e isoforms of PI3K. Cross-linking of the FcεRI by multivalent Ag is known to activate a Tyr kinase signaling cascade, which provides a direct molecular link to class IA PI3K signaling. Genetic or pharmacological inactivation of p110δ has been shown to lead to a substantial, but not complete, block in the allergic responses in mice. Surprisingly, genetic inactivation A 922500 959122-11-3 of p110γ in mice has been reported to lead to a complete block in passive cutaneous and systemic anaphylaxis responses in vivo. This is remarkable, given that the FcεRI Tyr kinase signaling pathway does not appear to provide a direct molecular link to this GPCRcoupled PI3K.
Evidence has been presented for p110γ being part of an auto/paracrine mechanism whereby exocytosed mast cell-derived GPCR agonists, initially released by an FcεRI-dependent Aloe-emodin inhibitor pathway, promote hyperactivation of mast cells through GPCR signaling to overcome inhibition by the lipid phosphatases SHIP and PTEN, which antagonize PI3K signaling. Differences in experimental procedures, especially when using model organisms such as mice, often make it difficult to directly compare data from different laboratories. We have therefore directly compared side-by-side the roles of the p110γ and p110δ isoforms of PI3K in mast cell signaling in vitro and in the allergic immune response in vivo. For this, we have used PI3K mutant mice on the same genetic background, as well as a panel of newly developed small molecule inhibitors against PI3K isoforms. We find that in vitro, both p110γ and p110δ are important for IgE/Ag-dependent mast cell activation.
In vivo, however, IgE/Agtriggered allergic responses appear to a large extent driven by p110δ and are not dependent on p110γ. These findings have implications for the ongoing development of small molecule-PI3K inhibitors for allergy and inflammation. Materials and Methods Mice Mice in which p110γ or p110δ have been inactivated have been described previously. Mice were backcrossed onto a C57BL/6 genetic background for 10 generations. Agematched, 6�?0-wk-old mice were used for all experiments. C57BL/6 mice were used for pharmacological experiments. All protocols involving live animals were approved by the United Kingdom Home Office and local ethical review committee. Small molecule inhibitors Compounds used were: TGX-155 ), IC87114 ), and AS-605240, AS-604850 and AS-252424.
Compound or vehicle ) were 4Abbreviations used in this paper: GPCR, G protein-coupled receptor; BMMC, bone marrow-derived mast cell; HSA, human serum albumin; i.d., intradermal; KO, knockout; PCA, passive cutaneous anaphylaxis; SCF, stem cell factor; WT, wild type; Tyr, tyrosine; PKB, protein kinase B. Ali et al. Page 2 J Immunol. Author manuscript; available in PMC 2009 February 16. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript administered per os 1 h before Ag challenge. PI3K inhibitors were tested at 30 mg/kg and administered 1 h before Ag challenge. Mast cell culture Mast cell precursors were isolated from bone marrow of 6-wk-old C57BL/6 male mice, as described , and maintained in RPMI 1640 medium containing 10% ultra-low IgG FBS , penicillin and streptavidin, glutamine and 20 ng/ml recombinant mouse stem cell factor , and 20 ng/ml IL-3 for at least 4 wk and with culture times not exceeding 8 wk.
Expression of FcεRI and Kit were confirmed by flow cytometry as described. Assessment of Akt/protein kinase B phosphorylation in mast cells in vitro For stimulations with adenosine or SCF, cells were starved for 3 h in serum- and cytokine-free medium. Cells were then treated with compound or 0.5% DMSO for 15 min, followed by stimulation with SCF or adenosine. Ce