Isoform-specific functions of phosphoinositide 3-kinases: p110 delta but not p110 gamma promotes optimal allergic responses in vivo

Abstract

The leukocyte-enriched p110gamma and p110delta isoforms of PI3K have been shown to control in vitro degranulation of mast cells induced by cross-linking of the high affinity receptor of IgE (FcepsilonRI). However, the relative contribution of these PI3K isoforms in IgE-dependent allergic responses in vivo is controversial. A side-by-side comparative analysis of the role of p110gamma and p110delta in mast cell function, using genetic approaches and newly developed isoform-selective pharmacologic inhibitors, confirms that both PI3K isoforms play an important role in FcepsilonRI-activated mast cell degranulation in vitro. In vivo, however, only p110delta was found to be required for optimal IgE/Ag-dependent hypersensitivity responses in mice. These observations identify p110delta as a key therapeutic target among PI3K isoforms for allergy- and mast cell-related diseases.

FIGURE 1

Impact of genetic inactivation of p110γ or p110δ on mast cell numbers and vascular permeability responses in vivo. A, Mast numbers in different anatomical locations. Depending on the anatomical location, the units for counting were as follows: ear dermis: per 5 mm length, beginning at the ear tip; stomach: per one complete sagittal section; and back dermis: unit is per 10 high power fields (×40 objective = ×400 magnification). Exclusively, dermal mast cells (those found among dermal collagen) were counted for each mouse; s.c. mast cells (those found among fat cells of the subcutis) were not counted, as thickness of the s.c. fat can vary greatly among animals depending on body condition. Data are presented as mast cell numbers expressed as % of WT (n = 5 for all genotypes). The mast cell distribution in δ^D910A mice has been published previously (17) and is presented here for comparative purposes. B, Effect of vasoactive stimuli on vascular leakage in PI3K mutant mice. Numbers of mice used were as follows: histamine: WT, γ^KO, and δ^D910A, n = 6 each; and mast cell extract: WT, n = 8; γ^KO, n = 6; and δ^D910A, n = 8.

FIGURE 2

Effect of p110γ or p110δ inhibition on adenosine-dependent Akt/PKB phosphorylation in mast cells and on adenosine-dependent vascular permeability. A (Left panel), γ^KO and δ^D910A BMMCs were stimulated with adenosine or vehicle (control) and Akt/PKB phosphorylation assessed by Western blotting, as described in Materials and Methods. A representative blot of two independent experiments is shown. Middle and right panels, BMMCs were pretreated for 30 min with varying concentrations of inhibitors, followed by adenosine stimulation for 1 min and immunoblotted for Akt/PKB (phospho-Ser473 or total). IC₅₀ values were determined by ratiometric analysis of immunoblots, for which Akt/PKB phosphorylation was calculated as the ratio between phosphorylated Akt/PKB and total Akt/PKB for each lane and expressed as the percentage of Akt/PKB phosphorylation in the absence of inhibitor (data not shown). A representative immunoblot of three independent experiments is shown. B, Impact of genetic inactivation of p110γ or p110δ on adenosine-induced PCA response in vivo. Number of mice used: WT and γ^KO, n = 10 each; and δ^D910A, n = 11. C, Impact of pharmacological inactivation of p110γ or p110δ on adenosine-induced PCA response in vivo. Number of WT mice dosed with AS605240, n = 9 or IC87114, n = 9.

FIGURE 3

Effect of p110γ or p110δ inhibition on SCF-dependent Akt/PKB phosphorylation and adhesion of mast cells. A, (Left panel) γ^KO and δ^D910A BMMCs were stimulated with SCF or vehicle (control) and Akt/PKB phosphorylation assessed by western blotting as described in Materials and Methods. A representative blot of two independent experiments is shown. (Middle and right panels), BMMCs were pretreated for 30 min with varying concentrations of inhibitors, followed by stimulation with SCF for 5 min and immunoblotted for Akt/PKB (phospho-Ser473 or total). IC₅₀ values were determined by ratiometric analysis of immunoblots (data not shown), as described in the legend to Fig. 2. A representative immunoblot of three independent experiments is shown. B, Impact of genetic inactivation of PI3K isoforms on SCF-dependent mast cell adhesion. The experiment shown is representative of five independent experiments. C, Impact of pharmacologic inactivation of PI3K isoforms on SCF-dependent mast cell adhesion. Graphs show data from a representative experiment done at least three (AS-2252424) or two (IC87114) times, with identical results.

FIGURE 4

Effect of p110γ or p110δ inhibition on IgE-dependent in vitro mast cell degranulation, Akt/PKB phosphorylation, and PCA response in vivo. A, Effect of genetic inactivation of p110γ or p110δ on IgE/Ag-induced BMMC degranulation in vitro. Graph shows the mean ± SD of the following number of independent experiments: WT, n = 10; δ^D910A, n = 10; and γ^KO, n = 8; done in quadruplicates. Mean ± SD spontaneous hexosaminidase release for experiments was as follows: WT, 8.23% ± 1.83 (n = 10); δ^D910A, 11.88% ± 1.9 (n = 10); and γ^KO, 8.0% ± 1.4 (n = 8). B, Effect of isoform-selective PI3K inhibitors on IgE/Ag-induced BMMC degranulation in vitro. Graph shows data from two independent experiments ± SEM. C, Impact of PI3K inhibitors on IgE/Ag-induced Akt/PKB phosphorylation upon 30-s (top panel) and 5-min (bottom panel) stimulation of IgE-sensitized mast cells with Ag. A representative blot from three independent experiments is shown. D, PCA response of WT and gene-targeted mice (WT, γ^KO, and δ^D910A; n = 11 each). E, PCA responses of WT mice treated with PI3K inhibitors. Numbers of mice used were as follows: left panel: IC87114 (p110δ inhibitor): vehicle, n = 8 and IC87114, n = 9; middle panel: AS-604850 and AS-252424 (p110γ inhibitors): vehicle, n = 9, AS-604850, n = 10, and AS-252424, n = 8; and right panel: TGX-155 (p110β inhibitor): vehicle, n = 10 and TGX-155, n = 10.