Targeting Janus Kinase 3 in Mast Cells Prevents Immediate Hypersensitivity Reactions and Anaphylaxis*

Janus kinase 3 (JAK3), a member of the Janus family protein-tyrosine kinases, is expressed in mast cells, and its enzymatic activity is enhanced by IgE receptor/FcεRI cross-linking. Selective inhibition of JAK3 in mast cells with 4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline) (WHI-P131) blocked the phospholipase C activation, calcium mobilization, and activation of microtubule-associated protein kinase after lgE receptor/FcεRI cross-linking. Treatment of IgE-sensitized rodent as well as human mast cells with WHI-P131 effectively inhibited the activation-associated morphological changes, degranulation, and proinflammatory mediator release after specific antigen challenge without affecting the functional integrity of the distal secretory machinery. In vivo administration of the JAK3 inhibitor WHI-P131 prevented mast cell degranulation and development of cutaneous as well as systemic fatal anaphylaxis in mice at nontoxic dose levels. Thus, JAK3 plays a pivotal role in IgE receptor/FcεRI-mediated mast cell responses, and targeting JAK3 with a specific inhibitor, such as WHI-P131, may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.

Acute allergic reactions, also known as immediate (type I) hypersensitivity reactions, including anaphylaxis with a potentially fatal outcome, are triggered by three major classes of proinflammatory mediators, namely preformed granule-associated bioactive amines (e.g. histamine and serotonin) and acid hydrolases (e.g. ␤-hexosaminidase), newly synthesized arachidonic acid metabolites (e.g. leukotriene (LT) 1 C 4 , prostaglandin D 2 , and platelet activating factor), and a number of proinflammatory vasoactive cytokines (e.g. tumor necrosis factor (TNF) ␣ and interleukin (IL)-6) (1, 2). These proinflammatory mediators are released from sensitized mast cells upon activation through the antigen-mediated cross-linking of their high affin-ity cell surface IgE receptors/Fc⑀RI (1,3,5). IgE receptor/Fc⑀RI is a multimeric receptor with ␣, ␤, and homodimeric ␥ chains (4). Both ␤ and ␥ subunits of the IgE receptor/Fc⑀RI contain ITAMs (immunoreceptor tyrosine-based activation motifs), which allow interaction with protein-tyrosine kinases (PTK) and PTK substrates via their Src homology 2 domains (4, 6, 7). The engagement of IgE receptors by antigen triggers a cascade of biochemical signal transduction events, including activation of multiple PTK (6,7). The activation of PTK and subsequent tyrosine phosphorylation of their downstream substrates have been implicated in the pathophysiology of type I hypersensitivity reactions (6,7). The elucidation of the PTK-dependent signal transduction events that lead to Fc⑀RI-mediated mast cell degranulation and mediator release may provide the basis for the rational design of potent mast cell inhibitors for prevention and treatment of allergic reactions.
Signal transducers and activators of transcription (STAT) are pleiotropic transcription factors that mediate cytokinestimulated gene expression in multiple cell populations (8,9). All STAT proteins contain a DNA-binding domain, a Src homology 2 domain, and a transactivation domain necessary for transcriptional activation of target gene expression. Janus kinases (JAK), including JAK1, JAK2, Tyk, and JAK3, are cytoplasmic PTK that play pivotal roles in initiation of cytokinetriggered signaling events by activating the cytoplasmic latent forms of STAT proteins via tyrosine phosphorylation on a specific tyrosine residue near the Src homology 2 domain (10). Tyrosine phosphorylated STAT proteins dimerize through specific reciprocal Src homology 2-phosphotyrosine interactions and translocate from the cytoplasm to the nucleus where they stimulate the transcription of specific target genes by binding to response elements in their promoters (11). Among the four members of the JAK family, JAK3 is abundantly expressed in lymphoid cells and plays an important role in normal lymphocyte development and function, as evidenced by qualitative and quantitative deficiencies in the B-cell as well as T-cell compartments of the immune system of JAK3-deficient mice (12,13) and development of severe combined immunodeficiency in JAK3-deficient patients (14,15). Besides lymphoid cells, nonlymphoid cells (16), including monocytes, megakaryocytes, endothelial cells, cancer cells, and, as we now report, mast cells also express JAK3, but no information is currently available regarding the physiologic function of JAK3 in these nonlymphoid cell populations.
In a recent study, we found that the IgE/antigen-induced degranulation and mediator release are substantially reduced with Jak3 Ϫ/Ϫ mast cells from JAK3-null mice that were generated by targeted disruption of Jak3 gene in embryonic stem cells (17), indicating that JAK3 plays a pivotal role in IgE receptor/Fc⑀RI-mediated mast cell responses both in vitro and in vivo. Here, we report that selective inhibition of JAK3 in mast cells with 4-(4Ј-hydroxylphenyl)-amino-6,7-dimethoxy-* 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.

