And Impaired Mast Cell Activation In

Impaired Mast Cell Activation in Gene-Targeted Mice Lacking the Serum- and Glucocorticoid-Inducible Kinase SGK1

This information is current as Malgorzata Sobiesiak, Ekaterina Shumilina, Rebecca S. of September 24, 2021. Lam, Florian Wölbing, Nicole Matzner, Susanne Kaesler, Irina M. Zemtsova, Adrian Lupescu, Naima Zahir, Dietmar Kuhl, Martin Schaller, Tilo Biedermann and Florian Lang J Immunol published online 11 September 2009

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 11, 2009, doi:10.4049/jimmunol.0803017 The Journal of Immunology

Impaired Mast Cell Activation in Gene-Targeted Mice Lacking the Serum- and Glucocorticoid-Inducible Kinase SGK11

Malgorzata Sobiesiak,2* Ekaterina Shumilina,2* Rebecca S. Lam,3* Florian Wo¨lbing,† Nicole Matzner,* Susanne Kaesler,† Irina M. Zemtsova,* Adrian Lupescu,* Naima Zahir,* Dietmar Kuhl,‡ Martin Schaller,† Tilo Biedermann,4† and Florian Lang4*

The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1؊/؊) and their wild-type littermates (sgk1؉/؉). The BMMC number as well as CD117, CD34, and Fc␧RI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the Fc␧RI receptor, Ca2؉ entry but not Ca2؉ release from intracellular stores was markedly impaired in sgk1؊/؊ BMMCs. The currents through Ca2؉-activated K؉ channels induced by Ag were significantly higher in sgk1؉/؉ Downloaded from BMMCs than in sgk1؊/؊ BMMCs. Treatment with the Ca2؉ ionophore ionomycin (1 ␮M) led to activation of the K؉ -channels in both genotypes, indicating that the Ca2؉-activated K؉ channels are similarly expressed and sensitive to acti vation by Ca2؉ in sgk1؉/؉ and sgk1؊/؊ BMMCs, and that blunted stimulation of Ca2؉-activated K؉ channels was secondary to decreased Ca2؉ entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1؊/؊ BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substan- http://www.jimmunol.org/ tially reduced in sgk1؊/؊ mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1؊/؊ than in sgk1؉/؉mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction. The Journal of Immunology, 2009, 183: 4395–4402.

ast cells express IgE receptors (Fc␧RI) and largely ac- pressed in all tissues tested (33). SGK1 has been shown to regulate count for IgE-dependent allergic reactions (1, 2), such a variety of ion channels (32). The kinase contributes to the reg- M as allergic rhinitis (3), asthma (4), anaphylaxis, and ulation of salt appetite (34), renal electrolyte excretion (35, 36), delayed-type hypersensitivity reactions (5–8). They release a va- and blood pressure (37). It participates in the regulation of intes- by guest on September 24, 2021 riety of cytokines and thus regulate the function of other inflam- tinal transport (38), peripheral glucose uptake (39), and insulin matory cells such as neutrophils and T cells (7, 9–14). Mast cell release from pancreatic ␤-cells (40). Several observations point to function is under tight regulation, involving activation of Ca2ϩ its pivotal role in fibrosing disease (41–45). Nothing is known, channels (15–22), Kϩ channels (15, 23–25), and ClϪ channels (18, however, about the influence of SGK1 on those ion channels, 19). Mast cell activation includes stimulation of PI3K (26–29), an which are decisive in the regulation of mast cell function. effect correlating with PI3K-sensitive ion channel activity (30). This study was performed to elucidate whether the PI3K-depen- Signaling molecules downstream of PI3K include the serum- dent ion channel regulation and function of mast cells similarly and glucocorticoid-inducible kinase SGK15 (31, 32), which is ex- involves SGK1. Thus, the function of mast cells has been studied in gene-targeted mice lacking SGK1 (sgk1Ϫ/Ϫ) and their wild-type littermates (sgk1ϩ/ϩ). *Department of Physiology, University of Tu¨bingen, Tu¨bingen, Germany; †Depart- ment of Dermatology, University of Tu¨bingen, Tu¨bingen, Germany; and ‡Center for Molecular Neurobiology, Institute for Molecular and Cellular Cognition, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Materials and Methods Animals Received for publication September 11, 2008. Accepted for publication July 23, 2009. The costs of publication of this article were defrayed in part by the payment of page All animal experiments were conducted according to the guidelines of the charges. This article must therefore be hereby marked advertisement in accordance American Physiological Society as well as the German law for the welfare with 18 U.S.C. Section 1734 solely to indicate this fact. of animals and were approved by local authorities. 