Impaired signaling via the high-affinity IgE receptor in Wiskott–Aldrich syndrome protein-deficient mast cells

Vadim I. Pivniouk1,5, Scott B. Snapper2, Alexander Kettner1, Harri Alenius1, Dhafer Laouini1, Hervé Falet3, John Hartwig3, Frederick W. Alt4 and Raif S. Geha1

1 Division of Immunology, Children’s Hospital, 2 Division of Gastroenterology, Massachusetts General Hospital, 3 Division of Hematology, Brigham and Women’s Hospital, and 4 Howard Hughes Medical Institute, Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA 5 Present address: Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724-5030, USA

Correspondence to: R. S. Geha; E-mail: raif.geha{at}tch.harvard.edu
Transmitting editor: S. J. Galli


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Wiskott–Aldrich syndrome protein (WASP) is the product of the gene deficient in boys with X-linked Wiskott–Aldrich syndrome. We assessed the role of WASP in signaling through the high-affinity IgE receptor (Fc{epsilon}RI) using WASP-deficient mice. IgE-dependent degranulation and cytokine secretion were markedly diminished in bone marrow-derived mast cells from WASP-deficient mice. Upstream signaling events that include Fc{epsilon}RI-triggered total protein tyrosine phosphorylation, and protein tyrosine phosphorylation of Fc{epsilon}RIß and Syk were not affected by WASP deficiency. However, tyrosine phosphorylation of phospholipase C{gamma} and Ca2+ mobilization were diminished. IgE-dependent activation of c-Jun N-terminal kinase, cell spreading and redistribution of cellular F-actin in mast cells were reduced in the absence of WASP. We conclude that WASP regulates Fc{epsilon}RI-mediated granule exocytosis, cytokine production and cytoskeletal changes in mast cells.

Keywords: allergy and immunology, cell degranulation, receptor-mediated signal transduction, Wiskott–Aldrich syndrome


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Allergic reactions are mediated by products released from mast cells and basophils following ligation of their high-affinity receptor for IgE (Fc{epsilon}RI) by allergen. These products include preformed mediators stored in mast cell granules as well as mediators and cytokines that are synthesized and released following Fc{epsilon}RI ligation. Fc{epsilon}RI expressed on mast cells is comprised of an IgE-binding {alpha} chain and two chains, ß and {gamma}, which function as signal-transducing subunits. Both of these chains contain an immunoreceptor tyrosine-based activation motif (ITAM) with paired tyrosine residues. The cross-linking of IgE bound to Fc{epsilon}RI with multivalent antigen (allergen) results in activation of Src family protein tyrosine kinase Lyn (1), and in phosphorylation of the tyrosines within ITAM motifs of Fc{epsilon}RIß and Fc{epsilon}RI{gamma} chains (2). This creates docking sites for the protein tyrosine kinase Syk and causes its activation. Lyn and Syk phosphorylate a number of targets that include phospholipase C (PLC){gamma}, linker for activation of T cells (LAT), the adaptor protein SLP-76, Vav-1 and Btk (26). PLC{gamma} activation generates inositol triphosphate (IP3) and diacylglycerol, which play a critical role in Ca2+ mobilization and in activation of protein kinase C, respectively. Fc{epsilon}RI cross-linking results in the activation of mitogen-activated protein kinase (MAPK) pathways that include extracellular signal regulated kinase, c-Jun N-terminal kinase (JNK) and p38 MAPK (79). JNK activates transcription factors such as c-Jun that regulate the expression of cytokine genes that include IL-6 and tumor necrosis factor (TNF)-{alpha} (10).

Study of mice with targeted gene mutations has demonstrated that mast cell degranulation and cytokine production are deficient in Syk–/– (11), btk–/– (3), SLP-76–/– (12), LAT–/– (13) and Vav-1–/– mice (14). A candidate link between these signaling molecules and downstream events that include actin cytoskeleton reorganization is the Wiskott–Aldrich syndrome protein (WASP). WASP is the product of the gene deficient in boys with X-linked Wiskott–Aldrich syndrome and is expressed in hematopoietic cells (15). WASP possesses a WASP homology domain 1, a Cdc42/Rac GTPase-binding domain, a proline-rich domain, a G-actin-binding verprolin homology domain, a cofilin homology domain and a C-terminal acidic segment that can interact with the actin polymerizing complex Arp2/3 (16,17). WASP may be linked to SLP-76 by the adaptor protein Nck (18) and by Vav-1 by virtue of its ability to bind Cdc42. WASP is thought to play an important role in linking cell-surface antigen receptor signaling and actin cytoskeleton reorganization (17). WASP is expressed in mast cells (19), and its phosphorylation increases after Fc{epsilon}RI cross-linking and is facilitated by activated Cdc42 (19).

