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The Journal of Immunology, 2008, 180, 7869-7877
Copyright © 2008 by The American Association of Immunologists, Inc.
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The Tec Family Kinase, IL-2-Inducible T Cell Kinase, Differentially Controls Mast Cell Responses1

Archana S. Iyer*,{dagger} and Avery August2,*,{dagger}

* Center for Molecular Immunology & Infectious Disease and Department of Veterinary & Biomedical Sciences, and {dagger} Immunology & Infectious Disease Graduate Program, Pennsylvania State University, University Park, PA 16802


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Tec family tyrosine kinase, IL-2-inducible T cell kinase (Itk), is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired Th2 cytokine secretion; however, they have increased circulating serum IgE, but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and Fc{epsilon}RI signaling. We report in this study that Itk null mice have reduced allergen/IgE-induced histamine release, as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, because the transfer of Itk null bone marrow-derived cultured mast cells into mast cell-deficient W/Wv animals is able to fully rescue histamine release in the W/Wv mice. Further analysis of Itk null bone marrow-derived cultured mast cells in vitro revealed that whereas they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-{alpha}, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the Fc{epsilon}RI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses; however, these cells express elevated levels of NFAT, which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells and IgE play an important role during allergic responses, including allergen-induced airway hyperreactivity and allergic asthma (1, 2). Cross-linking of IgE bound to high-affinity Fc{epsilon}RI with the cognate Ag releases proinflammatory mediators, which can result in activation and recruitment of other innate inflammatory cells and aid with the development of adaptive immune responses (1, 2, 3, 4, 5). Due to the importance of IgE in allergic asthma, there is significant interest in understanding the interplay of signals downstream of the Fc{epsilon}RI pathway. The allergic airway response follows a typical biphasic course: an early or immediate phase, which appears within minutes of exposure and resolves in 1–2 h (6). This is caused by allergen-induced cross-linking of the IgE molecules bound to Fc{epsilon}RI on mast cells, resulting in the release of preformed inflammatory mediators such as histamine, serotonin, and tryptase (2, 3). These vasoactive amines cause vasodilation, mucous secretion, and bronchoconstriction, which present as immediate airway hyperresponsiveness (AHR).3 The late-phase allergic responses begin 3–4 h after Ag provocation and peak at ~9 h, and the airway obstruction resolves within 12–24 h. T lymphocytes, basophils, and eosinophils are thought to be responsible in inducing the late-phase response, although the role of mast cells may not be ruled out (4, 7).

The Fc{epsilon}RI is a tetrameric receptor composed of an IgE-binding {alpha} subunit and β{gamma}2 signaling subunits. Engagement of Fc{epsilon}RI results in phosphorylation of Src kinase Lyn, followed by subsequent activation of Syk and tyrosine phosphorylation of adapter proteins such as linker for activation of T cells and non-T cell activation linker (8). These adapter proteins act as scaffolds to organize other signaling proteins such as PI3K, Tec kinases, SLP76, Grb2, Gads, and Vav-1, resulting in the activation of phospholipase C (PLC) {gamma}, protein kinase C, and Ras/MAPK pathway, and release of intracellular Ca2+ reserves (8, 9). PI3K plays a critical role in mast cell signaling and is required for the production of phosphoinositides, leading to the recruitment of Tec kinases and PLC{gamma} to the Fc{epsilon}RI-activated signaling complex (9, 10). Thus, the receptor-proximal events are amplified by the sequential activation of Src, Syk, and Tec family of tyrosine kinases and adapter molecules, resulting in a multimolecular signalosome that further activates multiple pathways leading to mast cell degranulation, activation of transcription factors, and cytokine production (8, 10).

The Tec family of protein tyrosine kinases is an important component of this multimolecular signalosome complex. Studies in T cells, B cells, and mast cells have shown that Tec kinases play a role in regulating the calcium pathway (10, 11). Three Tec family kinases, Bruton’s tyrosine kinase (Btk), IL-2-inducible T cell kinase (Itk), and Tec, have been reported to be expressed in mast cells (12, 13). Of these, Btk and Itk have been shown to be activated downstream of the Fc{epsilon}RI pathway (14, 15, 16). Btk and Itk are also major players in BCR and TCR signaling, respectively. Absence of Btk leads to defective B cell development and a reduction in B cell numbers (17, 18, 19, 20, 21, 22). Similarly, absence of Itk in T cells leads to defects in T cell development and function (11). In T cells, Itk is required for Ca2+ mobilization, activation of PLC{gamma}, MAPK, and NFAT family of transcription factors (20, 21, 22, 23). T cells from Itk–/– mice exhibit impaired cytokine secretion and reduced or absent Th2 differentiation (20, 22, 23, 24). This results in Itk–/– mice being significantly resistant toward developing allergic asthma in an OVA/Alum murine model (25, 26).

