Cross-linking the high-affinity IgE receptor, FcεRI, on mast cells activates signaling pathways leading to the release of preformed inflammatory mediators and the production of cytokines and chemokines associated with allergic disorders. Bone marrow-derived mast cells (BMMCs) from Lyn-deficient (Lyn−/−) mice are hyperresponsive to FcεRI cross-linking with multivalent Ag. Previous studies linked the hyperresponsive phenotype in part to increased Fyn kinase activity and reduced SHIP phosphatase activity in the Lyn−/− BMMCs in comparison with wild-type (WT) cells. In this study, we compared gene expression profiles between resting and Ag-activated WT and Lyn−/− BMMCs to identify other factors that may contribute to the hyperresponsiveness of the Lyn−/− cells. Among genes implicated in the positive regulation of FcεRI signaling, mRNA for the tyrosine kinase, Fyn, and for several proteins contributing to calcium regulation are more up-regulated following Ag stimulation in Lyn−/− BMMCs than in WT BMMCs. Conversely, mRNA for the low-affinity IgG receptor (FcγRIIB), implicated in negative regulation of FcεRI-mediated signaling, is more down-regulated in Ag-stimulated Lyn−/− BMMCs than in WT BMMCs. Genes coding for proinflammatory cytokines and chemokines (IL-4, IL-6, IL-13, CSF, CCL1, CCL3, CCL5, CCL7, CCL9, and MIP1β)are all more highly expressed in Ag-stimulated Lyn−/− mast cells than in WT cells. These microarray data identify Lyn as a negative regulator in Ag-stimulated BMMCs of the expression of genes linked to FcεRI signaling and also to the response pathways that lead to allergy and asthma.

Mastcells and basophils express the high-affinity IgE receptor (FcεRI) on their cell surfaces and are activated when multivalent Ag cross-links IgE-bound receptors. FcεRI cross-linking activates a series of tyrosine kinases, including Lyn, Syk, and Fyn, that in turn couple to signaling cascades that lead within minutes to the release of granule-associated inflammatory mediators such as histamine and proteases and within hours to the synthesis and secretion of cytokines and leukotrienes (1, 2). Prominent among the synthesized mediators are Th2 cytokines such as IL-4, IL-13, and TNF-α that exacerbate allergic responses and chemokines like RANTES that are involved in directing the infiltration of inflammatory cells into tissues (3, 4).

Previously, we and others have reported that Lyn−/− bone marrow-derived mast cells (BMMCs)4 are hyperresponsive to Ag and IL-3 stimulation (2, 5, 6, 7, 8). Our mechanistic studies linked this hyperresponsiveness in part to increased activity of the signal-promoting kinase, Fyn, and to decreased activity of the signaling inhibitor, SHIP, in Lyn−/− BMMCs (7). These studies on BMMCs, and earlier work with Lyn−/− B cells (9, 10, 11, 12, 13), all support a role for Lyn as a negative regulator of immunoreceptor signaling.

In this study, we used cDNA microarrays to study the expression profiles of wild-type (WT) and Lyn−/− BMMCs in response to FcεRI stimulation. We report that a number of genes encoding proteins involved in the positive regulation of FcεRI signaling via Fyn activation and Ca2+ regulation are expressed at higher levels in Ag-stimulated Lyn−/− than in WT BMMCs, while the gene for the negative regulator of FcεRI signaling, FcγRIIB, is down-regulated in the Lyn−/− cells. In addition, multiple cytokine and chemokine genes known to be induced upon cross-linking FcεRI in mast cells and basophils are expressed at 3- to 30-fold higher levels in Lyn−/− BMMCs than in WT BMMCs.

Monoclonal mouse anti-DNP IgE was purified from ascites as described in Liu et al. (14). Biotinylated anti-DNP IgE was prepared using EZ-Link Sulfo-normal horse serum-Biotin (Pierce). Biotinylated anti-CD117 (c-kit) mAb and the isotype control, biotinylated rat anti-mouse IgG2B, were purchased from Caltag Laboratories.

WT and Lyn knockout mice (12) on a C57BL/6 background were bred in specific pathogen-free facilities in the University of New Mexico Animal Research Facility (Albuquerque, NM). BMMCs were obtained by culturing bone marrows from 8- to 12-wk WT and Lyn−/− mice as described in Hernandez-Hansen et al. (6, 7). Briefly, BMMCs were obtained by culturing mouse bone marrow cells in RPMI 1640 medium, supplemented with 10% FCS (HyClone), 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 55 μM 2-ME, and 1 mM HEPES (complete RPMI medium) and 30% WEHI-3-conditioned medium. Culture reagents were from Invitrogen Life Technologies. Mast cell morphology, granularity, and differentiation were analyzed by toluidine blue-stained cytospin preparations and flow cytometry as described previously (6). By 6 wk, WT and Lyn−/− mast cells expressed similar levels of FcεRI and c-kit at their surfaces and were morphologically similar with respect to granule content. Microarray experiments were carried out on 6-wk-old mast cells. For stimulation, WT and Lyn−/− BMMCs were sensitized with 1 μg/ml anti-DNP IgE overnight (12 h) in complete RPMI medium without WEHI-3 medium. BMMCs were harvested, washed, and stained with trypan blue to verify viability. Viability was >95% for all experimental conditions. BMMCs were resuspended in complete RPMI medium and activated by the addition of 10 ng/ml DNP-BSA for 2 or 4 h at 37°C.

