Src-tyrosine kinase family; members of this family are critical for the signal transduction cascades triggered by all antigen receptors including by FcεRI, TCR, FcγRs, and the B-cell antigen receptor. Several genes coding for protein phosphatases were also upregulated, including several members of the dual specificity phosphatase family coding for DUSP1, DUSP2, and DUSP6 (Table. 1g). Moreover, we show here the upregulation of genes that code for oxidized low density lipoprotein receptor (OLR1), and for the low density lipoprotein receptor (LDLR) (Table. 1g), indicating a potential role for mast cells in cholesterol homeostasis. Of particular interest is the upregulation of the gene coding for the lipid kinase, sphingosine kinase 1 (SPHK1) (Table. 1g). We and others have previously reported that SPHK1 plays a critical role in the intracellular signaling pathways triggered by FcεRI in mast cells 2831, and coordinates several physiological responses triggered by activated mast cells.

We confirmed our microarrays findings by Real time PCR on selected genes such as IL-1β IL6, IL-8, MCP3, RANTES and SPHK1, utilizing an aliquot of the same RNA sample that was used for the microarray experiments (Figure 4). The results showed that the messenger RNA for the selected genes follows a similar pattern of expression to that observed with the oligo-DNA microarray experiment, thus confirming the results and the quality of the data obtained with the high-density microarrays.

Mast cell activation also results in the sustained de novo production of pro-inflammatory cytokines and chemokines both of which may contribute to the inflammation and pathology underlying allergic disease as well as in innate and acquired immunity. The amounts of these cytokines were measured by ELISA and depicted in Figure 5A. FcεRI-triggered generation of IL-1β, IL-6, IL-8, CCL7 (MCP3) and CCL5 (RANTES), whereas IgE sensitization alone triggered smaller amounts of IL-1β and MCP3, high amount of IL-8, and very less amounts of IL-6 and RANTES (Figure 5A).

We verified the differential expression of SPHK1 by Western blot analysis (Figure 5B). Its levels of expression were found to be consistent with that of microarray as well as Real Time PCR results. Thus, these data together with Real Time PCR data validate microarray results.

Mast cell activation via FcεRI triggers exocytosis of granules containing pre-formed inflammatory mediators in a tyrosine kinase and calcium dependent manner. Here we studied, whether monomeric-IgE alone, may activate FcεRI intracellular signaling pathways, leading to physiological responses of mast cells, by analyzing the overall tyrosine phosphorylation; fluctuations in cytosolic Ca²⁺ concentration; and degranulation by measuring β-hexosaminidase release (Fig. 6A,B &6C). We show here that in our experimental setting monomeric-IgE alone is not able to trigger any changes on the overall protein-tyrosine phosphorylation patterns compared with resting cells; nor was it able to trigger calcium release from internal stores; neither degranulation. On the other hand FcεRI-aggregation did indeed trigger all these responses (Fig. 6A,B &6C).

Human umbilical cord blood (CB) samples were collected from normal full-term deliveries of informed individuals with formal consents, meeting the Universality Institutional Review Board guidelines, for research using human samples. CD34⁺ haematopoietic progenitor-cells were harvested using MACS cell isolation kit (Miltenyi Biotec), following the manufacturer's instructions.

The isolated CD34⁺ haematopoietic progenitor-cells were cultured for 5-6-weeks in the presence of 100 ng/ml of Stem Cell Factor (SCF Cat: 300-07, PeproTec, Rocky Hill, NJ), and for the first week this was supplemented with 10 ng/ml of Interleukin-3 (IL-3 Cat: 200-03, PeproTec, Rocky Hill, NJ). Cells were shown to be differentiated by staining them for specific mast cell markers as follows: for mast cells chymase, with an anti-human chymase mAb (IgG1-MAB1254, Chemicon, Temecula, CA), and FITC-conjugated secondary antibody (anti-mouse IgG-FITC, Sigma-Aldrich, Singapore); for c-Kit with anti-human c-Kit mouse monoclonal PE-conjugated (Cat. No. 555714; clone YB5.B8, BD Biosciences – Pharmigen, Singapore), isotype control anti-mouse IgE-PE (Cat. No. 555749; clone MOPC-21, BD Biosciences – Pharmigen, Singapore); and for FcεRI cell-surface expression with anti-human FcεRI polyclonal (Rabbit-IgG-ab31494; Abcam, Cambridge, UK) and FITC-conjugated secondary antibody (anti-rabbit IgG-FITC, Sigma-Aldrich, Singapore) also used as isotype control, and analyzed immediately using a Coulter Epics-Elite ESP Flow Cytometer (Beckman, Germany). Purity was estimated at >97%. The differentiated mast cells were plated in 6 well plates and allowed to rest for 24 hr.