Reagents and Materials
Mice-Male BALB/c mice (6 -8 weeks old) were purchased from Charles River Laboratories (Wilmington, MA). Animals were caged in groups of five in a pathogen-free environment in accordance with the rules and regulations of U. S. Animal Welfare Act, and the National Institutes of Health. Animal care and the experimental procedures were carried out in agreement with institutional guidelines.

Mast Cell Cultures
RBL-2H3 Cells-RBL-2H3 cells were a gift from Dr. Reuben P. Siraganian (Laboratory of Microbiology and Immunology, National Institute of Dental Research, National Institute of Health). The cells were maintained as monolayer cultures in 75-and 150-cm 2 flasks in Eagle's essential medium supplemented with 20% fetal calf serum (4).
Human Mast Cells-Human fetal livers (16 -21 weeks of gestational age) were obtained from prostaglandin-induced abortuses. Subsequently, cell suspensions were prepared, and mononuclear cells were isolated from cell suspensions by centrifugation on Ficoll-Hypaque gradients as described (23). Isolated cells were cultured for 5 weeks in the presence of 50 ng/ml recombinant human stem cell factor and 2 ng/ml rhIL-4 (24). Culture medium was replaced with fresh medium once a week for the first 2 weeks and twice a week thereafter. At the end of the 5 weeks, the fetal liver-derived cell cultures contained Ͼ70% mast cells, based on tryptase staining (25). All the human tissue specimen were used following the guidelines of the Hughes Institute Institutional Review Board on the Use of Human Subjects in Research for secondary use of pathologic or surgical tissue.
Confocol Microscopy-Staining of mast cells with primary and secondary antibodies followed by confocal laser scanning microscopy was performed as described previously in detail (26). After staining with appropriate primary and secondary antibodies, cells were washed three times to remove unbound antibody. DNA labeling was performed by incubation of coverslips with Toto-3 (Molecular Probes, Eugene, OR) for 10 min. Excessive dye was washed with PBS plus O.1% Triton X-100. Cells were visualized under MRC 1024 laser scanning microscope after mounting with Vectashield (Vector laboratories, Inc., Burlingame, CA). The anti-JAK3 (27) and anti-tubulin (clone B-5-1-2; Sigma) antibodies were used according to standard procedures (26).
Stimulation of Mast Cells-RBL-2H3 cells were sensitized with monoclonal anti-DNP IgE antibody (0.24 mg/ml) for 1 h at 37°C in a 48-well tissue culture plate. Unbound IgE was removed by washing the cells with phosphate-buffered saline. After washing the PIPES-buffered saline containing 1 mM calcium chloride was added to the monolayers of the RBL-2H3 cells. The cells were challenged with 20 ng/ml DNP-BSA for 30 min at 37°C. The plate was centrifuged at 200 ϫ g for 10 min at 4°C. Supernatants were removed and saved. The cell pellets were washed with phosphate-buffered saline and solubilized in PIPES-buffered saline containing 0.1% Triton X-100.
Fetal liver-derived human mast cells were resuspended in tyrode buffer containing calcium and magnesium and challenged with anti-Fc⑀RI antibody 22E7 for 15 min. In some experiments fetal liverderived human mast cells were resuspended in culture medium at a cell density of 5 ϫ 10 6 /ml and sensitized with IgE (150 g/ml) for 3 h at 4°C. After sensitization the cells were washed with tyrode buffer containing FIG. 1. Expression and activation of JAK3 in mast cells after IgE receptor cross-linking. A, RBL-2H3 mast cells were stained with a polyclonal anti-JAK3 antibody and labeled with a fluorescein-labeled secondary antibody as well as the DNA-specific dye Toto-3 and visualized using confocal laser scanning microscopy. B, to study IgE/antigeninduced activation JAK3 in mast cells, RBL-2H3 mast cells were sensitized with monoclonal anti-DNP IgE and then challenged with DNP-BSA. Mast cells were lysed using a Nonidet-P40 lysis buffer prior to or 30 min after antigen challenge, and JAK3 immune complexes from these cell lysates were subjected to anti-phosphotyrosine (APT) Western blot (WB) analysis to examine the autophosphorylation of the JAK3 kinase (lanes 1 and 2). In parallel, JAK3 immune complexes were also examined by anti-JAK3 immunoblotting (lanes 3 and 4) to confirm that the increased tyrosine phosphorylation in APT blots was not due to differences in the amount of JAK3 immunoprecipitated (IP). calcium and magnesium and challenged with mouse monoclonal antihuman IgE (40 /ml) for 30 min at 37°C. To study the effects of the test compounds, mast cells were incubated with WHI-P131, WHI-P111, or WHI-P112 at the indicated concentrations or vehicle for 1 h prior to challenge.