WASP-deficient T cells proliferate poorly and are deficient in cap formation following TCR cross-linking (2022). The role of WASP in mast cell development and activation remains unknown. We have used mast cells derived from WASP-deficient mice to examine the role of WASP in Fc{epsilon}RI signaling. We show that degranulation, JNK activation, cytokine production and spreading on IgE-coated surfaces following Fc{epsilon}RI ligation are deficient in WASP–/– mast cells. These findings suggest that WASP plays an important role in linking Fc{epsilon}RI to granule exocytosis, JNK activation, cytokine secretion and the actin cytoskeleton.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The generation of WASP–/– mice has been described (21). WASP–/– mice were backcrossed onto the 129Sv background for five generations. WASP+/+ and WASP+/– littermates were found to have indistinguishable responses from 129Sv age-matched animals which were used as wild-type controls. Both WASP–/0 male and WASP–/– female mice used in this study are referred to as WASP–/–. Mice were housed under pathogen-free conditions according to institutional regulations.

Reagents and antibodies
Anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology (Waltham, MA). Antibodies against phospho-SAPK/JNK, phospho-p38, SAPK/JNK and p38 were from New England Biolabs (Beverly, MA). Anti-phospho-Erk and anti-Erk antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit anti-phospho-Akt and anti-Akt antibodies were from PharMingen (San Diego, CA). Cytochalasin D was from Calbiochem-Novabiochem (San Diego, CA).

Enumeration of mast cells in ear skin
Ears from WASP–/– and control mice were fixed in 2% paraformaldehyde, processed into 3-µm thick, paraffin-embedded sections and stained with toluidine blue. Slides were examined by light microscopy for determination of mast cell numbers. Mast cells in six randomly chosen fields were counted per slide (six slides total for both WASP–/– and control mice).

Blood histamine measurements
WASP–/– mice and wild-type controls were sensitized with 3 or 10 µg of mouse IgE anti-DNP mAb SPE-7 (Sigma, St Louis, MO) by i.v. injection in the retro-orbital vein. Twenty-four hours later the mice were challenged with i.v. injection of HSA-DNP (500 µg/mouse). Blood histamine levels were determined by competitive radioimmunoassay (Immunotech, Brea, CA) on 100 µl of plasma 1.5 and 5 min after antigen challenge according to the manufacturer’s instructions.

Derivation and characterization of bone marrow-derived mast cells (BMMC)
BMMC were derived as previously described (12). Briefly, bone marrow cells were cultured in WEHI-3 conditioned medium (WCM) as a source of IL-3 (23). Passages were made every week. To assess IgE binding, the cells were incubated with 1 µg/ml of mouse IgE (SPE-7), followed by biotinylated rat anti-mouse IgE and streptavidin–CyChrome (both from PharMingen). The cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Data on 5–10 x 105 viable cells (as determined by forward versus side scatter) were collected for each sample.

ß-Hexosaminidase release assay
ß-Hexosaminidase release assay was performed as previously described (12). BMMC (1 x 106) were incubated in WCM containing the indicated concentrations of rat monoclonal IgE anti-DNP (clone LO-DNP-30; Serotec, Oxford, UK) for 1 h on ice. After washing, pellets were resuspended in their original volume with WCM with 25 µg/ml F(ab')2 fragments of mouse anti-rat Ig (Jackson ImmunoResearch, West Grove, PA) and incubated for 2–30 min at 37°C. The cell pellets were lysed in their original volumes. Aliquots (5 µl) of supernatants and cell lysates were incubated for 30 min with 80 µl of substrate solution (1.3 mg/ml p-nitrophenyl-ß-D-2-acetamido-2-deoxyglucopyranozide in 0.1 M citrate, pH 4.5). The reaction was stopped by the addition of 200 µl of 0.2 M glycine (pH 10.7). OD was read at 405 nm in an ELISA reader. The percent release values were calculated by the formula [S/(S + P)] x 100, where S and P are the ß-hexosaminidase contents of the supernatant (S) and pellet (P) from each sample.