Total RNA was prepared from resting and activated WT and Lyn−/− BMMCs using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Further purification of RNA was done using the RNeasy mini kit clean-up protocol (Qiagen). A total of 10 μg of total RNA was converted to cDNA using the Superscript II RNA reverse transcriptase (Invitrogen Life Technologies). Double-strand cDNA was produced with an oligo(dT) primer containing the T7 RNA polymerase site at the 5′ end. The cDNA was labeled with biotinylated nucleotides directly with the ENZO in vitro labeling kit (Affymetrix) to produce antisense RNA. Labeled cRNA was fragmented and purity was checked using Agilent RNA chips (Agilent Technologies). A total of 15 μg of labeled cRNA was hybridized for 16 h to U74A murine Genechips (Affymetrix) containing 12,488 genes and expressed sequence tags (ESTs). A mixture of bacterial RNA (BioB, bioC, bioD, cre) with known concentrations (1.5, 5, 25, and 100 pM, respectively) was added to each chip hybridization mixture. Chips were washed, stained with PE-streptavidin and read with an HP Gene Array Scanner according to the manufacturer’s instructions.

All Affymetrix chips were scaled in the Affymetrix MAS 5.0 software to a target fluorescence of 500 and had scaling factors ranging from 2 to 15, indicating good probe preparation and hybridization. Filtering and statistical analyses of microarray data were performed using Genespring software version 6.0 (Silicon Genetics). For all statistical analyses, the experiment interpretation mode was set to the log of ratios and data were normalized per gene to the median of the appropriate unstimulated duplicate samples. For analysis of differential gene expression between stimulated WT and Lyn−/− BMMCs, data were normalized so that the baseline mRNA expression corresponded to the median of the stimulated WT BMMCs. To evaluate changes in gene expression, data were filtered in Genespring for genes expressing a 3-fold up or down change in expression when compared to the control samples. A Flag filter was applied to exclude those data in which a gene’s expression was absent or marginally present in at least half of the samples based on the Affymetrix algorithm. Finally, the one-way ANOVA filter (p < 0.05) was used to identify statistically significant differences in gene expression between resting and stimulated samples. For generation of heat maps, lists of regulated genes were clustered with the gene tree method by measuring similarity with smooth correlation. Statistical analyses were performed with two-way ANOVA to identify strain and/or treatment interactions for all eight samples. Statistical analyses were performed with two-way ANOVA to determine significant differences due to the kinase status (±Lyn), treatment (±Ag), and/or to the combination of kinase status and treatment for all eight samples. Variance was computed by applying the Cross-Gene Error Model when generating lists of regulated genes. The Benjamini-Hochberg false discovery rate was applied to the two-way ANOVA model as a multiple testing correction to control for occurrences of false positives that arise from performing multiple tests. The numbers of genes with a Benjamini-Hochberg adjusted p value <0.05 are listed in the Venn diagram.

RNA isolation and first double-strand cDNA synthesis were performed as described above. IL-13, Fyn, and actin primers and probes were designed using Primer Express Software according to guidelines recommended by Applied Biosystems for TaqMan Gold RT-PCR. Primers were used to amplify 100 ng of template cDNA (triplicate samples each of WT ± activation and Lyn−/−± activation) and produced single products when analyzed on 0.8% Tris acetate EDTA (TAE) agarose gels. PCR conditions used to amplify template DNA were as follows: 30 cycles of each 95°C for 48 s, 58°C for 45 s, 73°C for 3 min followed by a final 10-min extension at 72°C using a standard 50 μl PCR. The PCR amplification product obtained from each set of primers was purified from the agarose gel using the Qiaex II gel extraction kit (Qiagen). Fragment concentration was determined by measuring A260 absorbance of purified PCR fragments. These PCR fragments were then used to construct standard curves for real-time PCRs. Resultant cDNA used for microarray hybridization experiments served as a template for real-time quantitative PCR and was performed on an ABI 7700 Sequence Detection system (Applied Biosystems) in which a fluorescent reporter dye (FAM) was released and quantified during each specific replication of the template. Each PCR (25 μl) contained 100 ng of cDNA and was mixed with 2× TaqMan Universal PCR Master Mix (Applied Biosystems), gene-specific forward and reverse primers (15 μM), and dye-labeled oligonucleotide probes (2 μM). GAPDH primers and probes were from Applied Biosystems and were provided by Dr. C. Schwab (University of New Mexico, Albuquerque, NM). The forward and reverse primers used here were: for IL-13, 5′-CGCAAGGCCCCCACTAC-3′ and 5′-AGTTTTGTTATAAAGTGGGCTACTTCGA-3′; for Fyn, 5′-GGTTACATTCCCAGCAATTACGT-3′ and 5′-TGCGGCCAAGTTTTCCA-3′; for β-actin, 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCCGT-3′. The fluorogenic probes used for hybridization were as follows: for IL-13, 5′-(6FAM)-TCTCCAGCCTCCCCGATACCA-(carboxytetramethylrhodamine), [TAMRA]-3′; for Fyn, 5′-(6FAM)-TCCAGTTGACTCCATCCAGGCAGAAGA (TAMRA)-3′; for β-actin, 5′-(6FAM)-CACTGCCGCATCCTCTTCCTCCC-(TAMRA)-3′. PCR parameters were as recommended for the TaqMan Universal PCR master mix kit: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Triplicate samples of 2-fold serial dilutions (ranging from 1 × 104 molecules up to 2 × 106 molecules) of IL-13, Fyn, actin, or GAPDH fragments were assayed and used to construct the standard curves. Data were analyzed with ABI Prism 7000 SDS software version 1.0 and were normalized to the average copy number of GAPDH. Data are presented as fold induction with respect to unstimulated WT samples. Experiments were performed twice with triplicate samples (cDNA isolated from three separate cultures of BMMCs). Statistical analysis was performed with the Student unpaired t test using GraphPad Prism software.