Mediator Release Assays-Histamine content in cell free supernatants and in the solubilized cell pellets was estimated using a commercially available enzyme immunoassay (29). LTC 4 levels were estimated in cell-free supernatants by immunoassay (30). TNF␣ levels were estimated in cell-free supernatants using a standard cytotoxicity assay (31). In RBL-2H3 cells, ␤-hexosaminidase release was estimated in cell-free supernatants and Triton X-100 solubilized pellets as described (32). Tryptase levels were quantitated in cell free supernatants and pellets of fetal liver-derived human mast cells as described previously in detail (24).
Calcium Measurements-Calcium mobilization assays were performed as described earlier (35). RBL-2H3 cells were loaded with Fluo-3 and stimulated with DNP-BSA in presence and absence of WHI-P131 as described above. Calcium response was measured by an calcium imaging device (Universal Imaging Co., West Chester, PA) mounted onto a inverted microscope. The exitation wavelength was 485 nm, and the emission wavelength for detection was 535 nm. The fluorescent image of an individual cell was acquired by a CCD72 video camera (Dage-MTI Inc., Michigan City, IL) at the speed of 1 frame/s and digitized by computer.

FIG. 2. WHI-P131 prevents JAK3 but not SYK activation in mast cells after IgE receptor cross-linking.
To show that WHI-P131 prevents JAK3 activation in mast cells after IgE receptor cross-linking, RBL-2H3 cells were sensitized with monoclonal anti-DNP IgE, treated with either vehicle (A) or 30 M WHI-P131 (B) and then challenged with DNP-BSA. Cells were lysed, and JAK3 immune complexes were subjected to kinase assays in the presence of cold ATP followed by APT immunoblotting (upper panels in A and B) as well as to JAK3 Western blot (WB) analysis (lower panels in A and B) as described under "Experimental Procedures." To show the activation of SYK in mast cells following IgE receptor cross-linking, RBL-2H3 cells were sensitized with monoclonal anti-DNP IgE, left untreated (C), or treated with either vehicle or 30 M WHI-P131 (D) and then challenged with DNP-BSA. Cells were lysed at the indicated time points, and SYK immune complexes were subjected to kinase assays in the presence of cold ATP followed by APT immunoblotting (upper panels in C and D) as well as to SYK Western blot analysis (lower panels in C and D). Lanes C, base-line control. Cells were lysed at the indicated time points with ice-cold 20% perchloric acid and were assayed for Ins-1,4,5-P 3 levels using a radioligand competition assay (33,34). The data points represent the means Ϯ S.E. of pooled data obtained from two independent experiments, each performed in triplicate. *, p Ͻ 0.05 (Student's t test) compared with vehicle-treated controls that were stimulated with IgE/DNP-BSA under identical conditions in the same experiments.
Anaphylaxis Models-To examine the effect of WHI-P131 on passive cutaneous anaphylaxis in mice, dorsal sides of the ears of BALB/c mice were injected intradermally with 20 ng of DNP-IgE (left ears) or PBS (right ears) in 20 l of volume using a 30-gauge needle as described previously (39). After 20 h, mice were treated with WHI-P131 (10 or 25 mg/kg intraperitoneally) twice at 1-h intervals prior to the antigen challenge. Control mice were treated with an equal volume of vehicle. 30 min after the last dose of WHI-P131 or vehicle, mice were challenged with 100 g of antigen (DNP-BSA) in 200 l of 2% Evan's blue dye intravenously. Mice were sacrificed by cervical dislocation 30 min after the antigen challenge. For quantitation of the Evan's blue dye extravasation as a measure of anaphylaxis-associated vascular hyperpermeability, 8-mm skin specimens were removed from the ears of mice, minced in 2 ml of formamide and incubated at 80°C for 2 h in water bath to extract the dye. The absorbance was read at 620 nm. The data were expressed as plasma exudation indices (i.e. fold increase in optical density over PBS-treated ears at 620 nm).