The importance of Btk in mast cells has been well established through a number of studies by Kawakami and colleagues (14, 16, 27, 28, 29). Btk–/– mice have reduced anaphylaxis, and Btk-deficient cultured mast cells reveal defects in degranulation and cytokine production due to reduced PLC{gamma} activation, Ca2+ mobilization, and activation of the JNK pathway. By contrast, the role of Itk in mast cells is largely unknown. Itk is expressed and phosphorylated upon Fc{epsilon}RI triggering in mast cells (15). Recently, Forssell et al. (30) suggested that Itk–/– mice have defective mast cell degranulation in vivo. They also compared both Btk–/– and Itk–/– mice and found that the absence of Itk led to a more severe defect in mast cell degranulation in vivo than the absence of Btk. However, the use of Itk–/– mice and the OVA/Alum model to assess mast cell function complicates the interpretation of the results given the fact that these mice are known to have a well-established defect in Th2 cell effector functions that affects their response in this model (23, 24, 25, 26). In addition, it should be noted that there is less of a role for mast cells in the OVA/Alum model, with T cells playing a major role in the development of airway infiltration and hyperresponsiveness (31, 32).

In light of these recent findings and to understand the role of Itk in mast cells during allergic asthma (without interfering with T cell functions), we adopted a model to study allergen-induced mast cell-dependent early AHR. We report that Itk-deficient mice have reduced IgE/allergen-induced AHR and histamine release to systemic anaphylaxis. However, Itk–/– mast cells are not defective in degranulation because transfer of Itk–/– bone marrow-derived cultured mast cells (BMMCs) into mast cell-deficient W/Wv mice can rescue the histamine responses to Ag challenge. Our results support the idea that the impaired degranulation observed in vivo in Itk–/– mice is due to high levels of IgE observed in these mice. Further investigation of Fc{epsilon}RI signaling in Itk–/– BMMCs revealed that whereas they exhibit similar levels of degranulation, they secrete elevated levels of cytokines, including IL-13 and TNF-{alpha}. This split response in degranulation and cytokine secretion suggests that Itk may differentially regulate Fc{epsilon}RI signals in mast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Wild-type (WT) C57BL/6, WBB6F1/J-KitW/KitW-v (W/Wv), congenic WBB6F1, and B6.Cg-KitW-sh mice were obtained from The Jackson Laboratory. Itk–/– mice backcrossed to C57BL/6 background for greater than 10 generations were used for these experiments. Mice were kept in microisolater cages and provided with food and water ad libitum. All experiments were approved by the Office of Research Protection’s Institutional Animal Care and Use Committee at Pennsylvania State University.

Determination of AHR

Airway responsiveness was assessed using a Buxco whole body plethysmograph (Buxco Electronics). Five- to 8-wk-old WT and Itk–/– mice were injected i.p. with 1 µg of murine IgE anti-DNP (SPE-7; Sigma-Aldrich), 12 h before the intranasal administration of 50 µl of 1 mg/ml, DNP conjugated to human serum albumin (DNP-HSA; Sigma-Aldrich), or PBS alone. Conscious mice were placed into a chamber of the plethysmograph, and respiratory parameters in response to 10 mg/ml methacholine (Sigma-Aldrich) were measured for up to 1 h after Ag exposure at 10-min intervals. Airflow obstruction was expressed as enhanced pause.

Derivation and characterization of BMMCs

Bone marrow cells were obtained by flushing the femur and tibia bones of 6- to 10-wk-old mice, and cultured in RPMI 1640 medium supplemented with 10% FCS, 100 µM nonessential amino acids, 50 µM 2-ME, and murine rIL-3 (gift from S. Pullen, Boehringer Ingleheim Pharmaceuticals, Ridgefield, CT). Cells were passaged twice every week by replating the cells in fresh medium. After 4–5 wk of culture, >95% of the trypan blue-excluding viable cells were mast cells, as evidenced by high-affinity Fc{epsilon}RI expression by FACS analysis. To assess high-affinity Fc{epsilon}RI expression, cells were incubated with anti-mouse Fc{epsilon}RI PE (eBiosciences) and anti-c-Kit FITC. IgE binding to Fc{epsilon}RI was detected by incubating BMMCs with 1 µg/ml murine IgE for 1 h on ice, followed by one wash with PBS and incubation with anti-IgE PE (eBiosciences). The cells were then analyzed by flow cytometry.

Histamine analysis

WT and Itk–/– mice were injected with 1 µg of anti-DNP IgE i.p., 12 h before the i.v. administration of DNP-HSA (50 µg). Three minutes later, animals were sacrificed, and serum was collected and analyzed for histamine content using an ELISA (Beckman Coulter). Cultured BMMCs from WT and Itk–/– (5 x 106) were transferred i.v. into mast cell-deficient W/Wv mice, and after 9 wk the mice were analyzed for systemic anaphylaxis, as described above.

Analysis of degranulation in vitro

WT and Itk–/– BMMCs (1 x 106/ml) were factor starved overnight and sensitized in complete RPMI 1640 with 1 µg/ml anti-DNP IgE overnight. Cells were washed in Tyrode buffer (112 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.6 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (pH 7.5), 0.05% gelatin, and 0.1% glucose) and resuspended at 2 x 106/ml. BMMCs were stimulated with varying amounts of DNP-HSA (0–100 ng/ml) for 1 h at 37°C. For the hexosaminidase release assay, aliquots of supernatant and lysates were incubated with p-nitrophenyl-N-acetyl-β-D-glucosamide (Sigma-Aldrich). The reaction was terminated by the addition of Na2CO3-NAHCO3 buffer, and the OD was measured on a plate reader at 405 nm. Degranulation was expressed as percentage of release of total hexosaminidase.