Measurement of IL-6 and TNF-{alpha} secretion
BMMC (1 x 106) preloaded for 1 h with the indicated concentrations of rat IgE were incubated with 25 µg/ml F(ab')2 fragments of mouse anti-rat Ig, with ionomycin (10 µM) or with ionomycin (1 µM) plus PMA (20 ng/ml) for indicated periods of time in culture medium. Supernatants were assayed for their content of IL-6 or TNF-{alpha} using an IL-6 or TNF-{alpha} ELISA kit (both from R & D, Minneapolis, MN) respectively.

RT-PCR analysis of IL-6 gene expression
cDNA was synthesized from 10 µg of total RNA using Superscript II (Gibco/BRL, Carlsbad, CA) for 120 min at 37°C. The primers used to amplify cDNA for ß2-microglobulin and IL-6 were as described previously (24). To quantify mRNA, a fixed amount of reverse-transcribed cellular mRNA was co-amplified in the presence of serial dilutions of a multispecific internal plasmid control, pMUS3 (24), which contains nucleotide sequences of multiple cytokines. The dilution at which pMUS3-derived and cDNA-derived signals were of equivalent intensity was used to establish the relative amount of cytokine mRNA. The results are expressed as a ratio of cytokine cDNA to cDNA of the constitutively expressed ß2-microglobulin gene.

Western blotting and immunoprecipitation
Lysates prepared from 0.5 x 106 BMMC by boiling in SDS loading buffer were separated on a 9% gel by PAGE and transferred to nitrocellulose membrane. The blots were developed using indicated antibodies followed by Protein G linked to horseradish peroxidase (Bio-Rad, Hercules, CA) and ECL (Amersham, Piscataway, NJ) or the Super Signal Ultra system (Pierce, Rockford, IL) according to the manufacturer’s instructions.

For immunoprecipitation, cell lysates prepared in RIPA buffer were precleared for 2 h with normal rabbit serum coupled to Protein G–Sepharose beads, then immunoprecipitated overnight with rabbit antibody to Syk prepared in our laboratory or Fc{epsilon}RIß mAb (kind gift from Dr J. P. Kinet, Beth Israel Hospital, Boston) on Protein G–Sepharose beads. The immunoprecipitates were resolved by SDS–PAGE and transferred to nitrocellulose membranes. Blots were probed with anti-phosphotyrosine mAb 4G10 and developed with ECL as above. To ensure equal loading, blots were stripped and then reprobed with the appropriate antibody.

Measurement of [Ca2+]i
BMMC were loaded with Indo-1/AM as previously described (25). Cells were sensitized with rat IgE (LO-DNP-30) while loading with the dye and stimulated with anti-IgE antibody while monitoring Ca2+ content using a spectrofluorimeter (LS50; Perkin-Elmer Cetus, Norwalk, CT). Excitation wavelength was 354 nm, and emission wavelengths were 405 and 485 nm.

Time-lapse video microscopy and immunofluorescence microscopy
Glass coverslips were coated with 2.5, 10 and 25 µg/ml of mouse monoclonal IgE anti-ovalbumin (a gift of Dr Mamoru Kiniwa, Taiho Pharmaceutical, Saitama, Japan) overnight at 4°C. BMMC (1 x 106/ml) were allowed to attach to the coverslips for 30, 60 or 90 min at 37°C. Unattached cells were washed off. When indicated, BMMC were pre-incubated with cytochalasin D at 10 µM for 1 h on ice, washed twice and resuspended in culture medium. Coverslips with attached BMMC in culture medium were transferred to the warm stage of the Nikon Eclipse TE2000 microscope. Images were acquired with a CCD-300-RC charge-coupled device (Dage-MTI, Michigan City, IN) at x400 magnification. Frames were taken every 5 s for 20 min.