To induce passive systemic anaphylaxis, BALB/c mice were sensitized with 50 g of DNP-IgE intravenously. At 24 h DNP-BSA (2 mg) was administered intravenously with 0.5% Evan's blue (200 l). For assessment of vascular leak, animals were sacrificed 30 min after the antigen challenge, and their foot pads were examined for blue coloration. Histamine levels in plasma were measured 5 min after the antigen challenge. To this end, blood samples were obtained from the ocular venous plexus by retroorbital venupuncture, and histamine levels were determined by ELISA using a commercial kit (Immunotech, West Brook, ME) (29). For histopathologic evaluation of tissue mast cell degranulation, ears of mice were removed 1 h after the DNP-BSA injection and fixed in 10% buffered formalin. Processed thin sections (3-5 m) were stained with avidin-FITC (6.25 g/ml) for 2 h, washed with PBS to remove unbound dye, and then mounted in buffered glycerol, 30 mM triethylenediamine, pH 8.6 (40). In the murine model for antigen-induced active anaphylaxis, mice were sensitized with 2 mg of BSA in 200 l of aluminum hydroxide gel (Reheis Inc., Berkeley, NJ). 10 days later, anaphylactic shock was induced by the intravenous challenge of the mice with 200 g of BSA. Fig. 1A, JAK3 is abundantly expressed in RBL-2H3 mast cells. This finding prompted us to examine the potential involvement of JAK3 in IgE receptor/Fc⑀RI-mediated mast cell activation. The baseline tyrosine phosphorylation level of unstimulated RBL-2H3 cells varied from experiment to experiment. Cross-linking of the IgE receptors on RBL-2H3 mast cells that were previously sensitized by a monoclonal anti-DNP-IgE antibody with the specific antigen DNP-BSA resulted in enhanced tyrosine phosphorylation of JAK3 regardless of its base-line phosphorylation state (Figs. 1B and 2A).

Expression and IgE Receptor/Fc⑀RI-mediated Activation of Janus Kinase 3 in Mast Cells-As shown in
Inhibition of JAK3 in Mast Cells Abrogates IgE Receptortriggered Biochemical Signaling Events Downstream to Activation of SYK-We next set out to determine the effects of WHI-P131, a rationally designed JAK3-specific tyrosine kinase inhibitor, on JAK3 activation as well as other biochemical signal transduction events in mast cells. WHI-P131 inhibits JAK3 with an IC 50 value of 9 M, but it does not exhibit any detectable inhibitory activity against other protein-tyrosine kinases, including the structurally similar Janus kinases JAK1 and JAK2, SRC family tyrosine kinase LYN, ZAP/SYK family tyrosine kinase SYK, TEC family tyrosine kinase BTK, and receptor family tyrosine kinase IRK even at concentrations as high as 350 M (18,19). Treatment of the rat mucosal mast cell line RBL-2H3 with WHI-P131 abrogated JAK3 activation after IgE receptor cross-linking (Fig. 2, A and B). Notably, WHI-P131 did not prevent the robust SYK activation signal in RBL-2H3 mast cells after IgE receptor/Fc⑀RI cross-linking (Fig. 2, C  and D). Therefore, any biological consequences of JAK3 inhibition in WHI-P131-treated mast cells cannot be attributed to impaired SYK activation. These results also demonstrate that JAK3 in mast cells does not act upstream of SYK in the signal transduction cascade initiated by the engagement of the high affinity IgE receptor/Fc⑀RI, and its activation is not mandatory for activation of SYK. We also noted a slight difference in kinetics of activation of JAK3 versus SYK in that JAK3 tyrosine phosphorylation ( Fig. 2A) did not reach its maximum as rapidly as SYK tyrosine phosphorylation (Fig. 2C).
Ozawa et al. (32) have shown that activation of PLC␥ and downstream calcium mobilization are essential and sufficient signals for the secretory responses of RBL-2H3 cells to antigen stimulation. Previous studies with SYK-negative variants of RBL-2H3 cells and RBL-2H3 cells expressing truncated SYK proteins with a dominant negative function have clearly shown that SYK activation is essential for activation of PLC␥ (41)(42)(43). Therefore, we next sought to determine whether JAK3 could act downstream of SYK or cooperate with SYK in activation of PLC␥ and calcium mobilization. The PLC␥ activation in IgEsensitized RBL-2H3 mast cells was assayed by measuring the Ins-1,4,5-P 3 levels before and after engagement of the IgE receptor/Fc⑀RI with DNP-BSA in two independent experiments. As shown in Fig. 3, the IgE receptor engagement resulted in a rapid increase of the Ins-1,4,5-P 3 levels from 4.3 Ϯ 0.6 pmol/10 6 cells to 27.3 Ϯ 5.7 pmol/10 6 cells at 30 s after the stimulation and 31.2 Ϯ 1.3 pmol/10 6 cells at 60 s after the stimulation. In accordance with previous reports, piceatannol, a naturally occurring stilbene that selectively inhibits SYK by competing for its substrate-binding site (44), effectively blocked the Ins-1,4,5-P 3 production (Fig. 3). At 30 and 60 s after stimulation, the Ins-1,4,5-P 3 levels of piceatannol (30 g/ml, ϳ100 M)-treated RBL-2H3 cells were 5.7 Ϯ 1.6 pmol/10 6 cells and 6.8 Ϯ 1.0 pmol/10 6 cells, respectively. Notably, the JAK3 inhibitor WHI-P131 also blocked the Ins-1,4,5-P 3 production in RBL-  (Fig. 3). These results provided the first experimental evidence that inhibition of JAK3 in mast cells blocks the SYK-mediated activation of the PLC␥ signaling pathway that follows the stimulation of mast cells via their high affinity IgE receptor/Fc⑀RI. This evidence was further supported by our finding that WHI-P131 blocks the Ins-1,4,5-P 3 -mediated downstream calcium mobilization in mast cells. Specifically, RBL-2H3 cells were sensitized with IgE and loaded with Fluo-3 prior to stimulation with DNP-BSA. The intracellular calcium ion concentration reached a maximum within 2-3 min after stimulation, which is consistent with previous findings (45,46). WHI-P131 inhibited the calcium response in a concentration-dependent fashion with abrogation of calcium mobilization at 30 M (Fig. 4). Thus, JAK3 (similar to SYK) is essential for the IgE receptor/Fc⑀RI-mediated calcium mobilization in mast cells. As shown in Fig. 4, ionomycininduced IgE receptor-independent calcium responses were not affected by WHI-P131.