Analysis of skin mast cells

Skin sections from WT and Itk–/– mice were fixed overnight in 2% glutaraldehyde in 0.1 M cacodylate buffer. Samples were treated with 1% osmium tetraoxide in 0.1 M cacodylate buffer and 2% uranyl acetate and infiltrated with eponate resin. Ultrathin sections of the samples were placed on uncoated copper grid, treated with uranyl acetate and lead citrate, and analyzed with a transmission electron microscope at the Electron Microscope Facility of Pennsylvania State University. For toluidine blue staining, skin samples were stained with 0.1% toluidine blue for 1–2 min, washed, and observed under an Olympus BX51 microscope.

Analysis of surface IgE expression on mast cells in vivo

Splenocytes or peritoneal cells were stained with anti-c-Kit and anti-IgE (to detect surface-bound IgE and thus Fc{epsilon}R). Mast cells were identified by gating and c-Kit/IgE reactivity. To assess complete occupancy of IgE on mast cells, peritoneal cells were incubated with excess of IgE for 1 h. Samples were washed and stained with anti-c-Kit and anti-IgE before and after the treatment.

Analysis of B cells

Spleens were collected from WT and Itk–/– mice and analyzed by flow cytometry for expression of B220, IgM, and IgE using specific Abs.

Analysis of IgE levels

Dilutions of sera were analyzed for total IgE by IgE-specific ELISA (Southern Biotechnology Associates).

Cytokine analysis

BMMCs were factor starved and sensitized by overnight incubation with 1 µg/ml anti-DNP IgE mAb (SPE-7; Sigma-Aldrich). Cells were washed once in Tyrode buffer, resuspended in complete medium to 2 x 106 cells/ml. Cells were stimulated with DNP-HSA and supernatants collected after 8 h for TNF-{alpha}, or 24 h for IL-13 and analyzed by ELISA (R&D Systems). For analysis of stimulation with IgE alone, cells were treated as indicated above, and supernatants were collected after overnight incubation. IL-2, IL-4, TNF-{alpha}, and GM-CSF were analyzed using a Luminex multiplex system (Bio-Rad).

Calcium analysis

BMMCs were sensitized by an overnight incubation with 1 µg/ml anti-DNP IgE, washed in Tyrode buffer, and loaded with 2 µM fura 2-AM (Invitrogen). Bulk intracellular calcium levels were monitored with a Hitachi F-2000 spectrofluorimeter. Briefly, the fluorescence emission at 510 nm was recorded at excitation wavelengths of 340 and 380 nm. Intracellular Ca2+ was calculated from the 340/380 ratio, for the fura 2-Ca2+ complex. Analysis at baseline was acquired for 50–100 s before Fc{epsilon}RI cross-linking with 0–100 ng/ml DNP-HSA. Calcium analysis was continued for 300 s, followed by addition of 1 µM ionomycin (Sigma-Aldrich) to determine the peak population response using an additional 60-s data acquisition. Extracellular calcium response was determined by stimulating BMMCs with DNP-HSA in the absence of extracellular calcium at 50 s, followed by the addition of 2 mM Ca2+ at 100 s and 1 µM ionomycin at 400 s.

In vitro Fc{epsilon}RI stimulation

BMMCs were factor starved and sensitized by overnight incubation with 1 µg/ml anti-DNP IgE. Cells were washed once in Tyrode buffer, resuspended (2 x 106 cells/ml), and stimulated with Ag (100 ng/ml DNP-HSA) for the indicated time intervals. Cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Igepal CA-630, 50 mM Na pyrophosphate, 2 mM sodium vanadate, 50 mM sodium fluoride, 1 mM PMSF, 1 µg/ml leupeptin, and 2 µg/ml aprotinin) immediately after stimulation. Lysates were centrifuged at 4°C for 10 min. Cleared lysates were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (NEN Life Science Products). Membranes were blocked and incubated consecutively with primary Ab and HRP-conjugated secondary Ab, and immunoreactive proteins were visualized by ECL reagents (Amersham). The following Abs were used: anti-phospho-ERK, anti-ERK, anti-phospho-p38, and anti-p38 (Cell Signaling Technology); anti-phosphotyrosine (Santa Cruz Biotechnology); anti-actin (Sigma-Aldrich); and anti-NFAT1/NFATc2 and NFAT2/NFATc1 (Santa Cruz Biotechnology).

Real-time PCR analysis

Total RNA was extracted from BMMCs using TRIzol (Invitrogen), and cDNA was synthesized. Quantitative RT-PCR was performed in triplicate using primer sets specific to NFATs 1–2, with GAPDH used as a housekeeping gene (Applied Biosystems). Data were analyzed using the comparative threshold cycle method and normalized to GAPDH. The relative gene expression levels were then determined by comparing to the expression found in the WT BMMCs set at 1.