Immunofluorescence
Cells were spun down onto the glass slides (controls) or were allowed to attach to the IgE-coated coverslips for 60 min at 37°C, and fixed with 4% formalin solution (Sigma) in PBS at room temperature for 20 min, washed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS at room temperature for 5 min. Cells were washed twice with PBS and blocked with 2% BSA (Gibco/BRL) in PBS for 10 min, and then stained for actin. For actin staining, cells were incubated with 1 µM TRITC–phalloidin (Sigma) for 20 min at room temperature. Photographs were taken under a fluorescent microscope (Nikon Eclipse E800) using a CCD-300-RC camera (Dage-MTI). Images were processed using Adobe Photoshop.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BMMC develop normally in the absence of WASP
Mouse bone marrow cells cultured in the presence of IL-3 differentiate into mast cells (26). After 4 weeks of culture in IL-3-containing WCM, similar numbers and percentages of cells (~90%) from WASP–/– and wild-type mice were mast cells as evidenced by their capacity to bind IgE (Fig. 1A). Furthermore, the same proportion of cells contained metachromatic granules when stained with toluidine blue (data not shown). Expression of WASP and its homologue N-WASP was examined by western blotting. Figure 1(B) confirms that BMMC from WASP–/– mice do not express WASP. Both wild-type and WASP–/– BMMC expressed N-WASP (Fig. 1C). The kinetics of cell growth and differentiation of WASP–/– and wild-type BMMC were similar (data not shown). We also examined the number of tissue mast cells in WASP–/– mice. The number of mast cells in the ear skin of WASP–/– mice was comparable to that of controls [16.3 ± 3.8 (n = 6) in WASP–/– mice versus 18.9 ± 3.8 (n = 6) in controls].



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Fig. 1. In vitro development of mast cells in the absence of WASP. (A) Bone marrow cells from WASP–/– and wild-type mice were examined for IgE binding by FACS analysis after 4 weeks of culture in WCM. Cells were incubated with mouse IgE followed by biotinylated anti-mouse IgE and streptavidin–CyChrome (dashed line). For control staining, cells were incubated with biotinylated anti-mouse IgE and streptavidin–CyChrome in the absence of IgE (solid line). Similar results were obtained on four occasions using independently derived lines of BMMC. (B and C) Western blot analysis of WASP (B) and N-WASP expression (C) in BMMC.

 
IgE-mediated systemic histamine release is greatly diminished in WASP–/– mice
Adoptive transfer of IgE antibodies to normal mice primes them to undergo passive systemic anaphylactic reactions in response to i.v. challenge with specific antigen. IgE-mediated anaphylaxis is dependent on Fc{epsilon}RI signaling, as it is virtually absent in Fc{epsilon}RI{alpha}-deficient mice (27). IgE-mediated anaphylaxis is also dependent on mast cells, as it is diminished in mast cell-deficient W/Wv mice (28,29) and virtually absent in mast cell-deficient Sl/Sld mice (30). A major vasoactive mediator released by activated mast cells and basophils is histamine. Systemic anaphylaxis is associated with a dramatic increase in plasma histamine levels (31).

To evaluate the role of WASP in IgE-dependent systemic anaphylaxis, we passively sensitized six WASP–/– mice and six controls with 3 µg mouse IgE anti-DNP mAb. This was followed 24 h later by challenge with DNP-HSA. Plasma histamine content was determined before and 1.5 min after challenge with antigen (Fig. 2A). The plasma histamine level prior to antigen administration was comparable in wild-type and WASP–/– mice. Following antigen challenge, there was a marked rise in plasma histamine in control mice. The antigen-induced increase in plasma histamine was significantly reduced in WASP–/– mice. Similar results were obtained when 10 µg mouse IgE anti-DNP mAb was used for sensitization with plasma histamine measured at 5 min after DNP-HSA challenge (data not shown). These results suggest that WASP is essential for optimal IgE-mediated mast cell degranulation in vivo.