After differentiation, mast cells were plated in 6 well plates and allowed to rest for 24 hr. Cells in all wells, except the control well, were sensitized with human monomeric IgE (1 μg/ml, IgE, Cat: 30-AI05, Lot number A01071004, Fitzgerald, Concord, MA) overnight. FcεRI aggregation was carried out by incubating the cells with monoclonal mouse-anti-human IgE (1 μg/ml, Anti-human-IgE, Cat: MCA2115, clone 4C3, Serotec, Oxford, UK) at 37°C, for 2 hr, 6 hr and 12 hr. RNA was extracted from all the samples using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA). Integrity of RNA was checked by formamide gel electrophoresis; quantification of RNA was carried out by measuring the A_{260 nm}.

Labeling and hybridization was carried out as previously described 46. Briefly, 8 μg of total RNA from each sample was used to synthesize double stranded cDNA using T7-(dT24) oligonucleotide primer and Superscript Reverse transcriptase (Invitrogen). The resultant cDNA was purified and 1 μg of purified cDNA was labeled with biotin by transcription in vitro. The labeled cRNAs were, fragmented in the presence of metal ions and then hybridized to HG-Focus array (Affymetrix), following hybridization the gene chips were washed and stained after which the chips were scanned by Gene Array Scanner (Agilent technologies).

Data collection and analysis was carried out using MicroArray Suite 5.0 (MAS) (Affymetrix). The absolute data (signal intensity, detection call and detection P-value) were exported into GeneSpring v7.0 (Silicon Genetics, Redwood City, CA, USA) software for analysis by parametric test based on cross gene error model (PCGEM). The ANOVA approach has been used to find differentially expressed genes (P < 0.05). The Benjamini and Hochberg False Discovery Rate multiple testing correction was applied.

Agglomerative average-linkage hierarchical clustering of the five different experimental were obtained for selected groups of genes with Gene Spring 7.0 software (Silicon Genetics, Redwood City, CA, USA) using standard correlation as similarity matrix.

Real-PCR was performed, as previously described 46, using 1 μg of total RNA from the same samples used for microarray experiments. PCR was performed for transcripts of IL-1β (Primers: forward 5'-ATG GCA GAA GTA CCT AAG CTC GC-3', reverse 5'-ACA CAA ATT GCA TGG TGA AGT CAG TT-3'); IL-6 (Primers: forward 5'-ATG AAC TCC TTC TCC ACA AGC GC-3', reverse 5' GAA GAG CCC TCA GGC TGG ACT G-3'); IL-8 (Primers: forward 5'-ATG ACT TCC AAG CTG GCC GTG GCT-3', reverse 5'TCT CAG CCC TCT TCA AAA ACT TCT C-3'); MCP-3 (Primers: forward 5'-TCC AAG GCT TTA TGT TCA AA-3', reverse 5'-ACT GAA CTG AAA ACA AGC CA-3'); RANTES (Primers: forward 5'-GCT GTC ATC CTC ATT GCT ACT G-3', reverse 5'-TCG AAC TCC TGA CCT CAA GTG ATC-3'); and for SPHK1 (Primers: forward 5'-TGA ACC CGC GCG GCA AGG GC-3', reverse 5'-GGT CAG CCG GCG CCA TCC ACG-3').

For amplicon detection, the Light Cycler RNA Master SYBR Green Kit (Roche) was used as described by the manufacturer. PCRs were performed in a LightCycler^® instrument (Roche) as follows: reverse transcription at 61°C for 20 min, initial denaturation at 95°C for 2 min; amplification for 45–65 cycles of denaturation (95°C, 5s, ramp rate 20°C/s), annealing (optimal temperature, 5s, ramp rate 20°C/s) and extension (72°C, product length [bp]/25 s, ramp rate 2°C/s). A single online fluorescence reading for each sample was taken at the end of extension step. Quantitative results were expressed by identification of the second derivative maximum points, which marked the cycles where the second derivatives of the fluorescence signal curves are at maximum. These points were expressed as fractional cycle numbers. Then, these cycle numbers were plotted against the logarithm of the concentrations of serially 2-fold diluted standard samples to obtain a standard curve. The concentrations of unknown samples were calculated by extrapolation from this standard curve. Positive sample specificity was confirmed by determining the melting curve (95°C, 5s, ramp rate 20°C/s; 68°C, 15s, ramp rate 20°C/s; 95°C, 0s, ramp rate 0.1°C/s, continuous measurement).

Supernatants from control cells, cells sensitized, and cells following FcεRI aggregation, were collected and stored at -20C until use. IL-1β, IL-6, IL-8, MCP-3 and RANTES levels in the supernatants were evaluated using ELISA (R&D Systems Inc., MN, USA) following the manufacturer's instructions.

Western blots were carried out as previously done 31. Briefly, 40 μg of lysate for each sample was resolved on 10% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 μm nitrocellulose membranes.

For overall tyrosine phosphorytaion, the blots were probed using a specific monoclonal anti-phosphotyrosine primary antibody (P-Tyr, Sc-7020, Santa Cruz, CA, USA), and an anti-mouse HRP-conjugated secondary antibody (anti-mouse IgG-HRP, A-4416, Sigma). Bands were visualized using the ECL Western Blotting Detection System (Amersham, Singapore).