Supernatants were collected from resting and activated WT and Lyn−/− BMMCs and analyzed for IL-4 or IL-13 using a two-site sandwich ELISA as described previously (15). Abs specific for IL-4 (11B11, and biotin-BVD6-24G2) were from BD Pharmingen; Abs specific for IL-13 (38213.11, and biotin-BAF413) were from R&D Systems. Capture mAbs were bound to ELISA plates diluted in 0.1 M Na2HPO4 overnight at 4°C. Plates were washed, blocked with 1% BSA in PBS, and incubated overnight at 4°C with samples. Bound cytokines were detected by the addition of biotinylated mAbs followed by streptavidin-HRP (0.625 μg/ml final concentration for IL-13; 2.5 μg/ml final concentration for IL-4) and colorimetric substrate (ABTS for IL-4; tetramethylbenzidine (TMB; Sigma-Aldrich), for IL-13). OD405 was determined and cytokines were quantified by comparison to standard curves generated using rIL-4 (BD Pharmingen) and IL-13 (R&D Systems). Supernatants were analyzed with commercial ELISA kits for IL-6 (BD Biosciences) and for IL-2 and TNF-α (eBioscience) according to the manufacturer’s instructions. Detection limits for each cytokine assay were assigned as the lowest concentration in the linear portion of the standard curve. Measurements were made in duplicate using cells from three independent cultures of BMMCs.

Fig. 1 and Table I summarize the principal differences in gene expression between WT and Lyn−/− BMMCs under resting conditions. The data are strikingly similar between the duplicate samples, indicating that the technical procedures from RNA preparation to data acquisition and analysis are highly reproducible. Expression levels for the mRNAs encoding the receptors CXCR4 and insulin-like growth factor 2 receptor (IGF2R) are higher in Lyn−/− BMMCs than in WT BMMCs. In addition, annexin A1 that inhibits PLA2 activity in vitro and phospholipase A2 group VII are expressed at higher levels in Lyn−/− BMMCs. Levels of several other mRNAs are consistently lower in Lyn−/− BMMCs compared with WT BMMCs; in general, the down-regulated genes lack known roles in signal transduction pathways.

FIGURE 1.

The absence of Lyn alters the gene expression profile of resting mast cells. Two independent cultures of 6-wk-old WT and Lyn−/− BMMCs were incubated in complete RPMI without growth factors for 16 h. Cells were harvested, their viability was confirmed by trypan blue exclusion, and total mRNA was isolated. Biotinylated antisense cRNA was hybridized to Affymetrix U74A DNA chips for 16 h. DNA chips were washed and scanned with an Affymetrix scanner. Data were normalized so that the level of baseline mRNA expression corresponded to the median of unstimulated WT BMMCs. The heat map represents 14 genes that were up- or down-regulated by a factor of 3 or greater and was generated using a stringent filter scheme that incorporated the ANOVA filter. Each row corresponds to a single gene and each column represents an independent condition. Location of the gene and common name are provided. Changes in gene expression correspond to the color scale shown.

FIGURE 1.

The absence of Lyn alters the gene expression profile of resting mast cells. Two independent cultures of 6-wk-old WT and Lyn−/− BMMCs were incubated in complete RPMI without growth factors for 16 h. Cells were harvested, their viability was confirmed by trypan blue exclusion, and total mRNA was isolated. Biotinylated antisense cRNA was hybridized to Affymetrix U74A DNA chips for 16 h. DNA chips were washed and scanned with an Affymetrix scanner. Data were normalized so that the level of baseline mRNA expression corresponded to the median of unstimulated WT BMMCs. The heat map represents 14 genes that were up- or down-regulated by a factor of 3 or greater and was generated using a stringent filter scheme that incorporated the ANOVA filter. Each row corresponds to a single gene and each column represents an independent condition. Location of the gene and common name are provided. Changes in gene expression correspond to the color scale shown.

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Table I.

Significantly changed genes between unstimulated WT and Lyn−/− mast cellsa

Common NameDescriptionGenBankFold Change in Lyn−/− Cells
Receptors    
 CXCR4 Chemokine (CXC motif) receptor 4 Z80112 13.5 
 IGF2R Insulin-like growth factor 2 receptor U04710 4.4 
Lipid metabolism    
 PLA2G7 Phospholipase A2 group VII U34277 4.7 
Calcium regulation    
 ANXA1 EST, Annexin A1 AV003419 5.1 
 ANXA1 Annexin A1 (phospholipase A2 inhibitor activity) M69260 13.5 
Proteolysis and peptidolysis    
 EGFBP1 Epidermal growth factor binding protein type 1 M17979 11.8 
 KLK9 Major epidermal growth factor binding protein M17962 5.6 
Defense response    
 CD24A M1/69-J11d heat stable antigen M58661 0.26 
Transcription    
 KROX-24 Zinc finger protein M28845 0.32 
EST EST AW123567 0.22 
 ARCN1 Archain 1 A1853439 0.12 
 1810009A16Rik EST AV086272 0.19 
 D6Ertd365e EST AA796868 0.25 
Others    
 TDE1 Overexpressed in testicular tumors L29441 0.23 
Common NameDescriptionGenBankFold Change in Lyn−/− Cells
Receptors    
 CXCR4 Chemokine (CXC motif) receptor 4 Z80112 13.5 
 IGF2R Insulin-like growth factor 2 receptor U04710 4.4 
Lipid metabolism    
 PLA2G7 Phospholipase A2 group VII U34277 4.7 
Calcium regulation    
 ANXA1 EST, Annexin A1 AV003419 5.1 
 ANXA1 Annexin A1 (phospholipase A2 inhibitor activity) M69260 13.5 
Proteolysis and peptidolysis    
 EGFBP1 Epidermal growth factor binding protein type 1 M17979 11.8 
 KLK9 Major epidermal growth factor binding protein M17962 5.6 
Defense response    
 CD24A M1/69-J11d heat stable antigen M58661 0.26 
Transcription    
 KROX-24 Zinc finger protein M28845 0.32 
EST EST AW123567 0.22 
 ARCN1 Archain 1 A1853439 0.12 
 1810009A16Rik EST AV086272 0.19 
 D6Ertd365e EST AA796868 0.25 
Others    
 TDE1 Overexpressed in testicular tumors L29441 0.23 
a

Data were normalized to the median baseline expression of unstimulated WT cells and analyzed with the one-way ANOVA filter. One-way ANOVA analysis established that the fold change in Lyn−/− cells compared to WT cells was statistically significant (p < 0.05) for each mRNA.