Confocal staining

BMMCs were fixed with 4% paraformaldehyde before cytospin on lysine-coated slides. Cells were permeabilized in a buffer containing 0.1% Triton X-100 and 2% FBS in PBS for 30 min and stained with anti-NFAT1/NFATc2 and TOPRO-3 (Molecular Probes). Samples were visualized using confocal microscopy Olympus FV300.

Data analysis

Statistical evaluation was conducted using Student’s t test with a probability value, p ≤ 0.05, considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Itk–/– mice exhibit reduced responses to allergen-induced mast cell-dependent early AHR

We adopted a model of passive sensitization with Ag-specific IgE to monitor early AHR after the first exposure of allergen (33). In this model, mice are injected with Ag-specific IgE (anti-DNP IgE) and then administered the allergen (DNP-HSA) intranasally. This should lead to activation of mast cells in the airways, resulting in altered airway responsiveness to methacholine. Mice were exposed to an aerosol dose of methacholine at 10-min intervals after Ag challenge over a 1-h time period, and AHR was determined using a noninvasive whole body plethysmograph. The results show that WT mice respond to allergen exposure with an increase in AHR over time compared with control mice exposed to PBS alone (Fig. 1A). In addition, mast cell-deficient mice (KitW-sh mice) did not exhibit any increase in AHR upon exposure to Ag, confirming that the AHR response in this model is indeed mast cell dependent (data not shown).


Figure 1
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  		FIGURE 1. Reduced allergen/IgE-induced AHR and histamine response in Itk–/– mice. A, Anti-DNP-IgE was injected into WT mice, and 24 h later, mice were challenged intranasally with PBS or DNP-HSA. AHR was then determined every 10 min in response to 10 mM methacholine. {blacksquare}, PBS-challenged mice; {blacktriangleup}, DNP-HSA-challenged mice (n = 4–10; *, p < 0.05 vs PBS). B, Anti-DNP-IgE was injected into Itk–/– mice, and 24 h later, mice were challenged intranasally with PBS or DNP-HSA and AHR was determined. {square}, PBS-challenged mice; {triangleup}, DNP-HSA-challenged mice (n = 3–7). C, Anti-DNP-IgE was injected into WT or Itk–/– mice, and 24 h later, the mice were challenged i.v. with DNP-HSA. Animals were sacrificed 3 min later, and sera were collected and assayed for histamine content by ELISA (n = 8; *, p < 0.05 vs WT).

 
To understand the role of Itk in this mast cell-dependent physiological response during early AHR, we examined Itk–/– mice in this model (Fig. 1B). We found that Itk–/– mice did not exhibit any changes in AHR upon Ag exposure, suggesting that Itk–/– mice fail to generate an acute airway response despite the presence of circulating Ag-specific IgE.

Reduced histamine release in Itk–/– mice in response to Ag challenge

The reduced AHR observed in Itk–/– mice to intranasal challenge of Ag suggested that Itk may regulate the release of pharmacological mediators from mast cells in response to Fc{epsilon}RI triggering. Because histamine is a major pharmacological mediator secreted by mast cells, we determined histamine release in WT and Itk–/– mice following injection of anti-DNP IgE and systemic Ag challenge i.v. Fig. 1C demonstrates that whereas WT mice respond to Ag challenge with increased histamine levels in sera, Itk–/– had significantly reduced levels of histamine following Ag challenge. This result supports the observation of Forssell et al. (30) that Itk–/– mice exhibit reduced mast cell degranulation in vivo.

Itk–/– mast cells have normal tissue mast cells, but higher surface expression of Fc{epsilon}R

Examination of Itk–/– mice for mast cell numbers and morphology in skin, peritoneum, and lungs by toluidine blue and ultrastructural morphology by transmission electron microscopy (data shown for skin in Fig. 2, A–C) revealed no difference in mast cell distribution (WT = 40/mm2 vs Itk–/– = 38/mm2, p = 0.239, not statistically significant) within the tissue or ultrastructural morphology. Similar analysis of dissociated lungs from WT and Itk–/– mice suggested normal numbers of mast cells (data not shown). To determine the expression of Fc{epsilon}RI on the mast cells in Itk–/– mice, we analyzed the levels of IgE occupied Fc{epsilon}RI on peritoneal mast cells (Fig. 2D) with similar results observed in splenic mast cells (data not shown). Mast cells from Itk–/– mice were found to have higher levels of IgE occupying the Fc{epsilon}R than mast cells from WT mice, suggesting higher levels of Fc{epsilon}R on these cells. These results rule out a role for Itk in mast cell development, or that the lack of response in the Itk–/– mice was due to reduced mast cells or Fc{epsilon}R expression.


Figure 2
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  		FIGURE 2. Normal tissue mast cells, but increased expression of Fc{epsilon}R in Itk–/– mast cells. A, Sections of skin from WT and Itk–/– mice analyzed by toluidine blue staining. Arrows indicate mast cells, with the epidermis at the top of the figure (x20). B, Sections of skin from WT and Itk–/– mice analyzed by toluidine blue staining (x60). Inset, Amplified view of toluidine blue-stained mast cells. C, Electron microscope analysis of skin mast cells from WT and Itk–/– mice (representative images from at least three mice each). D, Fc{epsilon}R expression on peritoneal mast cells from 6- to 8-wk-old WT (shaded lines) or Itk–/– mice (open lines) (repeated at least four times).