We next set out to determine whether JAK3 and SYK might "cooperate" in generation of an optimal calcium signal in mast cells stimulated via IgE receptor/Fc⑀RI by combining WHI-P131 and piceatannol. To this end, we used suboptimal concentrations of WHI-P131 and piceatannol that only partially block the calcium response. As shown in Fig. 5A, WHI-P131 only partially blocked the calcium signal in IgE-sensitized mast cells after DNP-BSA stimulation when it was used at 1 M or 3 M concentrations. Similarly, 200 ng/ml piceatannol only partially inhibited the calcium mobilization (Fig. 5B). However, total abrogation of the calcium response was achieved when 1 M WHI-P131 was combined with 200 ng/ml piceatannol (Fig.  5B). Notably, this combination resulted in complete block of the calcium signal even at a 1:10 dilution (i.e. 0.1 M WHI-P131 plus 20 ng/ml piceatannol) (Fig. 5C). These findings indicate that JAK3 and SYK promote coincident and potentially cooperative signals in mast cells that both result in the PLC␥ activation and calcium mobilization following the engagement of the IgE receptor/Fc⑀RI. SYK has also been shown to be the initiator of another signaling pathway leading to the tyrosine phosphorylation and activation of MAPK p42 MAPK /ERK2 (and subsequently phospholipase A 2 ) in RBL-2H3 mast cells (41). Because of the observed effects of the JAK3 inhibitor WHI-P131 on SYK-dependent signaling events that lead to PLC␥ activation and calcium mobilization, we next examined the effects of WHI-P131 on MAPK activation in mast cells following the engagement of the IgE receptor/Fc⑀RI. As shown in Fig. 6, the IgE receptor engagement resulted in enhanced tyrosine phosphorylation of the MAPK in mast cells, and WHI-P131 blocked this response. Protein expression levels for MAPK or actin were not affected by WHI-P131 (Fig. 6). Thus, JAK3 appears to play an essential role for the IgE receptor-mediated MAPK activation in mast cells as well. Taken together, these in vitro JAK3 inhibitor studies provided biochemical evidence that JAK3 in mast cells is a key regulator of IgE receptor/Fc⑀RI-mediated signal transduction events.
