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Increased Expression of Genes Linked to FcRI Signaling and to Cytokine and Chemokine Production in Lyn-Deficient Mast Cells¹

Valerie Hernandez-Hansen^2,*, Julie D. J. Bard^*, Christy A. Tarleton^*, Julie A. Wilder, Clifford A. Lowell, Bridget S. Wilson^* and Janet M. Oliver^3,*

^* Department of Pathology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, NM 87131; Lovelace Respiratory Research Institute, Albuquerque, NM 87108; and Department of Laboratory Medicine, University of California, San Francisco, CA 94143

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cross-linking the high-affinity IgE receptor, FcRI, 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 FcRI 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 FcRI 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 (FcRIIB), implicated in negative regulation of FcRI-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 MIP1)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 FcRI signaling and also to the response pathways that lead to allergy and asthma.

	Introduction

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Mastcells and basophils express the high-affinity IgE receptor (FcRI) on their cell surfaces and are activated when multivalent Ag cross-links IgE-bound receptors. FcRI 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- 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 FcRI stimulation. We report that a number of genes encoding proteins involved in the positive regulation of FcRI 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 FcRI signaling, FcRIIB, is down-regulated in the Lyn^–/– cells. In addition, multiple cytokine and chemokine genes known to be induced upon cross-linking FcRI in mast cells and basophils are expressed at 3- to 30-fold higher levels in Lyn^–/– BMMCs than in WT BMMCs.

	Materials and Methods

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Abs and reagents

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.

Cell culture and activation conditions

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 FcRI 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.

Isolation and labeling of mast cell RNA

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.

DNA microarray analysis

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.

Quantitative real-time PCR

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 2x 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 -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 -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 x 10⁴ molecules up to 2 x 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.

Cytokine ELISA

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- (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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Different gene expression profiles of resting WT and Lyn^–/– 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.

View larger version (36K): [in this window] [in a new window]   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.

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.

View larger version (39K): [in this window] [in a new window]   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.

The Venn diagram (Fig. 2B) 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. 2B, left circle). Another 470 genes displayed a treatment effect (Fig. 2B, 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. 2B, 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 FcRI cross-linking in both WT and Lyn^–/– cells. A complete list of these genes is available at www.cellpath.unm.edu.

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 FcRI 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 FcRI 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 (MIP1), CCL5 (RANTES), CCL7, and CCL9 (MIP1) 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 FcRI cross-linking.

Validation of microarray data

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 FcRI (WT, 1.2- ± 0.1-fold; Lyn^–/–, 1.2- ± 0.2-fold, mean ± SEM, n = 6; p > 0.05), (Fig. 3C). Standard curves generated from the indicated cDNAs showed a quantitative relationship between cDNA copy number and fluorescence signal intensity (data not shown).

View larger version (17K): [in this window] [in a new window]   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.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Increased IL-4, IL-6, IL-13, IL-2, and TNF- 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- (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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The cross-linking of IgE-FcRI 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 FcRI 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 FcRI cross-linking but are deficient in the termination of signaling, resulting in prolonged biochemical responses (receptor phosphorylation, the activation of AKT, phospholipase C, 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 FcRI 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 FcRI 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 FcRI 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 FcRI-mediated degranulation (27).

In contrast, expression of 501 genes was increased or decreased at least 3-fold after 4 h of FcRI 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 FcRI cross-linking in the Lyn-deficient cells.

Among genes for signaling proteins, FcRI 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, FcRI cross-linking induced a greater down-regulation of mRNA for the IgG receptor, FcRIIB, in Lyn-deficient than in WT BMMCs. FcRIIB is well-recognized as a negative regulator of FcRI 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 FcRIIB (16, 17). Recent evidence that SHIP-deficient BMMCs degranulate spontaneously and are hyperresponsive to FcRI cross-linking suggests that SHIP also plays a constitutive role in the down-regulation of signaling (28). In this case, reduced levels of FcRIIB 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- and IL-2 mRNA levels in Lyn^–/– cells than in WT mast cells. However, ELISAs showed that Lyn^–/– BMMCs produce higher amounts of TNF- and IL-2 protein than WT cells. These results are consistent with previously published data (5). The discrepancies between mRNA levels and TNF- 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-, TNF-) 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 (MIP1), CCL4 (MIP1), CCL5 (RANTES), CCL7 (MCP-3), and CCL9 (MIP1) 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-B (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 FcRI 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- (36). Furthermore, SHIP negatively regulates IL-6 production in mast cells by inhibiting NF-B activity (36). In turn, Kitaura et al. (37), linked increased AKT activity to increased cytokine (IL-2 and TNF-) gene promoter activities via the activation of NF-B, 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 FcRI 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 FcRI-mediated activation of Lyn^–/– BMMCs results in greater increases in mRNAs encoding proteins in the FcRI 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 FcRI 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.

	Acknowledgments

 
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.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
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.).

² Current address: Respiratory Immunology and Asthma Program, Lovelace Respiratory Research Institute, Albuquerque, NM 87108.

³ Address correspondence and reprint requests to Dr. Janet M. Oliver, Department of Pathology, University of New Mexico, School of Medicine, Cancer Research Facility 201, 2325 Camino De Salud NE, Albuquerque, NM 87131. E-mail address: joliver{at}salud.unm.edu

⁴ Abbreviations used in this paper: BMMC, bone marrow-derived mast cell; WT, wild type; EST, expressed sequence tag.

Received for publication June 24, 2004. Accepted for publication September 29, 2005.

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