 
Itk–/– mice have increased levels of serum IgE leading to almost complete occupancy of IgE on Fc{epsilon}R in mast cells

We and others have noted that Itk–/– mice have increased levels of serum IgE, and that these mice can mount a normal Ag-specific IgE response in the OVA/Alum-induced model of allergic asthma (Fig. 3A) (24, 25, 30). Indeed, analysis of B cells in the spleen of Itk–/– mice for surface expression of IgM and IgE revealed that a large percentage of the B cells has undergone class switch to express IgE (IgE+/IgM–, WT = 1.01% vs Itk = 35.28, p < 0.05; Fig. 3B shows a representative analysis). Although the reason for this increased IgE level is unknown, it has been well documented that IgE binding to Fc{epsilon}RI enhances mast cell Fc{epsilon}RI expression both in vivo and in vitro (34, 35, 36). This might explain why we observe higher levels of IgE on the cell surface of Itk–/– mast cells in Fig. 2D. It also suggests that the high-level occupancy of Fc{epsilon}RI by the IgE molecules in the Itk–/– mast cells may preclude the newly delivered Ag-specific IgE from binding to the cell surface. To test this possibility, we analyzed Fc{epsilon}R occupancy on peritoneal mast cells from WT and Itk–/– mice before and after incubation with an excess of IgE in vitro. This should allow us to determine whether the Fc{epsilon}RIs on Itk–/– mast cells are occupied/saturated with IgE and thus are unavailable for binding to newly delivered Ag-specific IgE, provided to them in the experiments shown in Fig. 1. The results demonstrate that whereas WT peritoneal mast cells showed an increase in IgE binding after incubation, there was no difference in Itk–/– peritoneal cells (Fig. 3C). These data support the idea that in Itk–/– mice, the Fc{epsilon}RIs on mast cells are saturated with IgE, precluding the binding of new IgE and thereby reducing their responses to Ag-specific allergen challenge in vivo.


Figure 3
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  		FIGURE 3. Itk–/– mice have higher class switch to IgE and increased occupancy of the Fc{epsilon}R on their mast cells. A, Total serum IgE levels from WT and Itk–/– analyzed by ELISA (n = 6; *, p < 0.05). B, Splenic B cells from 6- to 8-wk-old WT (left) or Itk–/– mice (right) were analyzed for IgE/IgM expression by gating on B220+ cells (repeated at least three times). C, IgE binding to Fc{epsilon}RI on peritoneal mast cells from 6- to 8-wk-old WT mice (left) and Itk–/– mice (right) before (control, shaded lines) and after incubation with IgE Ab (IgE, open lines) (repeated twice).

 
Adoptive transfer of Itk–/– BMMCs into W/Wv mice rescued mast cell degranulation in these mice

The interaction of IgE with Fc{epsilon}RI is very stable (35, 36, 37). Indeed, Kubo et al. (36) have shown that IgE can be maintained on the surface of mast cells for several weeks. Because the high levels of IgE in Itk–/– may interfere with mast cell responses in vivo, we cultured BMMCs from WT and Itk–/– mice for further analysis. Examination of these cells for expression of Fc{epsilon}RI did not reveal any difference in IgE binding or Fc{epsilon}RI expression (Fig. 4, A and B), suggesting that BMMCs from Itk–/– develop normally. To analyze these cells in an in vivo context devoid of extraneous IgE, we transferred WT and Itk–/– BMMCs into mast cell-deficient W/Wv mice (38, 39). Nine weeks posttransfer (to allow for complete reconstitution of mast cells), we challenged the mice with Ag-specific IgE (anti-DNP IgE) and analyzed histamine release upon systemic Ag challenge with DNP-HSA. In contrast to our results with the Itk–/– mice, we found that Itk–/– BMMCs in W/Wv mice were fully able to respond and release histamine similar to WT BMMCs (Fig. 4C). These data indicate that Itk–/– BMMCs are capable of responding to IgE/Fc{epsilon}RI triggering in vivo, and that the high levels of IgE seen in these mice may be interfering with the passive sensitization process, leading to the observed apparent reduction in mast cell responses.


Figure 4
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  		FIGURE 4. The defect in histamine secretion in Itk–/– mice is rescued by transfer of Itk–/– BMMCs into W/Wv mice. A, BMMCs were expanded from bone marrow of WT and Itk–/– animals, as described in Materials and Methods. Surface expression of the high-affinity Fc{epsilon}R was then determined by IgE binding and flow cytometry. Open lines, control staining in the absence of addition of IgE; filled lines, staining in the presence of IgE. B, Surface expression of the high-affinity Fc{epsilon}R determined by flow cytometric analysis using anti-Fc{epsilon}RI Abs. C, WT and Itk–/– BMMCs were transferred into W/Wv mice and allowed to reconstitute for 9 wk. Histamine release was analyzed as described in Fig. 1 (n = 3; *, p < 0.05).