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Fig. 2. IgE-mediated degranulation in the absence of WASP. (A) Plasma histamine levels in IgE-anti-DNP sensitized mice before and 1.5 min after DNP-HSA challenge in WASP–/– mice (n = 5) and wild-type controls (n = 6). (B) Release of ß-hexosaminidase by WASP–/– and wild-type BMMC sensitized with the indicated concentrations of rat IgE and challenged with F(ab')2 mouse anti-rat Ig (25 µg/ml). IO = ionomycin (10 µM). Results represent the mean of indicated numbers of mice (A) and the mean of five experiments (B), each performed in duplicate. Bars represent mean ± SEM. **P < 0.01.

 
IgE-induced degranulation in vitro is impaired in WASP-deficient BMMC
Upon cross-linking of Fc{epsilon}RI-bound IgE by antigen, mast cells degranulate by releasing preformed mediators stored in their granules. The capacity of WASP–/– BMMC to degranulate upon Fc{epsilon}RI cross-linking was examined. BMMC were sensitized with rat IgE (0.05–5 µg/ml) for 1 h, washed and bound IgE was cross-linked with mouse anti-rat Ig (25 µg/ml), and the release of ß-hexosaminidase, an enzyme found in mast cell granules (32), was measured. ß-Hexosaminidase release was lower in WASP–/– BMMC compared to control wild-type BMMC at all concentrations of IgE antibody used and at 2, 5, 15 and 30 min (Fig. 2B and data not shown). Treatment of mutant BMMC with ionomycin resulted in normal degranulation, demonstrating that their cellular machinery for degranulation and their ß-hexosaminidase granule content are normal.

IgE-induced cytokine production requires WASP protein
In addition to degranulation, Fc{epsilon}RI cross-linking induces the secretion of a number of cytokines by mast cells, including IL-6 (33) and TNF-{alpha} (10). To assess the role of WASP in Fc{epsilon}RI-mediated cytokine release, BMMC were sensitized and challenged as above, and IL-6 and TNF-{alpha} release measured by ELISA 24 h later. WASP–/– BMMC secreted markedly less IL-6 and TNF-{alpha} than control BMMC at 6, 24 and 48 h after Fc{epsilon}RI cross-linking (Fig. 3A and B and data not shown). Furthermore, RT-PCR analysis demonstrated that IL-6 mRNA levels following Fc{epsilon}RI ligation were substantially lower in WASP–/– BMMC compared to controls (Fig. 3C and D). Ionomycin or ionomycin plus PMA, which bypass receptor signaling, induced equivalent levels of IL-6 expression, and IL-6 and TNF-{alpha} secretion by both WASP–/– and wild-type BMMC.




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Fig. 3. IgE-mediated cytokine release and gene expression by BMMC. WASP–/– and wild-type BMMC were sensitized and challenged as in Fig. 2. Release of IL-6 (A) and TNF-{alpha} (B) by BMMC was measured after 24 h of culture. Results shown are representative of three independent experiments, each performed in duplicate. (C) RT-PCR analysis of IL-6 mRNA. (D) Quantification of RT-PCR results from (C). Levels of IL-6 mRNA were normalized to that of ß2-microglobulin mRNA as described in Methods. IL-6 mRNA levels induced with 1 µM ionomycin (IO) are shown for comparison. For (B) and (C), results are representative of four independent experiments.

 
IgE-induced tyrosine phosphorylation of PLC{gamma} and Ca2+ mobilization are impaired in BMMC from WASP–/– mice
Like other ITAM-containing receptors, Fc{epsilon}RI signals through associated protein tyrosine kinases of the src and syk families (2). Both the pattern and the intensity of protein tyrosine phosphorylation following Fc{epsilon}RI ligation were comparable in wild-type and WASP-deficient BMMC (data not shown). More importantly, protein tyrosine phosphorylation of the Fc{epsilon}RIß chain and Syk, both of which are thought to lie upstream of WASP, was similar in WASP–/– BMMC and controls (Fig. 4A and B). Protein tyrosine phosphorylation of SLP-76 and Vav was also unaffected (data not shown). In contrast, tyrosine phosphorylation of PLC{gamma} was diminished in WASP–/– BMMC (Fig. 4C). Fc{epsilon}RI ligation activates phosphatidylinositol-3-kinase resulting in the generation of phosphatidylinositol-3,4,5-tris phosphate and subsequent phosphorylation and activation of Akt (34). Akt phosphorylation was similar in WASP–/– BMMC and controls (Fig. 4D).