To identify other differentially transcribed genes between WT and Lyn−/− BMMCs, we compared the gene expression profiles of eight separate samples representing two separate experiments for each of four conditions: resting and Ag-stimulated from both WT and Lyn−/− cells. Two-way ANOVA was used to identify genes whose expression levels were significantly altered based on kinase status (±Lyn), treatment (±Ag), or on the interaction of both parameters (kinase status and treatment). Through this analysis, we identified 501 gene products with a Benjamini-Hochberg adjusted p value <0.05, indicating their expression is statistically different due to the absence of Lyn, the presence of Ag, or the interaction of both parameters. Fig. 2 provides two alternative graphical representations of these results.

FIGURE 2.

Gene expression profiles in resting and Ag-stimulated WT and Lyn−/− mast cells. Total RNA was isolated from two independent cultures of 6-wk-old WT and Lyn−/− BMMCs without (−) and with (+) stimulation with 10 ng/ml DNP-BSA for 4 h. Labeled antisense cRNA was hybridized to DNA chips and read with an Affymetrix scanner. Data were normalized to the median baseline mRNA expression of unstimulated WT BMMCs and filtered as described in Materials and Methods. The resulting gene list was analyzed by two-way ANOVA and resulted in 501 statistically significant regulated genes that are depicted in the heat map (A). Changes in gene expression correspond to the color scale shown. The Venn diagram (B) is an alternative graphical representation of the same data. There are 501 genes that received a Benjamini-Hochberg adjusted p value <0.05 in the two-way ANOVA model. The Venn diagram shows the number of genes that had an adjusted p value < 0.05 for the kinase effect, treatment effect, and the interaction effect between the two groups.

FIGURE 2.

Gene expression profiles in resting and Ag-stimulated WT and Lyn−/− mast cells. Total RNA was isolated from two independent cultures of 6-wk-old WT and Lyn−/− BMMCs without (−) and with (+) stimulation with 10 ng/ml DNP-BSA for 4 h. Labeled antisense cRNA was hybridized to DNA chips and read with an Affymetrix scanner. Data were normalized to the median baseline mRNA expression of unstimulated WT BMMCs and filtered as described in Materials and Methods. The resulting gene list was analyzed by two-way ANOVA and resulted in 501 statistically significant regulated genes that are depicted in the heat map (A). Changes in gene expression correspond to the color scale shown. The Venn diagram (B) is an alternative graphical representation of the same data. There are 501 genes that received a Benjamini-Hochberg adjusted p value <0.05 in the two-way ANOVA model. The Venn diagram shows the number of genes that had an adjusted p value < 0.05 for the kinase effect, treatment effect, and the interaction effect between the two groups.

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The data on 501 gene products are shown in Fig. 2 A as a heat map that compares all eight samples, normalized to the median of the two unstimulated WT samples. The striking similarity between the duplicate samples is again evident. Gene expression is moderately altered between unstimulated samples and is greatly altered in Ag-stimulated samples, with almost as many genes showing decreased expression (blue) as increased expression (red).

The Venn diagram (Fig. 2 B) displays the numbers of these gene products whose p values are influenced by each parameter independently (kinase status or treatment) and also by the effect that each parameter has on the other. From a total of 145 genes that display a kinase effect, 27 are significant due to the kinase effect only while 2 genes displayed a kinase effect and an interaction effect (Fig. 2 B, left circle). Another 470 genes displayed a treatment effect (Fig. 2 B, right circle). Of those, 85 genes are affected by both kinase status and treatment but each factor affects gene expression independently of one another. As shown in Fig. 2 B, lower circle, there are 31 total genes that have p values <0.05 for each of the tests: kinase effect, interaction effect, and treatment effect. In addition, 14 genes are significant due to the treatment effect and interaction effect but not the kinase effect. Two genes that are not significant in either the kinase test or treatment test alone had a p value <0.05 for the interaction effect.

Table II organizes a subset of these 501 gene products into functional categories and shows their fold change in expression after FcεRI cross-linking in both WT and Lyn−/− cells. A complete list of these genes is available at 〈www.cellpath.unm.edu〉.

Table II.

Differentially regulated genes between resting and Ag-stimulated WT or resting and Ag-stimulated Lyn−/− mast cellsa