Effects of JAK3 Inhibition on in Vitro Mast Cell Responses-In a systematic effort aimed at elucidating the biologic consequences of JAK3 inhibition in mast cells, we first sought to determine whether the JAK3 inhibitor WHI-P131 could prevent the IgE receptor/Fc⑀RI-mediated activation of mast cells. Because the IgE receptor/Fc⑀RI-mediated activation of mast cells results in a distinct morphologic transformation with marked cell spreading due to membrane ruffling, microtubule formation, and actin polymerization (47), we evaluated the effects of WHI-P131 on the activation-associated transforma- After stimulation with DNP-BSA for 1 h, cells were fixed in cold methanol for 15 min followed by permeabilization with PBS containing 0.1% Triton X-100. Fixed cells were stained with a monoclonal antibody reactive with ␣-tubulin (clone B-5-1-2, Sigma) for 40 min at 37°C. After 3 times wash with PBS plus 0.1% Triton X-100, cells were incubated with a fluorescein-labeled secondary antibody (Zymed Laboratories Inc., San Francisco, CA) for another 40 min. Cells were washed three times to remove the unbound antibody. DNA labeling was performed by incubation of coverslips with Toto-3 (Molecular Probes, Eugene, OR) for 10 min. Excessive dye was washed with PBS plus 0.1% Triton X-100. Cells were visualized under MRC 1024 laser scanning microscope after mounting with Vectashield (Vector Laboratories, Inc., Burlingame, CA), as previously reported (38). tion of shape and surface topography of RBL-2H3 mast cells using confocal laser scanning microscopy (48). The vast majority (95%) of unstimulated RBL-2H3 mast cells exhibited a spindle shape with arborized extensions and longitudinally oriented bundles of microtubules (Fig. 7A). Activation of RBL-2H3 mast cells by cross-linking their IgE receptors/Fc⑀RI using IgE/antigen induced a dramatic cell spreading response, and 93% of cells assumed a flattened shape with a generalized microtubule organization throughout their cytoplasm (Fig. 7B). A 2-h incubation with the JAK3 inhibitor WHI-P131 (but not the unsubstituted parent dimethoxyquinazoline compound WHI-P258, which lacks JAK3 inhibitory activity) at a concentration of 30 M prevented the IgE/antigen-induced mast cell activation, as evidenced by markedly reduced spreading (23% flattened, 58% spindle shaped) and microtubule organization (Fig. 7, C-F). In contrast to WHI-P131, the bromo-substituted control dimethoxyquinazoline compound WHI-112, which does not inhibit JAK3, was unable to produce any significant effects on antigen-induced mast cell spreading or microtubule organization (Fig. 7, G and H). In parallel, we tested the effect of the compounds on the viability of RBL-2H3 cells (as assessed by trypan blue dye exclusion test) under these experimental conditions and found that they do not affect cell viability at concentrations as high as 300 M (data not shown).
To further examine the role of JAK3 in IgE receptor/Fc⑀RImediated mast cell activation and degranulation, we next as-  (n ϭ 3). Vehicletreated control RBL-2H3 cells released 72.0 Ϯ 0.2% of their total cellular ␤-hexosaminidase content after treatment with A23187. Notably, pretreatment or RBL-2H3 cells with 10 or 30 M WHI-P131 did not inhibit this calcium ionophore-induced degranulation. In parallel, IgEsensitized RBL-2H3 cells were also treated with WHI-P131 (3, 10, or 30 M) and then challenged with specific antigen, as described in the legend to Fig. 8. Vehicle-treated control RBL-2H3 cells released 34.7 Ϯ 5.3% of their total cellular ␤-hexosaminidase content after IgE receptor cross-linking.
treated control mast cells. The data points represent the means Ϯ S.E. obtained from three to six independent experiments. *, p Ͻ 0.05 compared with control as determined by Student's t test; **, p Ͻ 0.0001 compared with control as determined by Student's t test.
sessed the effects of the JAK3 inhibitor WHI-P131 on mast cell degranulation and mediator release induced by IgE/antigen. WHI-P111 and WHI-P112, which do not inhibit JAK3, were included as control compounds. RBL-2H3 mast cells were preincubated with increasing concentrations of the test compounds or vehicle for 1 h before challenge with antigen (DNP-BSA). Stimulation of RBL-2H3 mast cells using IgE/antigen resulted in release of significant amounts of ␤-hexosaminidase (45.l Ϯ 3.1% of the total cellular content), LTC 4 (11.3 Ϯ 1.3 ng/10 6 cells), and TNF␣ (160 Ϯ 33.0 pg/10 6 cells). Notably, WHI-P131 prevented mast cell degranulation and release of preformed granule-associated ␤-hexosaminidase (Fig. 8A) as well as release of the newly synthesized arachidonic acid metabolite LTC 4 , (Fig. 8B) and the proinflammatory cytokine TNF␣ (Fig. 8C) in a dose-dependent fashion with near to complete inhibition at Ն30 M. Unlike these JAK3 inhibitors, the control dimethoxyquinazoline derivatives WHI-P111 and WHI-P112 lacking JAK3 inhibitory activity did not inhibit mast cell degranulation or mediator release after IgE receptor/Fc⑀RI cross-linking (Fig. 8).
The functional integrity of the secretory machinery of mast cells responsible for the release of mediators can be evaluated using ionophores that result in an increase of the intracellular calcium concentration and mediator release independent of the IgE receptor-linked proximal signal transduction events. The observed inhibitory effects of WHI-P131 on IgE receptor/Fc⑀RImediated mast cell responses were not due to a functional impairment of the distal secretory machinery in mast cells, because WHI-P131 did not prevent degranulation (as measured by ␤-hexosaminidase release) of RBL-2H3 mast cells after treatment with the ionophore A23187 (0.5 M) at concentrations sufficient for inhibition of antigen-induced degranulation of IgE-sensitized RBL-2H3 cells (Fig. 9).