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Fig. 4. Protein tyrosine phosphorylation in BMMC in response to Fc{epsilon}RI cross-linking. WASP–/– and wild-type BMMC were sensitized with 5 µg/ml rat IgE, followed by cross-linking with 25 µg/ml F(ab')2 mouse anti-rat Ig. Cell lysates were examined at the indicated time points (0, 5 or 0, 5 and 15 min) for tyrosine phosphorylation of Fc{epsilon}RIß (A), Syk (B) and PLC{gamma} (C) by immunoprecipitatation with the indicated antibodies followed by western blotting with mAb 4G10. (D) Akt phosphorylation was examined by western blotting with phospho-Akt specific antibody. Equal loading was verified by western blotting with appropriate antibodies. Results shown are representative of at least two independent experiments.

 
Fc{epsilon}RI-induced phosphorylation and activation of PLC{gamma} causes a rapid rise in [Ca2+]i (2), which is important for both degranulation and cytokine release. Since mast cells of WASP-deficient mice are impaired in their capacity to release various mediators and phosphorylate PLC{gamma} following Fc{epsilon}RI cross-linking, we examined Ca2+ mobilization in these cells. BMMC were sensitized with rat IgE, loaded with a Ca2+-sensitive dye and then challenged with F(ab')2 fragments of anti-rat Ig. Ca2+ mobilization in response to Fc{epsilon}RI cross-linking was clearly diminished in WASP–/– BMMC (Fig. 5).



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Fig. 5. Ca2+ mobilization in BMMC in response to IgE cross-linking. Changes in the intracellular Ca2+ concentration monitored in Indo-1 AM-loaded WASP–/– (dashed line) and wild-type (solid line) BMMC sensitized with 5 µg/ml of rat IgE, and then challenged at the indicated time points with 2.5 and 0.25 µg/ml F(ab')2 mouse anti-rat Ig. Results are expressed as ratio of fluorescence intensities at 405 and 485 nm. Results shown are representative of four independent experiments.

 
IgE-induced phosphorylation of MAPK in WASP-deficient BMMC
Fc{epsilon}RI cross-linking induces phosphorylation and activation of the MAPK Erk, JNK and p38 in mast cells (2,8). The phosphorylation status of the MAPK Erk, JNK and p38 in WASP–/– BMMC following Fc{epsilon}RI cross-linking was examined by western blotting with antisera that recognize selectively these kinases in their phosphorylated state. Erk 2 (p42) was the major Erk isoform phosphorylated following Fc{epsilon}RI ligation in mast cells. Phosphorylation of Erk2 and p38 was minimally affected in WASP–/– mast cells (Fig. 6A and B). In contrast, phosphorylation of both p54 and p46 JNK isoforms was severely reduced in WASP–/– mast cells (Fig. 6C).



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Fig. 6. Activation of MAPK in WASP–/– and wild-type BMMC following Fc{epsilon}RI cross-linking. BMMC were stimulated for 0, 5, 15 and 30 min as described in the legend to Fig. 4, and lysed in the SDS–PAGE sample buffer. Aliquots of the same lysates were analyzed for phosphorylation of Erk1/2 (A), p38 MAPK (B) and SAPK/JNK (C) by western blotting with the corresponding phospho-specific antibodies. Lower panels represent the same membranes stripped and reprobed with kinase-specific antibodies as a control for the loading. Insets show relative phosphorylation of MAPK determined by densitometry and normalized for loading. Results shown are representative of three independent experiments.