1 This work was supported by Deutsche Forschungsgemeinschaft (Grants SFB 766, As described previously (36), a conditional targeting vector was gener- SFB 685 A6, Bi 696/5-1, and Bi 696/3-1), the International Graduate School (Grant ated from a 7-kb fragment encompassing the entire transcribed region on GRK 1302/1), The PI3K Pathway in Tumor Growth and Diabetes, and the FORTU¨ NE 12 exons. The neomycin resistance cassette was flanked by two loxP sites program of the University of Tu¨bingen. and inserted into intron 11. Exons 4–11, which code for the sgk1 domain, 2 Contributed equally to the work and thus share first authorship. were floxed by inserting a third loxP site into intron 3. Targeted R1 em- 3 bryonic stem cells were transiently transfected with Cre recombinase. A Current address: Department of Molecular Neurogenetics, Max Planck Institute of clone with a recombination between the first and third loxP site (type I Biophysics, Max-von-Laue Strasse 3, D-60438 Frankfurt am Main, Germany. recombination) was injected into C57BL/6 blastocytes. Male chimeras 4 Address correspondence and reprint requests to Dr. Florian Lang or Dr. Tilo Bied- were bred to 129/SvJ females. Heterozygous sgk1-deficient mice were ermann, Department of Physiology, University of Tu¨bingen, Gmelinstrasse 5, D-72076 Tu¨bingen, Germany. E-mail address: fl[email protected] or De- partment of Dermatology, University of Tu¨bingen, Liebermeisterstrasse 25, D-72076 bumin; TNCB, trinitrochlorobenzene; TRPV2, transient receptor potential cation Tu¨bingen, Germany. E-mail address: [email protected] channel, subfamily V, member 2; PDK1, phosphoinositide-dependent kinase. 5 Abbreviations used in this paper: SGK1, serum- and glucocorticoid-inducible kinase-1; BMMC, bone marrow-derived mast cell; DNP-HSA, DNP-human serum al- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803017 4396 IMPAIRED MAST CELL ACTIVATION IN sgkϪ/Ϫ MICE backcrossed to 129/SvJ wild-type mice for two generations and then in- holding potential to potentials between Ϫ115 mV and ϩ65 mV. The cur- tercrossed to generate homozygous sgk1Ϫ/Ϫ and sgk1ϩ/ϩ littermates. The rents were recorded with an acquisition frequency of 10 kHz and were 3 animals were genotyped by PCR using standard methods. The study has kHz low-pass ﬁltered. been performed in 6- to 8-wk-old female and male sgk1ϩ/ϩ and The pipet solution contained (in millimolar concentrations): 140 potas- Ϫ/Ϫ sgk1 mice. sium gluconate, 5 KCl, 1.2 MgCl2, 2 EGTA, 1.26 CaCl2 (pCa 7), 2 Na2ATP, and 10 HEPES/KOH (pH 7.2), and was used in combination with Culture of bone marrow-derived mast cells (BMMC) NaCl Ringer bath solution (in millimolar concentrations): 145 NaCl, 5

ϩ/ϩ Ϫ/Ϫ KCl, 2 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES-NaOH (pH 7.4). For some Femoral bone marrow cells from 6- to 8-wk-old sgk1 and sgk1 2ϩ naive mice were cultured for 4 wk in RPMI 1640 (Life Technologies) experiments, Ca -free Ringer solution was used that contained 1 mM EGTA. Where indicated, the Ag DNP-HSA (50 ng/ml; Sigma-Aldrich), the containing 10% FCS, 1% penicillin/streptomycin, 20 ng/ml IL-3 (R&D ␮ Systems), and 100 ng/ml concentrations of the c-kit ligand stem cell factor channel blockers clotrimazole (1.5 M; Sigma-Aldrich), apamin (500 nM; (Peprotech; Tebu-bio). BMMC maturation was confirmed by flow cytom- Sigma-Aldrich), and iberiotoxin (100 nM; Sigma-Aldrich), as well as the 2ϩ ␮ etry (FACSCalibur; BD Biosciences) using the following specific fluores- Ca ionophore ionomycin (1 M; Sigma-Aldrich) were added to the bath cence-labeled Abs: PE-labeled anti-Fc␧RI (eBioscience); allophycocyanin- solution. DNP-HSA, apamin, and iberiotoxin were prepared as stock so- labeled anti-CD117 (BD Biosciences); and FITC-labeled anti-CD34 (BD lutions in water, clotrimazole in ethanol, and ionomycin in DMSO. The Biosciences). For experiments, BMMCs were sensitized for 1 h with final concentrations of DMSO and ethanol were 0.01 and 0.005%, monoclonal mouse anti-dinitrophenyl mouse IgE (anti-DNP IgE, 10 ␮g/ml respectively. per 1 ϫ 106 cells, clone SPE-7; Sigma-Aldrich) in culture medium and then The offset potentials between both electrodes were zeroed before seal- challenged acutely with dinitrophenyl-human