For SPHK1 expression, the blots were probed using a rabbit polyclonal anti-SPHK1 primary antibody (anti-SPHK1, X1627P, Exalpha, MA, USA), and HRP-conjugated secondary antibody (anti-rabbit IgG-HRP, sc-2004, Santa Cruz, CA, USA). For loading control the blots were probed with a monoclonal anti α-tubulin (anti-α-tubulin, sc-5286, Santa Cruz, CA, USA), and an anti-mouse HRP-conjugated secondary antibody (anti-mouse IgG-HRP, A-4416, Sigma). Bands were visualized using the ECL Western Blotting Detection System (Amersham, Singapore), and quantified by densitometry analysis.

Cytosolic calcium was measured as described previously 31. Briefly, cells were loaded with 1 μg/ml Fura2-AM (Molecular Probes, Leiden, The Netherlands) in PBS, 1.5 mM Ca²⁺ and 1 % BSA. After removal of excess reagents by dilution and centrifugation (in PBS), the cells were resuspended in PBS containing 1.5 mM Ca²⁺ and 1 % BSA, for 30 min; or in PBS containing 1.5 mM Ca²⁺, 1 % BSA, and human-monomeric IgE (1 μg/ml) for sensitization, for 30 min. After removal of excess IgE by dilution and centrifugation (in PBS), the cells were resuspended in 1.5 mM Ca²⁺ supplemented PBS and warmed to 37°C in the cuvette; Unsensitazed cells were placed in the cuvette and cytosolic calcium was measured before and after the addition of monomeric IgE. IgE-sensitized cells were placed in the cuvette and FcεRI was crosslinked by addition of mouse-anti-human IgE (1 μg/ml). Fluorescence was measured at 340 and 380 nm.

Degranulation was measured using as previously described 31. Briefly, an aliquot of cells was resuspended in PBS containing 1.5 mM Ca²⁺ and 1 % BSA, and incubated with monomeric IgE for 30 min at 37°C. Another aliquot of cells was resuspended in PBS containing human-monomeric IgE (1 μg/ml) for sensitization, 1.5 mM Ca²⁺ and 1 % BSA, for 30 min. After removal of excess IgE by dilution and centrifugation (in PBS), the cells were resuspended in 1.5 mM Ca²⁺ supplemented PBS, and FcεRI was crosslinked by addition of mouse-anti-human IgE (1 μg/ml) to cells for 30 min at 37°C.

Following the incubation, 50 μl of supernatant, was incubated with 200 μl of 1 mM p-nitrophenyl N-acetyl-β-D-glucosaminide for 1 hr at 37°C. The total β-hexosaminidase concentration was determined by a 1:1 extraction of the remaining buffer and cells with 1% Triton X-100; a 50 μl aliquot was removed and analyzed as described. Reactions were quenched by addition of 500 μl of 0.1 M sodium carbonate buffer. The enzyme concentration was determined by measuring the OD at 400 nm. β-hexosaminidase release was represented as a percent of total enzyme.

To analyze the expression of the intracellular mast cell chymase, 1 × 10⁶cells were washed with ice cold PBS, fixed and permeabilised using the FIX and PERM reagents from Caltag (Caltag Laboratories, Burlingame, CA) as follows: after washing, samples were resuspended in 100 ul of Reagent A (Fixation medium) and incubated for 15 min at RT. The cells were then washed twice with ice-cold PBS, and resuspended in 100 ul of Reagent B (Permeabilization medium) and incubated for 15 min at RT. Cells were washed twice and resuspended in 100 μl of PBS/1% FBS and 5 μl of the anti-human chymase mAb (IgG1-MAB1254, Chemicon, Temecula, CA) was added and samples were incubated for 20 min at RT. Samples were washed twice in ice-cold PBS, then resuspended in PBS/1% FBS and 5 μl of FITC-conjugated secondary antibody (anti-mouse IgG-FITC, Sigma-Aldrich, Singapore) was added, and incubated in dark for an 30 min at RT. Samples were washed twice with ice-cold PBS and resuspended in 100 μl of PBS/1% FBS for immediate analysis.

To analyze the cell surface expression of cKit and FceRI, the samples were initially processed as above except that the permeabilisation step was omitted. For c-Kit the primary antibody was an anti-human c-Kit mouse monoclonal (MCA1841, clone 104D2, Serotec, Oxford, UK), and the secondary antibody was a FITC-conjugated (anti-mouse IgG-FITC, Sigma-Aldrich, Singapore). For FcεRI cell-surface expression, the cells were labeled with the primary anti-human FcεRI polyclonal (Rabbit-IgG-ab31494. Abcam, Cambridge, UK), and the secondary antibody was a FITC-conjugated (anti-rabbit IgG-FITC, Sigma-Aldrich, Singapore). All the samples were analyzed by flow cytometry was using a FACSCalibur machine (BD biosciences), and the data analysed using the Cell Quest™ Pro Software.