Common NameGene DescriptionGenBank Acc. No.Fold Change WT (+Ag)Fold Change Lyn−/− (+Ag)
Cytokine or chemokine     
 IL-4 Interleukin 4 X03532 0.7 18.2 
 IL-6 Interleukin 6 X54542 5.6 22 
 IL-13 Interleukin 13 M23504 8.7 81 
 TNF-α Tumor necrosis factor alpha D84196 13.4 12 
 M-CSF Macrophage colony-stimulating factor M21952 1.9 3.4 
 GM-CSF Granolocyte macrophage colony-stimulating factor X03020 0.9 43 
 IL-7R Interleukin-7 receptor M29697 1.4 3.6 
 CCL1 Secreted T cell protein, CCL1 M23501 10 102 
 CCL2 Platelet-derived growth factor-inducible protein M19681 2.7 2.5 
 CCL3 TCR L2G25B protein J04491 6.5 31 
 CCL5 RANTES AF065947 2.9 79 
 CCL7 Cytokine gene X70058 11 65 
 CCL9 Macrophage inflammatory protein-1 γ U49513 3.7 34 
 MIP1β Macrophage inflammatory protein-1 β X62502 2.3 8.6 
 CCR1 MIP1-α/RANTES receptor U29678 7.4 
 XCL1 Lymphotactin U15607 0.5 31 
Signal transduction     
 Fyn p59fyn M27266 5.7 9.7 
 CISH Cytokine SH2-containing protein D89613 2.2 5.9 
 FCγRIIB Low affinity IgG receptor IIB M31312 0.4 0.1 
Calcium regulation     
 CDH2 Neural cadherin (N-cadherin) M31131 3.4 
 SERCA2 Sarco(endo)plasmic reticulum calcium ATPase AF029982 3.5 7.7 
 CAM1 Calmodulin M19381 3.1 3.1 
 ITPR1 Inositol 1,4,5-triphosphate receptor type 1 X15373 2.2 5.8 
 PCD6 Inositol 1,4,5-triphosphate receptor type 1 M21530 1.2 3.6 
 SPHK1A Sphingosine kinase 1 a AF068748 1.1 33 
 PLSCR2 Phospholipid scramblase 2 AF015790 17.1 2.1 
G-protein signal transduction     
 C3AR1 Anaphylatoxin C3a receptor gene U77461 4.4 5.5 
 RGSR Retinally abundant regulator of G-protein signaling U94828 1.9 12 
 RAMP2 Receptor activity modifying protein 2 AJ250490 1.1 
Transcription factors     
 EGR2 Early growth response 2 M24377 39 32 
 MYC c-myc L00039 9.8 8.7 
 NF-ATC1 Transcription factor NF-ATc isoform a AF087434 1.8 6.2 
Others     
 LGALS3 Mac-2 Ag X16834 4.5 4.2 
 SCD1 Stearoyl-CoA desaturase M21285 35 19 
 CRABP2 Cellular retinoic acid-binding protein II M35523 12.8 77 
Common NameGene DescriptionGenBank Acc. No.Fold Change WT (+Ag)Fold Change Lyn−/− (+Ag)
Cytokine or chemokine     
 IL-4 Interleukin 4 X03532 0.7 18.2 
 IL-6 Interleukin 6 X54542 5.6 22 
 IL-13 Interleukin 13 M23504 8.7 81 
 TNF-α Tumor necrosis factor alpha D84196 13.4 12 
 M-CSF Macrophage colony-stimulating factor M21952 1.9 3.4 
 GM-CSF Granolocyte macrophage colony-stimulating factor X03020 0.9 43 
 IL-7R Interleukin-7 receptor M29697 1.4 3.6 
 CCL1 Secreted T cell protein, CCL1 M23501 10 102 
 CCL2 Platelet-derived growth factor-inducible protein M19681 2.7 2.5 
 CCL3 TCR L2G25B protein J04491 6.5 31 
 CCL5 RANTES AF065947 2.9 79 
 CCL7 Cytokine gene X70058 11 65 
 CCL9 Macrophage inflammatory protein-1 γ U49513 3.7 34 
 MIP1β Macrophage inflammatory protein-1 β X62502 2.3 8.6 
 CCR1 MIP1-α/RANTES receptor U29678 7.4 
 XCL1 Lymphotactin U15607 0.5 31 
Signal transduction     
 Fyn p59fyn M27266 5.7 9.7 
 CISH Cytokine SH2-containing protein D89613 2.2 5.9 
 FCγRIIB Low affinity IgG receptor IIB M31312 0.4 0.1 
Calcium regulation     
 CDH2 Neural cadherin (N-cadherin) M31131 3.4 
 SERCA2 Sarco(endo)plasmic reticulum calcium ATPase AF029982 3.5 7.7 
 CAM1 Calmodulin M19381 3.1 3.1 
 ITPR1 Inositol 1,4,5-triphosphate receptor type 1 X15373 2.2 5.8 
 PCD6 Inositol 1,4,5-triphosphate receptor type 1 M21530 1.2 3.6 
 SPHK1A Sphingosine kinase 1 a AF068748 1.1 33 
 PLSCR2 Phospholipid scramblase 2 AF015790 17.1 2.1 
G-protein signal transduction     
 C3AR1 Anaphylatoxin C3a receptor gene U77461 4.4 5.5 
 RGSR Retinally abundant regulator of G-protein signaling U94828 1.9 12 
 RAMP2 Receptor activity modifying protein 2 AJ250490 1.1 
Transcription factors     
 EGR2 Early growth response 2 M24377 39 32 
 MYC c-myc L00039 9.8 8.7 
 NF-ATC1 Transcription factor NF-ATc isoform a AF087434 1.8 6.2 
Others     
 LGALS3 Mac-2 Ag X16834 4.5 4.2 
 SCD1 Stearoyl-CoA desaturase M21285 35 19 
 CRABP2 Cellular retinoic acid-binding protein II M35523 12.8 77 
a

Data were normalized to the median baseline mRNA expression of either unstimulated WT mast cells or unstimulated Lyn−/− cells. The genes listed were selected from a list of 501 genes that resulted from analysis by two-way ANOVA.

Among genes involved in tyrosine kinase-coupled signaling, levels of Fyn mRNA are increased in both WT and Lyn−/− BMMCs after FcεRI cross-linking, with the increase being greater in Lyn−/− cells (Table II). In contrast, mRNA expression for the low-affinity IgG receptor FcγRIIB is significantly reduced after Ag stimulation in both WT cells and Lyn−/− cells, with the extent of down-regulation being greater in the Lyn−/− BMMCs. Recent studies implicate Fyn in Lyn-independent signaling in activated mast cells (2) and FcγRIIB in the negative regulation of FcεRI signaling (16, 17). Thus, both observations may be relevant to the hyperresponsiveness of Lyn−/− BMMCs. Several genes implicated in calcium regulation, including the type I inositol-1,4,5-trisphosphate receptor (Ins(1,4,5)P3 receptor 1), sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2), and sphingosine kinase-1, are also significantly up-regulated in Ag-stimulated Lyn−/− BMMCs compared with control cells and could contribute to cellular hyperresponsiveness.