 
Spreading of mast cells on IgE-coated slides require WASP protein
T cells spread and extend protrusions when exposed to an anti-TCR–CD3-coated surface. This is dependent on actin cytoskeleton reorganization which is mediated by WASP (3537). Fc{epsilon}RI ligation has been shown to induce actin cytoskeletal changes in the RBL-2H3 mast cell line (38). We examined whether normal murine BMMC spread and extend protrusions when exposed to an IgE-coated surface. Wild-type mast cells adhered to coverslips coated with 25 µg/ml IgE, but not to untreated coverslips (data not shown). Following attachment, wild-type BMMC spread and extended membrane protrusions (Fig. 7A and B). Pre-incubation with cytochalasin D reduced spreading (from 72 ± 7.3 to 36 ± 11% at 1 h) and inhibited protrusion formation (Fig. 7B), suggesting that actin polymerization is important in these processes. WASP–/– mast cells adhered normally to IgE-coated coverslips (data not shown). However, they had a significantly reduced ability to spread (Fig. 7A) and were impaired in their ability to form protrusions (Fig. 7B). Similar data was obtained using concentrations of coating IgE of 2.5 and 10 µg/ml (data not shown).




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Fig. 7. Spreading of BMMC on IgE-coated glass slides. (A) Quantification of spreading, as percentage of adherent cells at 30, 60 and 90 min. Cells were counted in 10 fields/coverslip under high power (x400). Results represent the mean ± SEM. P values shown were determined by Student’s t-test. (B) Representative frames of wild-type (left), cytochalasin D-treated wild-type (middle) and WASP–/– BMMC (right) incubated for 1 h on IgE-coated coverslips. (C) F-actin distribution in wild-type (left panels) and WASP–/– BMMC (right panels) fixed on uncoated coverslips in cytospin preparations (upper panels), and after incubation for 1 h on coverslips coated with IgE (25 µg/ml). Results shown are representative of three independent experiments.

 
We compared the actin cytoskeleton of wild-type and WASP-deficient mast cells by intracellular staining for F-actin. The morphology of resting wild-type and WASP–/– BMMC was similar (Fig. 7C). Following incubation for 1 h on IgE-coated coverslips, wild-type mast cells spread and exhibited prominent membrane ruffles (Fig. 7C). In contrast, the majority of WASP–/– mast cells failed to spread, and their morphology and F-actin distribution remained similar to those of resting cells. The small fraction of WASP–/– cells that spread exhibited membrane ruffles (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we show that BMMC from WASP–/– mice are impaired in their ability to secrete both early and late mediators, and to spread following Fc{epsilon}RI cross-linking. This identifies WASP as an important signaling molecule which links Fc{epsilon}RI to mediator release and actin cytoskeletal changes.

Mouse mast cell development is known to be driven by IL-3 (26) and other cytokines such as stem cell factor (39), but is thought to be independent of Fc{epsilon}RI signals, as it proceeds normally in Fc{epsilon}RI{alpha}–/– mice (27). We found that WASP is not essential for mast cell growth and differentiation as the numbers of tissue mast cells in ears of WASP–/– mice and the development of mast cells from bone marrow of WASP–/– mice in vitro were normal (Fig. 1A).

Following IgE antibody sensitization and antigen challenge WASP–/– mice exhibited a significantly reduced rise in plasma histamine levels compared to wild-type controls (Fig. 2A). Since IgE-mediated histamine release in vivo is dependent on Fc{epsilon}RI (27), our results strongly suggest that WASP is a critical component of the Fc{epsilon}RI signaling pathway. The residual histamine response of WASP–/– mice in passive IgE-mediated anaphylaxis could be due to residual signaling via Fc{epsilon}RI. This may possibly involve N-WASP, which we found to be expressed by mast cells (Fig. 1C).

Degranulation and cytokine secretion are respectively early and late events triggered by Fc{epsilon}RI ligation (40). The signaling pathways leading to the release of these mediators are likely to be distinct. Fc{epsilon}RI-mediated mast cell degranulation and cytokine release were deficient in WASP–/– BMMC. This was evidenced by their decreased ability to release ß-hexosaminidase (Fig. 2B), and produce IL-6 and TNF-{alpha} (Fig. 3) following IgE cross-linking. These results place WASP in a pivotal position in Fc{epsilon}RI-mediated degranulation and cytokine gene expression.