We next set out to examine the effects of WHI-P131 on IgE receptor/Fc⑀RI-mediated degranulation and mediator release from human mast cells. To this end, we cultured fetal liverderived human mast cells in the presence of stem cell factor and IL-4 for 5 weeks. IgE-sensitized human mast cells were exposed to vehicle or increasing concentrations of WHI-P131 for 30 min. Human mast cells store the mast cell-specific protease ␤-tryptase in their secretory granules (Fig. 10A), and the release of ␤-tryptase by degranulation is a specific marker for human mast cell activation (49). The Fc⑀RI receptors of fetal liver-derived human mast cells were cross-linked with anti-IgE, and the resulting mast cell degranulation (i.e. ␤-tryptase release) (24) and LTC 4 release (30) were quantitated. WHI-P131 inhibited the release of ␤-tryptase (Fig. 10B) as well as LTC 4 (Fig. 10C) from IgE/antigen stimulated human mast cells in a dose-dependent fashion.
Taken together, these in vitro JAK3 inhibitor studies pro- vided evidence that JAK3 in mast cells is a key regulator of IgE receptor/Fc⑀RI-mediated responses. The ability of the JAK3 inhibitor WHI-P131 to inhibit mast cell degranulation as well as mediator release after IgE receptor/Fc⑀RI cross-linking prompted us to further evaluate the potential of this compound as an anti-allergic agent.
Effects of the JAK3 Inhibitor WHI-P131 on in Vivo Mast Cell Responses-Increased vascular permeability induced by mast cell mediators, such as histamine and leukotrienes, is a hallmark of anaphylaxis (37,44). Therefore, we first examined the effect of the JAK3 inhibitor WHI-P131 on vascular permeability in a well characterized murine model of passive cutaneous anaphylaxis (39). WHI-P131 inhibited the IgE/antigen induced plasma exudation (as measured by extravasation of systemically administered Evan's blue dye) in mice that had been presensitized with antigen-specific IgE by 70% at the 25 mg/kg nontoxic dose level, which is 10 times lower than its lethal dose (LD) 10 (i.e. the dose level that results in death of 10% of treated mice) (Fig. 11A). Next, we studied the effect of WHI-P131 on passive systemic anaphylaxis in mice (39,50,51). Mice were sensitized intravenously with 50 g of anti DNP-IgE. 24 h later, drug-or vehicle-treated animals were challenged with 2 mg of DNP-BSA systemically in presence of 0.5% Evan's blue dye to document the increased vascular permeabilily. Plasma FIG. 11. Prevention of anaphylaxis in mice using the JAK3 inhibitor WHI-P131. A, the effects of WHI-P131 on anaphylaxis-associated vascular hyperpermeability were examined by evaluating the cutaneous extravasation of albumin-bound Evan's blue dye in mice (n ϭ 12), and the plasma exudation indices were determined for vehicle-treated as well as WHI-P131-treated mice, as described under "Experimental Procedures." To study the effect of WHI-P131 on anaphylaxis, IgE-sensitized mice were injected with two consecutive doses of 10 or 25 mg/kg WHI-P131 at 90 min before and 30 min before the antigen challenge, respectively. Mice were then challenged with 100 g of DNP-BSA in 2% Evan's blue dye, and the plasma exudation indices (fold increase in optical density over PBS-treated ears) were determined. The data points represent the means Ϯ S.E. The mean A 620 nm vehicle-treated ears was 0.22 Ϯ 0.04 before and 0.92 Ϯ 0.05 after the IgE/antigen challenge. *, p Ͻ 0.05 compared with vehicle-treated controls. B, plasma extravasation during systemic anaphylaxis was evaluated as described under "Experimental Procedures." B.1, foot pad of a control mouse after intravenous injection of Evan's blue dye alone. B.2, foot pad of a vehicle-treated mouse after coadministration of DNP-BSA and Evan's blue. B.3, foot pad of a WHI-P131-treated mouse after coadministration of DNP-BSA and Evan's blue. C, for histopathologic evaluation of mast cell degranulation, ears were removed 1 h after the antigen challenge from vehicle-treated as well as WHI-P131-treated mice and formalin-fixed thin sections (3-5 m) of ears from were stained with Avidin-FITC. D, blood histamine levels of sensitized mice after the antigen challenge. Blood was collected by retro-orbital bleeding, and histamine levels were measured by ELISA. Histamine levels are expressed as nM. The histamine levels in the blood of PBS-treated mice and IgE/antigen-stimulated mice were 493 Ϯ 131 and 7527 Ϯ 2102 nM, respectively. The results are the means Ϯ S.E. (n ϭ 3). E, to study the effect of JAK3 inhibitor WHI-P131 on fatal anaphylaxis in mice, BALB/c mice were sensitized with 100 mg/kg bovine serum albumin in 200 l of the adjuvant aluminum hydroxide gel (Reheis, Inc., Berkeley, NJ), which favors the production of IgE in response to the presented antigen. 