A consistent trend of higher up-regulation in activated Lyn−/− mast cells extends to other genes that may contribute to signaling pathway activities. These include genes involved in G protein-coupled signal transduction and cellular retinoic acid-binding protein II (CRABP2) (Table II). Additionally, expression of mRNA for at least one transcription factor, the NF-ATc isoform, is up-regulated in Lyn−/− mast cells. In contrast, phospholipid scramblase 2 (PLSCR2) mRNA is only up-regulated 2.1-fold in Lyn−/− mast cells compared to 17.1-fold in WT cells.

The most striking differences noted between the two cell types is in the expression of cytokine and chemokine genes known to be up-regulated upon FcεRI aggregation in mast cells and basophils. In general, cytokine and chemokine gene expression is the same in WT and Lyn−/− BMMCs under resting conditions. As an exception, the expression of CXCR4 is increased in unstimulated Lyn−/− mast cells (Table I). Following FcεRI cross-linking (Table II), mRNA levels for the Th2 cytokines IL-4 and IL-13 are up-regulated 18.2- and 81-fold in Lyn−/− BMMCs, respectively, compared to 0.7- and 8.7-fold in WT mast cells. In addition, mRNA levels for all chemokines of the CC family, CCL1, CCL3, CCL4 (MIP1β), CCL5 (RANTES), CCL7, and CCL9 (MIP1γ) are significantly higher in activated Lyn−/− mast cells than in WT cells. As an exception, mRNA for the platelet-derived growth factor-inducible protein (CCL2) is up-regulated in both cell types to similar levels upon FcεRI cross-linking.

Microarray data were validated for selected genes by either real-time quantitative PCR or ELISAs. Fig. 3 shows the mRNA expression of Fyn, IL-13, and actin relative to that of unstimulated WT cells. Fyn and IL-13 mRNA expression increased in activated BMMCs (WT, 4.9- ± 0.9-fold; Lyn−/−, 8.7- ± 0.2-fold, mean ± SEM, n = 6; p < 0.05) and (WT, 5.9- ± 2.4-fold; Lyn−/−, 36.5- ± 3.0-fold; mean ± SEM, n = 6, p < 0.005), respectively (Fig. 3, A and B). In contrast, there was no significant difference in actin mRNA expression after cross-linking FcεRI (WT, 1.2- ± 0.1-fold; Lyn−/−, 1.2- ± 0.2-fold, mean ± SEM, n = 6; p > 0.05), (Fig. 3 C). Standard curves generated from the indicated cDNAs showed a quantitative relationship between cDNA copy number and fluorescence signal intensity (data not shown).

FIGURE 3.

Transcription of IL-13 and Fyn genes in WT and Lyn−/− mast cells is differentially regulated by Ag stimulation. Quantitative detection of Fyn (A), IL-13 (B), and actin (C) mRNA transcripts in resting and activated WT or Lyn−/− BMMCs. The copy number of the target gene was normalized using the average copy number of GAPDH as an internal standard. Data were then normalized so that the level of baseline mRNA expression corresponded to the median of unstimulated WT BMMCs. Data are presented as the mean fold induction ± SEM obtained from two independent experiments, each performed in triplicate. Mean values significantly different from WT levels are indicated: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.005, Student’s t test.

FIGURE 3.

Transcription of IL-13 and Fyn genes in WT and Lyn−/− mast cells is differentially regulated by Ag stimulation. Quantitative detection of Fyn (A), IL-13 (B), and actin (C) mRNA transcripts in resting and activated WT or Lyn−/− BMMCs. The copy number of the target gene was normalized using the average copy number of GAPDH as an internal standard. Data were then normalized so that the level of baseline mRNA expression corresponded to the median of unstimulated WT BMMCs. Data are presented as the mean fold induction ± SEM obtained from two independent experiments, each performed in triplicate. Mean values significantly different from WT levels are indicated: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.005, Student’s t test.

Close modal

Fig. 4 shows that cytokines are increased at the protein level in Ag-stimulated Lyn−/− mast cells. Lyn−/− BMMCs secrete significantly more IL-4 than control cells at 4 h after FcεRI cross-linking (Fig. 4,A). Similarly, FcεRI-mediated release of both IL-6 and IL-13 is significantly higher in Lyn−/− BMMCs at 2 and 4 h of activation (Fig. 4, B and C).

FIGURE 4.

Increased IL-4, IL-6, IL-13, IL-2, and TNF-α protein secretion in Ag-stimulated Lyn−/− mast cells. WT and Lyn−/− BMMCs were sensitized and challenged as described in Materials and Methods. Release of IL-4 (A), IL-6 (B), IL-13 (C), IL-2 (D), and TNF-α (E) by mast cells was measured. Results shown are representative of three independent experiments, each performed in duplicate. Mean values significantly different from WT levels are indicated: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.005, Student’s t test.

FIGURE 4.

Increased IL-4, IL-6, IL-13, IL-2, and TNF-α protein secretion in Ag-stimulated Lyn−/− mast cells. WT and Lyn−/− BMMCs were sensitized and challenged as described in Materials and Methods. Release of IL-4 (A), IL-6 (B), IL-13 (C), IL-2 (D), and TNF-α (E) by mast cells was measured. Results shown are representative of three independent experiments, each performed in duplicate. Mean values significantly different from WT levels are indicated: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.005, Student’s t test.

Close modal

Previous studies suggest that both IL-2 and TNF-α production are increased in Ag-stimulated Lyn−/− BMMCs (5). Both Ag-stimulated WT and Lyn−/− cells secrete very little IL-2, however, we note a slight increase in IL-2 production in Lyn−/− BMMCs after 4 h of Ag stimulation compared with WT mast cells (Fig. 4,D). We confirmed that Lyn−/− mast cells secrete more TNF-α at both 2 and 4 h of activation than WT cells (Fig. 4 E).