Our studies of early events in Fc{epsilon}RI signaling revealed that total protein, Fc{epsilon}RIß, Syk, and Akt phosphorylation was grossly intact in the absence of WASP, but that PLC{gamma} phosphorylation was diminished (Fig. 4). This suggests that WASP is acting downstream of Src and Syk, and of phosphatidylinositol-3-kinase which generates phosphatidylinositol-3,4,5-tris phosphate, a mediator that is essential for activation of Akt. Consistent with the decreased PLC{gamma} phosphorylation, calcium mobilization in response to Fc{epsilon}RI cross-linking in WASP–/– BMMC was clearly diminished (Fig. 5). It is likely that diminished calcium mobilization in WASP–/– BMMC contributes to their impaired degranulation and cytokine secretion. Activation of PLC{gamma} and subsequent calcium mobilization are dependent on LAT, SLP-76 and Tec family kinases. In T cells, WASP was reported to be a component of a multimolecular complex that includes SLP-76, Nck and Vav (41), and thus may be important for optimal activation of PLC{gamma}, possibly by virtue of its ability to nucleate actin polymerization which may provide a scaffold for a LAT–SLP-76–Tec kinase–PLC{gamma} complex. Our observation that tyrosine phosphorylation of SLP-76 and Vav was unaffected in WASP-deficient BMMC suggests that WASP may act downstream of these proteins.

Fc{epsilon}RI cross-linking induces phosphorylation and activation of the MAPK Erk, JNK and p38 in mast cells (79). We found that Fc{epsilon}RI-mediated JNK phosphorylation was decreased in WASP–/– BMMC (Fig. 6C). Normal JNK activation following TCR ligation has been reported in WASP–/– T cells (22). The discrepancy between this result and ours may be due to differences in cell types, receptors (Fc{epsilon}RI versus TCR) and/or mouse lines examined. WASP could potentially modulate JNK activation via its effects on actin polymerization, as JNK activation has been reported to be regulated by the actin cytoskeleton (42). Activation of JNK plays an important role in the expression of the cytokine IL-6 (43,44) and TNF-{alpha} (10) in mast cells. Thus, it is likely that impaired JNK activation in WASP–/– BMMC may underlie their decreased ability to produce IL-6 and TNF-{alpha} following Fc{epsilon}RI cross-linking.

We have found that normal BMMC adhere to IgE-coated coverslips, and undergo dramatic shape changes that include spreading and formation of ruffles. These changes are similar to those observed in T cells incubated on anti-CD3-coated surfaces (45) and are dependent on actin polymerization since they are inhibited by cytochalasin D. WASP-deficient mast cells adhered to IgE-coated plates, but were severely impaired in their ability to spread and form ruffles (Fig. 7). These results suggest that WASP links Fc{epsilon}RI signaling to actin cytoskeleton reorganization in mast cells. They also raise the possibility that WASP may play a direct role in granule movement and fusion with the plasma membrane. Fc{epsilon}RI signaling causes a redistribution of cellular F-actin with cortical depolymerization and with centripetal accumulation of F-actin, which is associated with granule exocytosis (46). Loss of the Cdc42–WASP–actin polymerization pathway may result in impaired F-actin redistribution and may contribute to defective degranulation in WASP–/– BMMC. The fact that the calcium ionophore ionomycin induced normal degranulation in these cells does not rule out a role for WASP in granule movement, because non-physiologically high calcium concentrations may circumvent a physiologic role for WASP in this process.

Our results demonstrate that WASP is essential for optimal Fc{epsilon}RI-induced degranulation and cytokine production in mast cells. Since IgE-dependent mast cell mediator release plays a critical role in the pathophysiology of allergic diseases, our results also suggest a novel potential approach to the treatment of these diseases.


    Acknowledgements
 
We thank Drs Lewis Cantley and Lucia Rameh for helpful suggestions. This work was supported by NIH grant AI-35714, and by grants from Baxter Healthcare, Aventis and Olsten Corporations to R. S. G. F. W. A is supported by a grant from the Howard Hughes Medical Institute.


    Abbreviations
 
BMMC—bone marrow-derived mast cells

Fc{epsilon}RI—high-affinity receptor for IgE

ITAM—immunoreceptor tyrosine-based activation motif

IP3—inositol 1,4,5-triphosphate

JNK—c-Jun N-terminal kinase

LAT—linker for activation of T cells

MAPK—mitogen-activated protein kinase

PLC—phospholipase C

TNF—tumor necrosis factor

WASP—Wiskott–Aldrich syndrome protein

WCM—WEHI-3 conditioned medium


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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