serum albumin (DNP-HSA, ing, and the potentials were corrected for liquid junction potentials as es- 50 ng/ml; Sigma-Aldrich). timated according to the method of Barry and Lynch (48). The original whole-cell current traces are depicted without further filtering and currents Determination of mast cell numbers in the ear of the individual voltage square pulses are superimposed. The applied volt- ages refer to the cytoplasmic face of the membrane with respect to the Downloaded from Anesthetized mice were euthanized by cervical dislocation, and the skin extracellular space. The inward currents, defined as flow of positive charge was cleansed with 70% ethanol. Ears were cut off at the base, fixed in 4% from the extracellular to the cytoplasmic membrane face, are negative cur- paraformaldehyde overnight, and finally embedded in paraffin. Tissue sec- rents and depicted as downward deflections of the original current traces. tions 3–4 ␮m thick taken from the middle of the ears were prepared, deparaffinized, and stained with toluidine blue. Mast cell numbers of at ␤-Hexosaminidase release and IL-6 production least three slices per ear were determined using a Zeiss Axiovert 200 mi- ␤ ϫ 6 croscope with a LD Achroplan ϫ40 lens in bright-field mode. For the -hexosaminidase release assay, mature BMMCs (0.3 10 per well) were seeded in a 96-well plate in fresh medium with 10 ␮g/ml anti- http://www.jimmunol.org/ Peritoneal lavage DNP IgE Ab for 1 h. Afterwards, cells were washed in Tyrode’s buffer (Sigma-Aldrich) and challenged with DNP-HSA (50 ng/ml) or for positive Anesthetized mice were euthanized by cervical dislocation, and the ab- control with 100 ng/ml PMA (Sigma-Aldrich) and 1 ␮M ionomycin for 15 dominal skin was cleansed with 70% ethanol. Sterile 0.9% NaCl (4 ml) was min at 37°C. Supernatant (20 ␮l) and 2 mM p-nitrophenyl-N-acetyl-␤-D- then instilled into the peritoneum. The abdomen was massaged gently for glucosaminide (20 ␮l; Sigma-Aldrich) diluted in 0.2 M citrate buffer, pH 1 min and then opened with sterile scissors. Recovered peritoneal lavage 4.5, were mixed and incubated for2hat37°C. The reaction was terminated ϫ fluid was centrifuged at 2000 g for 5 min, the cell pellets were resus- by the addition of 60 ␮l of 1 M Tris buffer, pH 9.0, and the optical density pended in RBC lysis buffer for 1 min and recentrifuged, and then the cell was measured at 405 nm in an ELISA microplate reader. pellet was resuspended in PBS. Slides were prepared as thick blood films Data are shown as the percent of the total release (0.1% Triton X-100) and fixed with 4% paraformaldehyde. After staining with toluidine blue, at corrected for spontaneous release. by guest on September 24, 2021 least 600 cells/slide were counted. Alternatively, peritoneal lavage fluid The concentration of IL-6 released from mast cells, which were sensi- was analyzed by FACS analysis. tized with 1 ␮g/ml IgE for6hat37°C and then stimulated with DNP-HSA Measurement of intracellular Ca2ϩ concentration (50 ng/ml) for 30 min, 6 h, or 24 h, was measured by ELISA according to the instructions of the manufacturer (BD Biosciences). Intracellular Ca2ϩ measurements were performed as described (46) using fura 2-AM. Fluorescence measurements were conducted with an inverted Passive systemic anaphylaxis/Ag-induced anaphylaxis and phase-contrast microscope (Axiovert 100; Zeiss). Cells were excited alter- histamine serum concentrations natively at 340 and 380 nm, and the light was deflected by a dichroic mirror ␮ ϳ ␮ into the objective (Fluar 40ϫ/1.30 oil; Zeiss). Emitted fluorescence inten- Mice were sensitized with 2 g/1 g weight ( 50 g per mouse) anti-DNP IgE sity was recorded at 505 nm; data were acquired using specialized com- by i.p. application. The next day, mice were challenged with either DNP-HSA ϳ ␮ ␮ puter software (Metafluor; Universal Imaging). As a measure of the in- ( 120 g/mouse or 4.8 g/1 g weight) or PBS. Body temperature was mon- crease of cytosolic Ca2ϩ activity, the slope and peak of the changes in the itored before and each 7 min after Ag challenge with a BAT-10 type T ther- 340 nm:380 nm ratio were calculated for each experiment. mocouple thermometer and a RET-3 rectal probe for mice (Physitemp Instru- BMMCs were sensitized with IgE at 37°C either with 10 ␮g/ml for 1 h ments; distributed by World Precision Instruments) using a Duo18 data or with 100 ng/ml overnight and subsequently