The cross-linking of IgE-FcεRI complexes with multivalent Ag activates mast cell signaling pathways leading to the release of a wide range of proinflammatory cytokines, chemokines, and other mediators associated with allergic responses. Activated mast cells release IL-4, IL-13, and other Th2 cytokines that enhance the IgE response by stimulating the differentiation of Th2 lymphocytes and promoting class switching to IgE in B cells, leading to increased production of IgE and resensitization of FcεRI on mast cells (reviewed in Ref.18).

Previously, we showed that BMMCs from Lyn−/− mice divide faster than WT cells in response to growth-promoting cytokines (IL-3 and stem cell factor) and undergo less apoptosis when cytokine is withdrawn (6). We and others (2, 5, 7, 8, 19) have also shown that Lyn−/− BMMCs are slow to initiate responses to FcεRI cross-linking but are deficient in the termination of signaling, resulting in prolonged biochemical responses (receptor phosphorylation, the activation of AKT, phospholipase Cγ, ERK, and other signaling enzymes) and exaggerated physiological responses (calcium mobilization, secretion, cytokine production, and integrin activation). Resolving previous controversy (reviewed in Refs.6 and 7), these results now provide a consensus view of Lyn as both a kinetic accelerator and negative regulator of FcεRI signaling in mast cells. Previous work has also provided partial insight into the mechanism of the hyperresponsiveness of Lyn−/− BMMCs. Specifically, increased basal and Ag-induced Fyn activity is thought to contribute to signal initiation in Lyn-deficient cells (7, 8), while the loss of Ag-induced SHIP activation provides at least a partial explanation for the failure of signal termination in Lyn−/− cells (7).

Previous groups have used microarray analyses to explore transcriptional profiles induced by FcεRI cross-linking in normal and secretion-impaired rodent and human mast cells and basophils (20, 21, 22, 23, 24, 25, 26). Here, microarray analysis was used to discover new properties of both unstimulated and Ag-stimulated Lyn-deficient BMMCs that might provide further insight into their hyperresponsive phenotype.

Relatively few genes showed strongly different expression levels between unstimulated WT and Lyn−/− BMMCs and none could be clearly linked to the enhanced early responses (calcium mobilization, integrin activation, degranulation, and others) to FcεRI cross-linking. The chemokine receptor, CXCR4, is a possible exception. CXCR4 was strongly up-regulated in Lyn−/− BMMCs. Ligation of G protein-coupled receptors can prime mast cells for increased FcεRI-mediated degranulation (27).

In contrast, expression of 501 genes was increased or decreased at least 3-fold after 4 h of FcεRI cross-linking. Of these genes, more were up-regulated than down-regulated. In general, the extent of up-regulation was greater in Lyn−/− than in WT BMMCs. Thus, there is strong potential for a transcriptional component to the enhanced late responses (cytokine and chemokine production and others) to FcεRI cross-linking in the Lyn-deficient cells.

Among genes for signaling proteins, FcεRI cross-linking induced a greater up-regulation of the tyrosine kinase, Fyn, and of a series of genes implicated in calcium regulation in Lyn-deficient than in WT BMMCs. In combination with earlier evidence for increased Fyn activity, perhaps linked in part to delayed activation of Csk-binding protein (Cbp), in Lyn−/− BMMCs (7, 8), these data support the hypothesis that increased signaling through the recently discovered Fyn-mediated pathway can contribute to the persistent hyperresponsiveness of Lyn−/− BMMCs.

Conversely, FcεRI cross-linking induced a greater down-regulation of mRNA for the IgG receptor, FcγRIIB, in Lyn-deficient than in WT BMMCs. FcγRIIB is well-recognized as a negative regulator of FcεRI signaling when the two receptors are co-cross-linked. The mechanism involves the recruitment of the inositol phosphatase, SHIP, to the membrane via its association with phosphorylated ITIMs present in FcγRIIB (16, 17). Recent evidence that SHIP-deficient BMMCs degranulate spontaneously and are hyperresponsive to FcεRI cross-linking suggests that SHIP also plays a constitutive role in the down-regulation of signaling (28). In this case, reduced levels of FcγRIIB in Lyn-deficient BMMCs may help to maintain the reduced levels and activity of membrane-associated SHIP and the elevated levels of membrane phosphatidylinositol 3,4,5-trisphosphate (PI (3,4,5)P3) characteristic of these cells (7).

Among the cytokines expressed after 4 h of Ag stimulation in BMMCs, our data show that levels of mRNA and protein for IL-4, IL-6, and for IL-13 are all significantly higher in Lyn−/− than in WT BMMCs. Both IL-4 and IL-13 proteins are elevated in the lungs of asthmatic patients, and are thought to be central regulators of this disease. In mice, recent studies suggest that IL-13 may be more directly involved in mediating allergic responses than IL-4 (29, 30, 31, 32). Thus, Ag-exposed Lyn-deficient BMMCs clearly develop a cytokine profile consistent with a predisposition to allergy and asthma.

We failed to see greater increases in TNF-α and IL-2 mRNA levels in Lyn−/− cells than in WT mast cells. However, ELISAs showed that Lyn−/− BMMCs produce higher amounts of TNF-α and IL-2 protein than WT cells. These results are consistent with previously published data (5). The discrepancies between mRNA levels and TNF-α and IL-2 protein production may be attributed to mRNA instability. We also did not observe robust production of IL-2 in either WT or Lyn−/− BMMCs as was reported by Kawakami et al. (5). These different results very likely reflect differences in the time that mast cells were stimulated: our measurements were made after 4 h of activation, while the previous group made measurements after 20 h. Likewise, Nishizumi and Yamamoto (19) reported that Lyn deficiency does not affect the production of cytokines (IL-4, IL-5, IL-6, TNF-α, TNF-β) when BMMCs are stimulated with Ag for 2.5 h. We, too, found rather little differences in IL-4 or IL-6 production when WT and Lyn−/− BMMCs were stimulated for 2 h, even though the differences were substantial after 4 h (Fig. 4).