 
Itk–/– BMMCs have normal degranulation, but enhanced cytokine secretion

We next analyzed these BMMCs for their responses to Ag-Fc{epsilon}RI triggering in vitro. Itk–/– BMMCs responded to Fc{epsilon}RI stimulation with similar levels of degranulation to WT BMMCs (Fig. 5A), confirming the in vivo data with the WWv mice that degranulation is not affected in the absence of Itk. Ag-IgE stimulation of mast cells results in secretion of a number of cytokines, including TNF-{alpha} and IL-13 (2, 34, 40). To determine whether cytokine secretion is altered in the absence of Itk, BMMCs were incubated overnight with IgE and then stimulated with Ag, and the supernatants were analyzed for IL-13 and TNF-{alpha}. The results show that Itk–/– BMMCs secreted more IL-13 and TNF-{alpha} than WT BMMCs (Fig. 5B). We also compared the cytokine responses upon stimulation with Ag to that seen upon stimulation with anti-IgE. In both cases, the Itk–/– BMMCs secreted more IL-13 and TNF-{alpha} compared with the WT BMMCs (Fig. 5C).


Figure 5
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  		FIGURE 5. Normal degranulation response, but enhanced cytokine secretion by Itk–/– BMMCs in response to Fc{epsilon}RI triggering. A, WT or Itk–/– BMMCs were left untreated (control) or coated with anti-DNP IgE (IgE), followed by stimulation with increasing doses of DNP-HSA. Supernatants were analyzed for β-hexosaminidase as a measure of degranulation. Results are expressed as a percentage of total cellular β-hexosaminidase (n = 3; *, p < 0.05 vs WT). B and C, WT or Itk–/– BMMCs were left untreated (control), or coated with anti-DNP IgE (IgE), followed by stimulation with 100 ng/ml DNP-HSA (B and C) or anti-IgE (C). Supernatants were analyzed by ELISA for IL-13 (n = 3; *, p < 0.05 vs WT) or TNF-{alpha} (n = 3; *, p < 0.05 vs WT). D, WT or Itk–/– BMMCs were incubated overnight with anti-DNP IgE alone, and supernatants were analyzed for the indicated cytokines (n = 3; *, p < 0.05 vs WT). E, Fc{epsilon}RI expression on BMMCs after overnight incubation with IgE.

 
Several studies have indicated that IgE/Fc{epsilon}RI interaction on mast cells in the absence of Ag can also result in the production of cytokines (41, 42, 43, 44, 45). We therefore examined cytokine secretion under conditions in which the BMMCs were exposed to IgE alone in the absence of Ag, and analyzed the supernatants for a number of cytokines. We found that Itk–/– BMMCs secreted elevated levels of IL-2, IL-4, TNF-{alpha}, and GM-CSF, whereas these cytokines were not detected from WT BMMCs under these conditions (Fig. 5D). These data indicate that unliganded IgE can trigger Itk–/– BMMCs for cytokine secretion, which is more pronounced than that seen in WT BMMCs. Unliganded IgE alone can also cause increased expression of Fc{epsilon}RI on BMMCs, and mast cells that have undergone this process are more sensitive to increased cytokine production and degranulation in response to sensitization, in part due to the elevated levels of Fc{epsilon}RI (45). We therefore examined the expression of the Fc{epsilon}RI on the surface of the BMMCs after incubation with anti-IgE-DNP overnight. There was no difference in the expression of Fc{epsilon}RI between WT and Itk–/– BMMCs under these conditions, ruling out the possibility that increased expression of Fc{epsilon}RI might contribute to increased sensitivity (Fig. 5E). These observations indicate that the absence of Itk makes BMMCs more responsive to IgE/Fc{epsilon}R-stimulated cytokine secretion, suggesting that Itk may play a negative role in modulating cytokine responses through the Fc{epsilon}RI on mast cells.

Fc{epsilon}RI-mediated signaling in Itk–/– BMMCs

To determine the molecular basis for this response, we analyzed select biochemical responses to Fc{epsilon}RI triggering in BMMCs. Itk-deficient T cells have reduced calcium mobilization upon TCR stimulation (20), and so we analyzed WT and Itk–/– BMMCs for the calcium response to Fc{epsilon}RI triggering at various concentrations of Ag DNP-HSA (5, 10, and 100 ng/ml) (Fig. 6A). Our data show that Itk–/– BMMCs exhibit slightly higher calcium response than WT BMMCs upon stimulation with various concentrations of Ag. However, analysis of peak calcium responses revealed no significant differences in this parameter (Fig. 6B). In addition, we also analyzed extracellular calcium entry and found that there was no significant difference between the WT and Itk–/– BMMCs (Fig. 6C). These data suggest that contrary to T cells, in mast cells Itk is not essential for calcium response through the Fc{epsilon}RI.