loaded with fura 2-AM (2 recording system (World Precision Instruments) during the midportion of the ␮M; Molecular Probes) for 20 min at 37°C. Intracellular Ca2ϩ was mea- light phase of the light cycle. Mice were placed with the tail raised and the sured before and following addition of DNP-HSA to IgE-sensitized Vaseline petroleum jelly-covered probe was inserted a standardized distance of BMMCs in the absence or presence of extracellular Ca2ϩ. 2 cm until a stable temperature reading was obtained. Baseline temperature For intracellular calibration purposes, ionomycin (10 ␮M) was applied was measured after habituating mice to rectal probe insertion. Ambient room at the end of each experiment. Experiments were performed with Ringer temperature was 23°C, and the animals were exposed to a 12-h light and 12-h solution containing (in millimolar concentrations): 125 NaCl, 5 KCl, 1.2 dark cycle (7 a.m.–7 p.m.). Data are expressed as a change in body temperature following treatment (⌬°C). Thirty minutes after induction of an anaphylactic MgSO4, 2 CaCl2,2Na2HPO4, 32 HEPES, 5 glucose, pH 7.4. To reach nominally Ca2ϩ-free conditions, experiments were performed using Ca2ϩ- reaction, blood was taken for the analysis of histamine plasma concentration. free Ringer solution containing (in millimolar concentrations): 125 NaCl, Histamine was measured by ELISA according to the instructions of the manufacturer (IBL-Hamburg). 5 KCl, 1.2 MgSO4,2Na2HPO4, 32 HEPES, 0.5 EGTA, 5 glucose, pH 7.4. Patch clamp Trinitrochlorobenzene (TNCB)-induced contact hypersensitivity reaction Patch clamp experiments have been performed at room temperature in voltage-clamp, fast-whole-cell mode (47). The cells were continuously per- TNCB (Sigma-Aldrich) was used to induce delayed-type hypersensitivity re- fused through a flow system inserted into the dish. The bath was grounded actions, which are strictly dependent on hapten-specific memory T cells and via a bridge filled with NaCl Ringer solution. Borosilicate glass pipets (2- mast cells and are associated with a strong infiltrate of polymorphic neutrophils to 4-MOhm tip resistance; GC 150 TF-10; Harvard Apparatus) manufac- (10, 49). These memory T cells lead to contact hypersensitivity reactions when tured by a microprocessor-driven DMZ puller (Zeitz) were used in com- the hapten is applied to the skin of sensitized mice (9). The mice were sensi- bination with a MS314 electrical micromanipulator (MW; Ma¨rzhaüser). tized with 5% TNCB (n ϭ 6) (80 ␮l of a 4:1 mixture of acetone-olive oil) at The currents were recorded by an EPC-9 amplifier (Heka) using Pulse the shaved abdomen; size, 2 ϫ 2 cm. One week later, mice were challenged software (Heka) and an ITC-16 interface (Instrutech). Whole-cell currents with 1% TNCB (20 ␮l of a 1:9 mixture of acetone-olive oil) on both sides of were determined as 10 successive 400-ms square pulses from the Ϫ35 mV the ear. In the 5% TNCB solution acetone is an irritant, whereas it is used The Journal of Immunology 4397 Downloaded from

FIGURE 1. Maturation of BMMCs from sgk1ϩ/ϩ and sgk1Ϫ/Ϫ mice. A, Original dot plots of CD117-, CD34- and Fc␧RI-positive BMMCs from ϩ/ϩ Ϫ/Ϫ sgk1 and sgk1 mice. Numbers depict the percent of cells in the ϩ ϩ Ϫ Ϫ FIGURE 2. Mast cell number in sgk1 / and sgk1 / mice. A, Original respective quadrant, acquired within the mast cell gate. B, Percent of mast ϩ ϩ ϩ ϩ dot plots of CD117 Fc␧RI mast cells from the peritoneum of sgk1 / http://www.jimmunol.org/ cells in primary culture. Mean percent (ϮSE; n ϭ 6 individual BMMC Ϫ/Ϫ ϩ/ϩ Ϫ/Ϫ (left) and sgk1 (right) mice. Values are the percent of cells in the re- cultures) of sgk1 (f) and sgk1 (Ⅺ) BMMCs acquired within the ϩ ϩ spective quadrants. B, Percent of CD117 Fc␧RI mast cells (ϮSE) ana- mast cell gate. APC, Allophycocyanin. ϩ ϩ lyzed by FACS analysis in the peritoneum from eight sgk1 / (f) and four sgk1Ϫ/Ϫ (Ⅺ) mice. C, Percent of toluidine blue-positive mast cells (ϮSE) among leukocytes in the peritoneum from eight sgk1ϩ/ϩ (f) and six solely as a solvent in the 1% TNCB solution. Specific ear swelling was de- sgk1Ϫ/Ϫ (Ⅺ) mice. D, Number of mast cells (ϮSE) in skin, analyzed by termined by measuring ear thickness with a micrometer (Oditest; Kroeplin) staining of ear sections with toluidine blue. Mean mast cell numbers of before and 4, 8, 12, and 24 h after TNCB challenge. Data are expressed as a toluidine blue-positive cells in 1 