10 days later, mice were treated with two doses of WHI-P131 (45 mg/kg) or vehicle 30 min apart and then challenged with an intravenous injection of the 10 mg/kg BSA. Cumulative proportions of mice surviving anaphylaxis-free are shown according to the time after the antigen challenge. Life table analysis and statistical comparisons using the log-rank test were performed, as previously reported (26,37). exudation was assessed by blue coloring of foot pads 30 min after the antigen challenge (50). Vehicle-treated control mice showed a marked blue coloring of their foot pads after antigen challenge, but no significant blue coloring was observed in mice pretreated with the JAK3 inhibitor WHI-P131 (Fig. 11B). We also assessed mast cell degranulation in histologic sections of ears by examining their fluorescence intensity after staining with avidin-FITC. Avidin specifically binds to heparin, the major proteoglycan in the granules of connective tissue mast cells (40). The fluorescence intensity of the stained mast cells is proportional to the amount of heparin, and therefore degranulation reduces the fluorescence intensity. Whereas the IgE/ antigen challenge resulted in a marked reduction of fluorescence intensity of avidin-FITC-stained tissue mast cells of control mice consistent with degranulation-associated depletion of heparin, no reduction in fluorescence intensity was observed for mast cells from WHI-P131-pretreated mice (Fig.  11C). Because a major vasoactive mediator released from activated mast cells is histamine and systemic anaphylaxis in humans and rodents has been associated with a significant increase in blood histamine levels (39, 50), we obtained blood samples from mice 5 min after the antigen challenge to determine their plasma histamine levels (29). As expected, the antigen challenge resulted in marked elevation of plasma histamine levels, but pretreatment with the JAK3 inhibitor WHI-P131 substantially reduced this histamine response (Fig. 11D). These results demonstrate that WHI-P131 is capable of preventing passive cutaneous and systemic anaphylaxis by blocking mast cell degranulation in vivo.
We next tested the efficacy of WHI-P131 in a model of IgE/ antigen-induced active systemic anaphylaxis. To this end, mice were first injected with BSA in an aluminum hydroxide gel to trigger a BSA-specific IgE response. 10 days later, these BSAsensitized mice were rechallenged with this antigen to induce anaphylaxis. 8 of 15 (53%) BSA-sensitized mice that were treated with WHI-P131 prior to antigen challenge survived without any signs of anaphylaxis, whereas 12 of 12 control mice (100%) developed anaphylaxis within 45 min after antigen challenge (p Ͻ 0.0001 by log-rank test; Fig. 11E).
In summary, our studies detailed herein provide experimental evidence that targeting JAK3 in mast cells with WHI-P131, a potent and specific inhibitor of JAK3 (18,19), abrogates mast cell degranulation and release of allergic mediators in vitro and, at nontoxic dose levels, prevents IgE receptor/Fc⑀RI-mediated anaphylactic reactions, including fatal anaphylactic shock, in vivo. Based on our own data presented herein and previous reports regarding the known function of Syk in IgE receptor/Fc⑀RI-mediated responses (7,44), we speculate that JAK3 and Syk may cooperate in initiation of mast cell-mediated hypersensitivity reactions. To our knowledge, this is the first report indicating the existence of an important cross-talk between the SYK-and JAK3-dependent signal transduction pathways in mast cells.
Studies employing chimeric receptors and chimeric JAKs support the notion that JAKs act primarily as conduits of signal transmission by an autoritative cytokine receptor (27,52). Recent studies suggest that individual JAKs may also have distinct functions and promote unique signals by selectively recognizing specific substrates (27,53). Janus kinase JAK3 has been shown to play an important role for lymphocyte development, activation, and cytokine responsiveness (10,12). The present study expands our knowledge of JAK3 functions and reveals that JAK3 has essential and nonredundant functions for the full signaling capacity of the high affinity IgE receptor on mast cells. This investigation extends earlier studies on the role of PTK in mast cell responses (4, 6, 7) and offers new evidence supporting the therapeutic potential of PTK inhibitors in the treatment of allergic disorders. Because of its in vivo potency and lack of systemic toxicity, the JAK3-specific PTK inhibitor WHI-P131 may offer the basis for new and effective treatment as well as prevention programs for mast cell-mediated hypersensitivity reactions in clinical settings.