Among the chemokines, mRNAs coding for CCL1, CCL3 (MIP1α), CCL4 (MIP1β), CCL5 (RANTES), CCL7 (MCP-3), and CCL9 (MIP1γ) are all up-regulated in Ag-stimulated Lyn−/− BMMCs compared with WT BMMCs. The greater induction of both RANTES and its receptor CCR1 may contribute to the hyperresponsiveness of Lyn−/− BMMCs via a potential autocrine signaling mechanism. Previous studies have demonstrated a central role for chemokines in mediating multiple aspects of the asthmatic response. Chemokines induce B cell Ab class switching (33). In addition, IL-13 is a potent inducer of chemokines (eotaxins) in airway epithelial cells (31, 34) and current models suggest that coordinated interactions between IL-13 and chemokines are importantly involved in the pathogenesis of asthma (35).

Increased expression of cytokine and chemokine mRNA is likely the consequence of increased activity of transcription factors. Our data show that mRNA coding for at least one transcription factor, the cytoplasmic NF-AT (NF-ATC, also known as NFATC1 and NFAT2) is induced 3-fold more in Lyn−/− BMMCs than in WT BMMCs (Table II). In addition, Ag-stimulated Lyn−/− BMMCs express slightly higher levels of NF-κB (Lyn−/−, 1.6-fold; WT, 0.8-fold), and slightly lower levels of c-Jun/activator protein-1 (AP-1) (Lyn−/−, 2-fold; WT, 4-fold). Studies of the phosphorylation and/or activation of these transcription factors are needed to know if increased levels are linked to increased activities of transcription factors.

The linkage between increased FcεRI signaling and increased gene transcription in Lyn−/− BMMCs is not known with certainty. However, we note that biochemical studies published previously linked reduced SHIP activation (discussed above) to increases in AKT and Ras/MAPK pathway activities (7, 28). Ag-stimulated SHIP−/− BMMCs also produce increased levels of various proinflammatory cytokines including IL-4, IL-5, IL-6, IL-13, and TNF-α (36). Furthermore, SHIP negatively regulates IL-6 production in mast cells by inhibiting NF-κB activity (36). In turn, Kitaura et al. (37), linked increased AKT activity to increased cytokine (IL-2 and TNF-α) gene promoter activities via the activation of NF-κB, NF-AT, and AP-1 in Lyn−/− BMMCs. Additionally, Monticelli et al. (38) recently described roles for NF-AT proteins in the regulation of IL-13 gene transcription in BMMCs. Thus, the increased levels of mRNA for multiple cytokines and chemokines in Lyn−/− BMMCs may result directly from their increased capacity to signal from AKT to the activation of transcription factors.

The 33-fold up-regulation of sphingosine kinase 1 in Ag-stimulated Lyn−/− BMMCs may also contribute to increased chemokine production. Sphingosine kinase is activated upon FcεRI cross-linking in RBL-2H3 cells and mast cells and is linked to calcium mobilization (39, 40, 41). Additionally, the product of sphingosine kinase, sphingosine-1-phosphate (S1P), acts as a ligand for G-protein coupled chemokine receptors (42). S1P levels are increased in bronchoalveolar lavage fluid from lungs of asthmatics after challenge with allergen (43) and can lead to a heightened production of chemokines in RBL-2H3 mast cells (44).

Our results in Lyn−/− BMMCs differ from results obtained in a recent study of gene expression in Lyn-deficient DT40 chicken B cells, where the absence of Lyn led to down-regulation of numerous genes encoding proteins involved in BCR signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal organization (45). One major difference between Lyn-deficient DT40 cells and BMMCs is that the Lyn−/− DT40 cells have no other Src kinase family members, whereas the Src kinase, Fyn, increases in both activity (7) and transcript levels (Fig. 3, Table II) in Ag-stimulated Lyn−/− BMMCs.

In conclusion, the FcεRI-mediated activation of Lyn−/− BMMCs results in greater increases in mRNAs encoding proteins in the FcεRI signaling pathway, greater decreases in mRNAs encoding a negative regulator of signaling and greater increases in mRNAs encoding Th2 cytokines and chemokines and key transcription factors than occur in control cells. All of these differences are likely to contribute to the hyperresponsiveness of Lyn−/− mast cells and to the greater predisposition of Lyn−/− mice to the allergic phenotype as indicated by the higher numbers of skin and peritoneal mast cells, higher serum IgE levels, increased levels of circulating histamine, and increased in vivo expression of surface FcεRI on the mast cells of Lyn−/− mice in comparison with WT littermates (6, 9, 10, 12, 19). Recently, Beavit et al. (46) confirmed that Lyn−/− mice develop severe, persistent asthma suggesting a role for Lyn as an important negative regulator of Th2 immune responses. Overall, our analysis suggests a key role for Lyn in setting the thresholds for mast cell signaling and response pathways, thus determining predisposition to allergic responses.

Microarray experiments were performed using the resources of the Keck-University of New Mexico (UNM) Genomics Resource in the UNM Cancer Research and Treatment Center. We thank Dr. Gavin Pickett for training with Genespring software, James A. Aden for interpretation of statistical analyses generated with Genespring, Marilee Morgan for technical assistance and reagents, and Dr. Chris Schwab for generously providing GAPDH primers and probes.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health (NIH) Grants RO1 GM49814 and P50 HL56384 and by a minority graduate fellowship from NIH/National Institute of General Medical Sciences (to V.H.-H.).

4

Abbreviations used in this paper: BMMC, bone marrow-derived mast cell; WT, wild type; EST, expressed sequence tag.

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