view field (ϫ400) as calculated from 18 change in ear swelling comparing to thickness before treatment (⌬␮m), cor- ϩ ϩ Ϫ Ϫ view fields from 9 sgk1 / (f) and 6 sgk1 / (Ⅺ) mice. APC, rected for irritation reaction in control mice (n ϭ 4). by guest on September 24, 2021 Allophycocyanin. Statistics Ϯ Data are provided as means SE; n represents the number of independent (Fig. 3B, right). Stimulation of the BMMCs with Ag in Ca2ϩ-free experiments. All data were tested for significance using Student’s unpaired 2ϩ two-tailed t test or ANOVA (Dunnet’s test), where applicable. Results with solution allowed an estimate of Ca release from intracellular p Ͻ 0.05 were considered statistically significant. stores (Fig. 3C). This release was not significantly different between the genotypes (Fig. 3D). Results In patch clamp experiments, Ag stimulation of BMMCs led to a As illustrated in Fig. 1, BMMCs express CD117, CD34, and rapid increase of Kϩ-selective conductance. Fig. 4A represents Fc␧RI, i.e., receptors typically expressed by mast cells. No signif- original current traces obtained before (control) and after stimula- icant difference in receptor abundance was observed between tion of IgE-sensitized sgk1ϩ/ϩ BMMC with Ag in a typical ex- BMMCs from SGK1 knockout mice (sgk1Ϫ/Ϫ) and their wild-type periment. Current-voltage relationships (I/V curve; Fig. 4B) and littermates (sgk1ϩ/ϩ). current conductances, calculated by linear regression (slope) of the The number of mast cells in the peritoneum analyzed either by I/V curve (Fig. 4C), demonstrate that the current activated by Ag FACS analysis (Fig. 2, A and B) or by toluidine blue staining (Fig. was dependent on the presence of extracellular Ca2ϩ. Removal of 2C) as well as the number of mast cells in the skin, analyzed by Ca2ϩ from the bath solution significantly blunted the activation of staining of ear sections with toluidine blue (Fig. 2D), was similar the current (Fig. 4, B and C). The current was inhibited by 1.5 ␮M in sgk1ϩ/ϩ and sgk1Ϫ/Ϫ mice. clotrimazole (Fig. 4C) but was not sensitive to apamin (500 nM) or According to fura-2 fluorescence, IgE-dependent activation of iberiotoxin (100 nM; data not shown). The same current could be BMMCs was followed by an increase in cytosolic Ca2ϩ concen- induced by the Ca2ϩ ionophore ionomycin (1 ␮M, Fig. 4F). The tration. The increase was significantly steeper in sgk1ϩ/ϩ than in current was thus carried by Ca2ϩ-activated Kϩ channels, which Ϫ/Ϫ sgk1 BMMCs (Fig. 3, A and B). Accordingly, the maximal have recently been identified as KCa3.1 (46) and are known to be fluorescence ⌬ratio (peak) and the slope of the ratio (⌬ratio/time) activated following Fc␧RI cross-linking in human mast cells (18, upon stimulation with Ag were significantly smaller in sgk1Ϫ/Ϫ 24). The current induced by Ag in sgk1Ϫ/Ϫ BMMCs was signifi- BMMCs than in sgk1ϩ/ϩ BMMCs (Fig. 3B). Routinely, the cells cantly smaller than in wild-type cells (Fig. 4, D and E). Following were sensitized with 10 ␮g/ml IgE for 1 h, but in one set of ex- treatment of the cells with ionomycin, the Kϩ channels were ac- periments a lower concentration of IgE was used (100 ng/ml over- tivated in both genotypes. The maximal current tended to be night). In this case, stimulation with Ag increased cytosolic Ca2ϩ slightly higher in sgk1ϩ/ϩ as in sgk1Ϫ/Ϫ BMMCs (Fig. 4, F and concentration to an extent similar to that of the higher IgE con- G), a difference, however, that was not statistically different. Thus, centration. At the lower IgE concentrations, the values were again Ca2ϩ-activated Kϩ channels are similarly expressed and are significantly different between sgk1Ϫ/Ϫ and sgk1ϩ/ϩ BMMCs similarly sensitive to activation by Ca2ϩ in sgk1ϩ/ϩ and sgk1Ϫ/Ϫ 4398 IMPAIRED MAST CELL ACTIVATION IN sgkϪ/Ϫ MICE

