Blunted IgE-mediated activation of mast cells in mice lacking the serum- and glucocorticoid-inducible kinase SGK3

Abstract

Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 (sgk3(-/-)) and their wild-type littermates (sgk3(+/+)). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular Ca(2+) and entry of extracellular Ca(2+). Influx of extracellular Ca(2+) but not Ca(2+) release from intracellular stores was significantly blunted in sgk3(-/-) BMMCs compared with sgk3(+/+) BMMCs. Antigen stimulation further led to a rapid increase of a K(+)-selective conductance in sgk3(+/+) BMMCs, an effect again blunted in sgk3(-/-) BMMCs. In contrast, the Ca(2+) ionophore ionomycin activated K(+) currents to a similar extent in sgk3(-/-) and in sgk3(+/+) BMMCs. β-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in sgk3(-/-) BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in sgk3(-/-) compared with sgk3(+/+) mice. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk3(-/-) than in sgk3(+/+) mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function.

Fig. 1.

Maturation of bone marrow mast cells (BMMCs) from sgk3^+/+ and sgk3^−/− mice. A: original dot plots of CD117-, CD34-, and FcεRI-positive BMMCs from sgk3^+/+ and sgk3^−/− mice. Numbers depict the percentage of cells in the respective quadrant, acquired within the mast cell gate. B: frequency of mast cells in primary culture. Mean percent (± SE; n = 6 individual BMMC cultures) of sgk3^+/+ (open bars) and sgk3^−/− (closed bars) BMMCs acquired within the mast cell gate. C: ear conche sections of sgk3^+/+ (top) and sgk3^−/− (bottom) mice stained with toluidine blue for mast cell detection (mast cells are indicated by black arrows). D: number of mast cells (±SE) in skin, analyzed by staining of ear conche sections with toluidine blue. Mean mast cell numbers of toluidine blue-positive cells in one area (×200 magnification) as calculated from 10 different areas on different slices per conch of 4 sgk3^+/+ (open bar) and 3 sgk3^−/− (closed bar) mice (P = 0.43, two-tailed unpaired t-test).

Fig. 2.

Antigen-induced Ca²⁺ entry into BMMCs from sgk3^+/+ and sgk3^−/− mice. A: representative original tracings showing the fura-2 fluorescence ratio of 340 over 380 nm in fura-2/AM-loaded BMMCs from sgk3^+/+ and sgk3^−/− mice before and following addition of antigen (Ag, 50 ng/ml). At the end of each experiment, ionomycin (10 μM) was added for calibration. For quantification of the Ca²⁺ entry into the BMMCs, the slope (Δratio/ms) and peak (Δratio) were calculated following addition of Ag as indicated in the figure. B: means (±SE) of the slope (left) and peak (right) of the fluorescence ratio change for sgk3^+/+ (n = 9, open bars) and sgk3^−/− (n = 8, closed bars) BMMCs following stimulation with Ag (50 ng/ml). *P < 0.05 and **P < 0.01, significant difference between both groups (two-tailed unpaired t-test). C: representative original tracings showing the ratio of 340/380 nm fura-2 fluorescence in fura-2-loaded sgk3^+/+ and sgk3^−/− BMMCs before and after addition of Ag (50 ng/ml) in the absence of extracellular Ca²⁺. To reach a Ca²⁺-free environment, EGTA (0.5 mM) was added to the Ca²⁺-free bath solution. D: means (±SE) of the slope (left) and peak value (right) of the fluorescence ratio change for sgk3^+/+ (n = 5, open bars) and sgk3^−/− (n = 5, closed bars) BMMCs upon stimulation with antigen in a Ca²⁺-free solution.

Fig. 3.

Antigen-induced K⁺ currents are reduced in sgk3^−/− BMMCs. A: representative whole cell currents from sgk3^+/+ (left) and sgk3^−/− (right) BMMCs elicited by 200-ms pulses ranging from −115 to +65 mV in 20-mV increments from a holding potential of −35 mV. Currents were recorded in standard NaCl bath solution 3 min after stimulation with either Ag (50 ng/ml, top) or ionomycin (1 μM, bottom). The dotted line indicates the zero current value. B: mean current-voltage relationships (±SE, n = 7) in sgk3^+/+ (open symbols) and sgk3^−/− (closed symbols) BMMCs before (control, squares) and 3 min after stimulation with antigen (Ag, 50 ng/ml, triangles). C: mean whole cell conductance (± SE) of sgk3^+/+ (open bars) and sgk3^−/− (closed bars) BMMCs as recorded in B before (control) and after stimulation with either Ag (50 ng/ml) or ionomycin (1 μM). Data were calculated by linear regression between −55 and +5 mV. *P < 0.05, significant difference between sgk3^+/+ and sgk3^−/− cells (ANOVA); ###P < 0.001, significant difference from sgk3^+/+ cells under control conditions (ANOVA); §§§P < 0.001, significant difference from sgk3^−/− cells under control conditions (ANOVA).

Fig. 4.

Degranulation of antigen-stimulated sgk3^−/− and sgk3^+/+ BMMCs. β-Hexosaminidase release from cultured sgk3^−/− BMMCs (closed bars) and their wild-type littermates sgk3^+/+ (open bars) stimulated for 15 min with 50 ng/ml antigen (±SE, n = 5 individual experiments). Release in the supernatant was calculated as percentage of total cellular (0.1% Triton X-100) β-hexosaminidase. The stimulated β-hexosaminidase release in each experiment was corrected for the spontaneous release. *Significant difference between genotypes (P < 0.05; two-tailed unpaired t-test).

Fig. 5.

Systemic anaphylactic reaction in sgk3^+/+ and sgk3^−/− mice. A: changes in body temperature (Δ°C) of sgk3^−/− mice (n = 5, closed squares) and their wild-type littermates sgk3^+/+ (n = 6, open squares) following induction of anaphylaxis (±SE). Mice were given intraperitoneal anti- dinitrophenyl (DNP) IgE (30 μg) and challenged with 100 μg DNP- human serum albumin (HSA) after 5 h. B: arithmetic means (± SE) of maximal changes in body temperature (Δ°C) of sgk3^−/− mice (n = 5, closed bars) and their wild-type littermates sgk3^+/+ (n = 6, open bars) following induction of anaphylaxis. *Significant difference between genotypes (P < 0.05; two-tailed unpaired t-test). C: serum histamine levels 30 min after induction of anaphylaxis in sgk3^−/− mice (closed bar) and their wild-type sgk3^+/+ littermates (open bar) (n = 3). **Significant difference between genotypes (P < 0.01; two-tailed unpaired t-test).