Figure 6
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  		FIGURE 6. Calcium responses to Fc{epsilon}RI triggering in Itk–/– BMMCs. A, WT or Itk–/– BMMCs were loaded with anti-DNP IgE overnight, loaded with fura 2, and then stimulated with the indicated concentration of DNP-HSA, and calcium responses were analyzed. Data expressed as fold increase compared with baseline signals. B, Peak fold increase in calcium response of two to four experiments was averaged and plotted vs concentration of antigenic stimulation (A and B are representative of at least 15 experiments). C, WT or Itk–/– BMMCs were coated with anti-DNP IgE overnight, loaded with fura 2, then stimulated with 100 ng/ml DNP-HSA in the absence of extracellular calcium at 50 s; 2 mM calcium was added at 100 s, and ionomycin was added as positive control at 400 s.

 
Analysis of Fc{epsilon}RI-triggered tyrosine phosphorylation of cellular proteins revealed that BMMCs from WT and Itk–/– showed similar overall activation pattern (Fig. 7A). Further analysis of the Erk/MAPK and p38 MAPK pathways also revealed no difference in the activation of these kinases (Fig. 7, B and C). Thus, the absence of Itk does not grossly affect signaling pathways downstream of the Fc{epsilon}RI.


Figure 7
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  		FIGURE 7. Fc{epsilon}RI-mediated signaling in Itk–/– BMMCs. WT or Itk–/– BMMCs were loaded with anti-DNP IgE overnight, then stimulated with 100 ng/ml DNP-HSA for the indicated time periods. Cells were then lysed, and total cell lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with anti-phosphotyrosine Abs (A), anti-phospho-ERK (B), or anti-phospho-p38 (C) (experiments repeated at least three times).

 
Elevated NFAT expression and activation in Itk–/– BMMCs

The NFAT family of transcription factors plays a critical role in inducing cytokine synthesis. Indeed, NFATs 1 and 2 (NFATc2 and c1) play important roles in mast cell cytokine production of IL-13 and TNF-{alpha} triggered by the Fc{epsilon}RI (40, 46, 47). We therefore analyzed mRNA levels of various NFAT isoforms in the WT and Itk–/– BMMCs by quantitative RT-PCR. We found that Itk–/– BMMCs have higher levels of NFAT1/NFATc2 and NFAT2/NFATc1 compared with WT BMMCs (Fig. 8A, top). Analysis of NFAT2/NFATc1 expression by Western blot revealed that Itk–/– BMMCs express higher levels of this transcription factor (Fig. 8A, bottom). To determine whether the NFAT in Itk–/– BMMCs was activated, we examined WT and Itk–/– BMMCs for NFAT nuclear localization. We found that in unstimulated WT cells, NFAT1/NFATc2 was largely cytoplasmic, whereas in Itk–/– cells, there was significant nuclear localization of NFAT (Fig. 8B). These data suggest that the observed higher levels of cytokine secretion in the absence of Itk may be due to elevated NFAT expression and activation.


Figure 8
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  		FIGURE 8. Increased NFAT expression and activation in Itk–/– BMMCs. A, Top, mRNA from WT or Itk–/– BMMCs was analyzed for the expression of the indicated NFAT isoforms by quantitative RT-PCR (n = 3; experiment done on two separate batches of BMMCs; *, p < 0.05 vs WT). Bottom, Nonstimulated WT or Itk–/– BMMCs were analyzed for expression of NFAT2/NFATc1 by Western blot. B, Nonstimulated WT or Itk–/– BMMCs were analyzed for localization of NFAT1/NFATc2 by confocal microscopy (green = NFAT1; red = DNA) (repeated twice).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we used a mast cell-specific early airway responsiveness model and found that Itk–/– mice exhibit reduced AHR and histamine secretion in response to Ag challenge. However, these initial results are in agreement with the published report by Forssell et al. (30), suggesting that Itk–/– mice may be defective in mast cell degranulation following passive sensitization. Our data reported in this study have determined that Itk–/– mast cells are unable to respond in vivo due to saturating levels of IgE occupancy on Fc{epsilon}RI, because transferring Itk–/– BMMCs into mast cell-deficient W/Wv mice resulted in normal levels of histamine release. In addition, in vitro experiments using different concentrations of Ag reveal no difference in degranulation between WT and Itk–/– BMMCs. These experiments strongly suggest that Itk may not be essential for Fc{epsilon}RI-induced degranulation. Previously, Forssell et al. (30) compared WT, Btk–/–, and Itk–/– mice using a mast cell-dependent passive cutaneous sensitization model, and found reduced mast cell degranulation in Itk–/– mice, suggesting that in vivo, Itk deficiency is more detrimental to mast cell degranulation than Btk deficiency. Itk–/– mice have elevated levels of IgE, and the binding of IgE to Fc{epsilon}RI on mast cells is very stable interaction and lasts for several weeks (25, 36). Our observation of elevated Fc{epsilon}RI occupancy on Itk-deficient mast cells in vivo thus supports the idea that the Itk-deficient mast cells have reduced degranulation in vivo because the Fc{epsilon}RIs are already occupied/loaded with saturating levels of IgE, which interferes with the binding of newly synthesized IgE or passive administration of IgE.