FIGURE 3. Ag-induced Ca2ϩ entry into BMMCs from sgk1ϩ/ϩ and sgk1Ϫ/Ϫ mice. A, Representative traces showing the ratio of 340:380 nm Fura-2 fluorescence in fura 2-AM-loaded BMMCs before and following acute addition of 50 ng/ml Ag. At the end of each experiment, ionomycin (10 ␮M) was added for calibration. For quantification of the Ca2ϩ entry into the BMMCs, the slope (‚ ratio/s, gray line) and peak (‚ ratio, dotted lines) were calculated following addition of Ag. B, Means Ϯ SE) of the peak (top) and slope (bot- tom) of the fluorescence ratio change for sgk1ϩ/ϩ (n ϭ 8; f) and sgk1Ϫ/Ϫ (n ϭ 9; Ⅺ) BMMCs sensitized with either 10 ␮g/ml IgE for1h(left) or 100 ng/ml IgE overnight (right) following stimulation with Ag (50 ng/ p Ͻ 0.01) indicate significant Downloaded from) ءء p Ͻ 0.05) and) ء .(ml difference between both groups (two-tailed unpaired t test). C, Representative traces showing the fura-2 fluorescence ratio before and after addition of Ag (50 ng/ml) in the absence of extracellular Ca2ϩ in fura 2-AM- loaded BMMCs. To create a Ca2ϩ-free environment, EGTA (0.5 mM) was added to the Ca2ϩ-free bath solution. D, Means Ϯ SE of the peak value (left) and slope http://www.jimmunol.org/ (right) of the fluorescence ratio change for sgk1ϩ/ϩ (n ϭ 6; f) and sgk1Ϫ/Ϫ (n ϭ 4; Ⅺ) BMMCs upon stimulation with Ag in Ca2ϩ-free solution. by guest on September 24, 2021

BMMCs. Accordingly, the blunted activation by Ag results largely severe impairment of in vivo mast cell function in from decreased Ca2ϩ entry into sgk1Ϫ/ϪBMMCs. SGK1-deficient mice. We also tested the ability of sgk1ϩ/ϩ and sgk1Ϫ/Ϫ BMMCs to Contact hypersensitivity responses to TNCB are prototypic T cell undergo degranulation upon stimulation with Ag via the Fc␧RI mediated delayed type hypersensitivity responses but involve several receptor. BMMCs release ␤-hexosaminidase, an enzyme stored in types of immune cells including mast cells (10). The ear thickness mast cell granules (50). Without stimulation, the release was 8.5 Ϯ following TNCB challenge of sensitized mice shows an early imme- 1.5% (n ϭ 5) in sgk1ϩ/ϩ BMMCs and 7.9 Ϯ 1.4% (n ϭ 5) in diate response, steadily increases, and displays a peak at ϳ24–48 h sgk1Ϫ/Ϫ BMMCs, values not significantly different between ge- declining thereafter. We show here that there is no decrease in contact notypes. Activation by Ag (50 ng/ml) or by ionomycin (1 ␮M) and hypersensitivity responses in sgk1Ϫ/Ϫ compared with sgk1ϩ/ϩ mice PMA (100 ng/ml) stimulated degranulation in both genotypes. The at 24 h indicating unequivocal sensitization and T cell mediated de- effect was, however significantly smaller in sgk1Ϫ/Ϫ BMMCs than layed type hypersensitivity responses in both mouse strains (Fig. 7). in sgk1ϩ/ϩ BMMCs (Fig. 5A). The early Ag-induced release of The increase of ear thickness at 4 and 8 h was significantly reduced in IL-6 (30 min of stimulation with Ag) was also significantly blunted sgk1Ϫ/Ϫ mice, indicating that the early release of preformed mast cell in sgk1Ϫ/Ϫ cells (Fig. 5B). However, no difference between the mediators is also blunted in this type of immune response (Fig. 7 and two genotypes was observed when the cells were stimulated with Ref. 51). Ag for a longer time (6 or 24 h; Fig. 5B). These data confirm that SGK1 is a critical component of the signaling cascade leading to Discussion release of mast cell mediators. The present study reveals a role of SGK1 in the regulation of ion To evaluate whether the observed alterations in sgk1Ϫ/Ϫ channel activity and function of mast cells. Distinct functional dif- BMMCs were also relevant for mast cell function in vivo, we ferences of mast cells are seen between gene-targeted mice lacking triggered passive systemic anaphylaxis. To this end, sgk1ϩ/ϩ and SGK1 (sgk1Ϫ/Ϫ) and their wild-type littermates (sgk1ϩ/ϩ). Defi- sgk1Ϫ/Ϫ mice were sensitized with anti-DNP IgE i.p. After over- ciency of SGK1 blunts Ca2ϩ entry, the currents through Ca2ϩ- night rest, mice received DNP-HSA or saline as a control by i.p. activated Kϩ channels and release of mast cell mediators. Most injection, and body temperature was monitored over time. As importantly, the anaphylactic reaction was strongly impaired in shown in Fig. 6, A and B, the decrease in body temperature fol- sgk1Ϫ/Ϫ mice. lowing Ag treatment was substantially decreased in sgk1Ϫ/Ϫ mice. In theory, the blunted anaphylactic reaction may have been due Serum histamine levels measured 30 min after induction of the to a decreased number of mast cells. We did not observe significant anaphylactic reaction were significantly lower in sgk1Ϫ/Ϫ than in differences of mast cell numbers between sgk1ϩ/ϩ and sgk1Ϫ/Ϫ sgk1ϩ/ϩ mice (Fig. 6C). Thus, these observations indeed point to mice, but the data do not rule out a minor reduction of mast cell The Journal of Immunology 4399 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Ca2ϩ-activated Kϩ current in sgk1ϩ/ϩ and sgk1Ϫ/Ϫ BMMCs upon Ag-IgE-induced stimulation. A, Representative whole-cell currents from sgk1ϩ/ϩ BMMCs elicited by 400-ms pulses ranging from Ϫ115 to ϩ65 mV in 20-mV increments from a holding potential of Ϫ35 mV. Currents were recorded in standard NaCl bath solution before (control) and 2 min after stimulation with 50 ng/ml Ag. Dotted line, zero current value. B, Mean I-V relationships (ϮSE; n ϭ 5–11) in sgk1ϩ/ϩ BMMCs before (control, Œ) or 3 min after stimulation with Ag added either to Ca2ϩ-free NaCl bath solution (Ag/ϪCa2ϩ, Ⅺ)or1mM Ca2ϩ-containing standard NaCl bath solution (Ag/ϩCa2ϩ, F). C, Mean whole-cell conductance in paired experiments (ϮSE; n ϭ 5) as recorded in B before or 3 min after stimulation with Ag added either to Ca2ϩ-free or 1 mM Ca2ϩ-containing standard NaCl bath solution, and after inhibition with 1.5 ␮M clotrimazole. -Significant difference from respective value following addition of the Ag to Ca2ϩ ,ء .Data were calculated by linear regression between Ϫ95 and ϩ45 mV containing NaCl bath (p Ͻ 0.05; ANOVA). D, Mean I-V relationships (ϮSE, n ϭ 11–13) of currents in sgk1ϩ/ϩ (closed symbols) and sgk1Ϫ/Ϫ (open symbols) BMMCs before (control, triangles) or 3 min after (circles) stimulation with Ag (50 ng/ml) added to standard NaCl bath solution. E, Mean whole-cell conductance (Ϯ SE) of sgk1ϩ/ϩ (f) and sgk1Ϫ/Ϫ (Ⅺ) BMMC, as recorded in D before and after stimulation with Ag. Data were calculated by linear regression between Ϫ95 ;p Ͻ 0.001; ANOVA). F, Mean I-V relationships (ϮSE ,ءءء ,p Ͻ 0.01 ءء) ,significant difference from Ag-stimulated sgk1ϩ/ϩ cells ,ءءء and ءء .and ϩ45 mV n ϭ 9–12) of currents in sgk1ϩ/ϩ (closed symbols) and sgk1Ϫ/Ϫ (open symbols) BMMCs before (control, triangles) or 2 min after (ionomycin, circles) stimulation with ionomycin (1 ␮M) added to standard NaCl bath solution. G, Mean whole-cell conductance (ϮSE; n ϭ 9–12) of sgk1ϩ/ϩ (f) and sgk1Ϫ/Ϫ (Ⅺ) BMMCs, p Ͻ 0.001), significant) ءءء .as recorded in F before and after stimulation with ionomycin. Data were calculated by linear regression between Ϫ95 and ϩ45 mV difference from control sgk1ϩ/ϩ cells; ## (p Ͻ 0.01), significant difference from control sgk1Ϫ/Ϫ cells; ANOVA. number in sgk1Ϫ/Ϫ mice. However, the in vitro experiments dem- inflammatory mediators (15–22, 52). Stimulation of mast cells onstrate that the blunted anaphylactic reaction is at least partially is paralleled by the activation of Ca2ϩ-activated Kϩ channels due to defective function of sgk1Ϫ/Ϫ mast cells. (15, 23–25, 46). Kϩ channels maintain the cell membrane The activation of Ca2ϩ channels is critically important for potential, which establishes the electrical driving force for Ca2ϩ the regulation of mast cell function, such as for the release of entry (53). 4400 IMPAIRED MAST CELL ACTIVATION IN sgkϪ/Ϫ MICE Downloaded from

FIGURE 6. Systemic anaphylactic reaction in sgk1ϩ/ϩ and sgk1Ϫ/Ϫ mice. A, Arithmetic means (ϮSE) of changes in body temperature (⌬°C) of sgk1Ϫ/Ϫ mice (n ϭ 8, Ⅺ) and their wild-type sgk1ϩ/ϩ littermates (n ϭ 7, f) following induction of anaphylaxis. Mice were given i.p. anti-DNP IgE http://www.jimmunol.org/ (ϳ50 ␮g per mouse or 2 ␮g/1 g weight) and challenged with DNP-HSA ␤ ϩ/ϩ FIGURE 5. -Hexosaminidase and IL-6 release from sgk1 and (ϳ120 ␮g per mouse or 4.8 ␮g/1 g weight) after overnight rest. B, Arith- Ϫ/Ϫ ␤ Ϯ ϭ sgk1 BMMCs. A, Mean -hexosaminidase release ( SE; n 5 indi- metic means (ϮSE) of maximal changes in body temperature (⌬°C) of Ϫ/Ϫ Ⅺ vidual experiments) from cultured sgk1 BMMCs ( ) and their wild- sgk1Ϫ/Ϫ mice (n ϭ 8, Ⅺ) and their wild-type littermates sgk1ϩ/ϩ (n ϭ 7, ϩ/ϩ f Significant difference between ,ءء .type sgk1 littermates ( ) stimulated for 15 min with 50 ng/ml Ag or f) following induction of anaphylaxis ␮ ␤ 100 ng/ml PMA and 1 M ionomycin. The stimulated -hexosaminidase genotypes (p ϭ 0.002; two-tailed unpaired t test). C, Serum histamine release in each experiment was corrected for the spontaneous release. levels 30 min after induction of anaphylaxis in sgk1Ϫ/Ϫ mice (Ⅺ) and their Ͻ ء Ͻ ءء (p 0.01) and (p 0.05), significant difference between genotypes, wild-type sgk1ϩ/ϩ littermates (f). Data represent the results obtained from two-tailed unpaired t test. B, Mean IL-6 release (ϮSE; n ϭ 3 individual three mice, and from each mouse three individual probes were analyzed. by guest on September 24, 2021 Ϫ/Ϫ Ⅺ -p Ͻ 0.05 two-tailed un ,ء ;Significant difference between genotypes ,ء experiments) from cultured sgk1 BMMCs ( ) and their wild-type ϩ/ϩ f sgk1 littermates ( ) sensitized for 6 h with