Analysis of the role of Btk in mast cells using BMMCs has shown that the absence of Btk leads to reduced calcium responses to Fc{epsilon}RI triggering in mast cells (28, 29, 48, 49). We do not observe any difference in both intracellular calcium response and extracellular calcium influx in the Itk–/– BMMCs, suggesting that Btk, but not Itk, is critical for this process in mast cells. Further biochemical analysis showed that the absence of Itk leads to normal ERK and p38 MAPK activation, suggesting that contrary to T cell signaling, the activation of these pathways may be independent of Itk in mast cells (20, 21, 29, 49).

Interestingly, we find increased cytokine secretion of TNF-{alpha} and IL-13 by Itk–/– BMMCs, suggesting that Itk may play a negative role in Fc{epsilon}RI regulation of these cytokines. The absence of Btk has been reported to cause defective or reduced histamine release as well as secretion of cytokines like TNF-{alpha}, IL-6, and GM-CSF. We find increased expression as well as nuclear localization of NFAT in the Itk–/– BMMCs, suggesting a mechanism for the observed increase in cytokine secretion. It is well established that NFAT is a major transcription factor that regulates the expression of TNF-{alpha} and IL-13 (40, 46, 50). Two isoforms of NFAT, NFAT1/NFATc2 and NFAT2/NFATc1, have been shown to regulate transcription of TNF-{alpha} and IL-13 in mast cells both individually or in combination, and mast cells deficient in NFAT1/NFATc2 and NFAT2/NFATc1 have been shown to be severely impaired in the production of TNF-{alpha} and IL-13 (47). These data support the idea that increase in NFAT levels may contribute to a mechanism for increased cytokine.

It should be noted that retroviral mediated expression of Itk into Btk-deficient mast cells has been shown to partially rescue histamine, TNF-{alpha}, IL-6, and GM-CSF secretion in these cells (16). We confirmed that Itk–/– mast cells express equivalent levels of Btk to WT mast cells (data not shown). These data therefore raise the question as to whether the two Tec family kinases differentially regulate signaling pathways in response to Fc{epsilon}RI triggering in mast cells. Altogether, these experiments indicate that Btk may play a more important role in amplifying signals from Fc{epsilon}RI in mast cells; however, the increased cytokine response in Itk–/– BMMCs implies that Itk may have a role in either enhancing or regulating the Fc{epsilon}RI signaling. Alternatively, because both Itk and Btk share similar substrates, it is possible that in the absence of Itk, Btk has less competition for substrates and is therefore more effective at signaling, leading to elevated cytokine secretion.

Our data also suggest that Itk–/– mice behave in part similar to mice lacking the Src kinase Lyn with regard to mast cell responses. Lyn was originally reported to be a positive regulator of mast cells because Lyn–/– mice were hyporesponsive to Fc{epsilon}RI stimuli in vivo (51, 52). However, it was subsequently found that Lyn–/– mice have high levels of IgE, and that Lyn–/– BMMCs were hyperresponsive to Fc{epsilon}RI triggering (53, 54). It is now well established that Lyn regulates Fc{epsilon}RI signaling both positively and negatively, by activating Syk, and recruiting the phosphatase SHIP, respectively (55).

In summary, our data indicate that whereas Itk is expressed in both T cells and mast cells, it functions differentially in these cells. In T cells, Itk–/– T cells exhibit defects in calcium increase and ERK/MAPK activation. Itk is also critical for T cell development, activation, and cytokine production, particularly IL-2 and Th2, such as cytokines IL-4 (11). By contrast, we have shown that Itk does not affect these pathways in mast cells, but may play a negative role in the production of cytokines in these cells, without affecting the ability of these cells to degranulate. It is possible that Itk plays a more critical role in T cell function due to levels of expression in these cells, whereas in mast cells, it may play a more specialized role. Alternatively, Itk may play negative roles for cytokine production in both cell types, but this role is more dominantly revealed in mast cells, where the absence of Itk results in deregulation of transcription factors such as NFAT, which set the stage for deregulated expression of these cytokines. These results also suggest that signaling pathways regulated by Itk have cell type-specific functions.


   Acknowledgments
 
We thank members of the Center for Molecular Immunology & Infectious Disease at Pennsylvania State University for helpful comments, and E. Kunze, N. Bem, S. Magargee, Dr. D. Grove, and M. Potter for technical assistance. We also thank Dr. G. Ning and the Electron Microscope Facility at Pennsylvania State University for help with electron microscope sections.


   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 Grants AI051626 and AI065566 to A.A. The Center for Molecular Immunology & Infectious Disease is supported in part by a grant from the Pennsylvania Department of Health. Back

2 Address correspondence and reprint requests to Dr. Avery August, Center for Molecular Immunology & Infectious Disease, Department of Veterinary & Biomedical Sciences, Pennsylvania State University, 115 Henning Building, University Park, PA 16802. E-mail address: axa45{at}psu.edu Back

3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; BMMCs, bone marrow-derived cultured mast cells; Btk, Bruton’s tyrosine kinase; HSA, human serum albumin; Itk, IL-2-inducible T cell kinase; PLC, phospholipase C; WT, wild type. Back

Received for publication September 25, 2007. Accepted for publication April 7, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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