IgE and then stimulated paired t test. either for 30 min with Ag (50 ng/ml, left)or6hor24h(right). Each -p Ͻ 0.001), two-tailed un) ءءء ;experiment was performed in duplicate paired t test. though SGK1 is a stimulator of a wide variety of Kϩ channels, it is apparently not required for the activation of mast cell Kϩ channels by ionomycin. Along those lines, under control conditions In mast cells, PI3K has been shown to regulate cell proliferation, capacitive Ca2ϩ entry is similar in sgk1ϩ/ϩ lung fibroblasts and adhesion, and migration, as well as Ag-IgE-induced degranulation sgk1Ϫ/Ϫ lung fibroblasts (66). However, the capacitive Ca2ϩ entry and cytokine release (26). Moreover, PI3K was suggested to target is down-regulated by serum deprivation and up-regulated by IGF-1 the transient receptor potential cation channel, subfamily V, member 2 (TRPV2) Ca2ϩ channel in these cells (54). SGK1 has previously been shown to increase the cell membrane abundance and activity of the Ca2ϩ channel TRPV5 (55, 56). It is conceivable that SGK1 has a similar stimulating effect on TRPV2. SGK1-dependent Ca2ϩ entry further affects the activation of Ca2ϩ-activated Kϩ channels following Ag exposure. Kϩ channel activation following ionomycin stimulation is, however, similar in sgk1ϩ/ϩ and sgk1Ϫ/Ϫ BMMCs. Thus, SGK1 blunts the Ag-induced activation of those channels largely by compromising Ca2ϩ entry with only little or no direct effect on the Ca2ϩ-activated Kϩ channels. In other cells, SGK1 has been shown to activate a wide variety of different Kϩ channels (40, 55, 57–64). The present study does not rule out Ca2ϩ-independent effects of SGK1 on Kϩ channels and other functions of mast cells.

Despite the broad role of SGK1 in channel regulation, the phe- ϩ ϩ FIGURE 7. Contact hypersensitivity response to TNCB in sgk1 / and notype of the SGK1 knockout mouse is amazingly mild (32). PI3K sgk1Ϫ/Ϫ mice. Mean increase in ear thickness (⌬␮m, from 4 h-24 h) (ϮSE, activates phosphoinositide-dependent kinase (PDK1), which in n ϭ 6) of sgk1Ϫ/Ϫ mice (Ⅺ) and their wild-type littermates sgk1ϩ/ϩ (f) turn activates the three SGK and the three protein kinase B/Akt during TNCB-induced contact hypersensitivity reaction, corrected for irri- Signiﬁcant difference from sgk1ϩ/ϩ mice ,ء .isoforms (65). The ability of SGK1 to stimulate distinct carriers tation reaction in control mice and channels is shared by several of those isoforms (32). Even (p Ͻ 0.05; two-tailed unpaired t test). The Journal of Immunology 4401

ϩ ϩ Ϫ Ϫ and dexamethasone in sgk1 / fibroblasts but not in sgk1 / fi- 15. Bradding, P., Y. Okayama, N. Kambe, and H. Saito. 2003. Ion channel gene broblasts (66). As was expected, knockout of PDK1 is not com- expression in human lung, skin, and cord blood-derived mast cells. J. Leukocyte Biol. 73: 614–620. patible with survival of the animals (67). Reduced expression of 16. Buess, M., O. Engler, H. H. Hirsch, and C. Moroni. 1999. Search for oncogenic PDK1 in PDK1 hypomorphic mice results in remarkably de- regulators in an autocrine tumor model using differential display PCR: identifi- Ϫ/Ϫ cation of novel candidate genes including the calcium channel mtrp6. Oncogene creased size of the animals (67). The size of sgk1 mice is, 18: 1487–1494. however, normal, indicating that SGK1 could be replaced by the 17. Dernick, G., d. T. Alvarez, and M. Lindau. 2003. Exocytosis of single chromaffin other five PDK1-dependent SGK and protein kinase B/Akt iso- granules in cell-free inside-out membrane patches. Nat. Cell Biol. 5: 358–362. 18. Duffy, S. M., W. J. Lawley, E. C. Conley, and P. Bradding. 2001. Resting and forms. One of those kinases is apparently SGK3, given that growth activation-dependent ion channels in human mast cells. J. Immunol. 167: is delayed in SGK3 knockout mice (68). Along those lines, PDK1- 4261–4270. dependent transport regulation is not abrogated in sgk1Ϫ/Ϫ mice 19. Duffy, S. M., M. L. Leyland, E. C. Conley, and P. Bradding. 2001. Voltage- dependent and calcium-activated ion channels in the human mast cell line under control conditions (32). Apparently, SGK1 is not vital for HMC-1. J. Leukocyte Biol. 70: 233–240. housekeeping functions but is critically important under defined 20. Kahr, H., R. Schindl, R. Fritsch, B. Heinze, M. Hofbauer, M. E. Hack, stress conditions (32). Thus, at least in theory, pharmacological M. A. 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