y U test with results of analysis and animal numbers presented in the relevant figure legends. The differences between wild-type and mutant animals or untreated and treated groups were statistically not significant if p 0.05 , significant if p 0.05 , very significant if A-769662 p 0.01 , and extremely significant if p 0.001. In vitro data were analyzed by nonparametric t test. GraphPad Prism software was used for all statistical analysis. Results Mouse lines used in this study were as follows. Mice which lack expression of p110γ as a consequence of gene deletion/knockout are referred to as γKO. Mice expressing a germline mutation encoding a kinase-dead version of p110δ are referred to asδD910A. Both mouse lines were backcrossed onto the C57BL/6 genetic background for 10 generations.
For genetic studies, Nutlin-3 the WT control mice were derived from inter-crosses of mice heterozygous for the p110 mutations. C57BL/6 WT mice from commercial breeders were used for pharmacological experiments. Isoform-selective PI3K inhibitors and their IC50 for the different PI3Ks are listed in Table I. In vivo doses for each inhibitor were established previously taking into account pharmacokinetic profiles. p110δ activity is important for the development or maintenance of tissue site-specific mast cell population We previously reported that genetic inactivation of p110δ leads to a reduction in mast cell numbers in specific tissues, such as the dermis of the ear and the submucosal and muscularis layers of the stomach. Mast cell numbers in other tissues, such as the dermis of the back and the mucosa layer of the stomach, were unaffected ; Fig.
1A). We have now also assessed the impact of p110γ deletion on mast cell numbers and found comparable mast cell numbers in γKO and WT mice at all anatomical sites assessed, in line with previously published data on a more limited set of tissues. Only the dermis of the back skin showed a minor reduction of toluidine blue-positive mast cells in p110γKO mice. These data Ali et al. Page 4 J Immunol. Author manuscript; available in PMC 2009 February 16. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript show that p110δ, unlike p110γ, has an impact on mast cell differentiation, which should be taken into account when interpreting studies using δD910A mice.
Inactivation of p110γ or p110δ does not affect vascular responsiveness to proinflammatory stimuli Recently, evidence has been presented for the presence of p110γ and p110δ in endothelial cells and vascular smooth muscle cells. Given that allergic responses in p110γ and p110δmutant mice have been assessed by leakage of Evans blue out of the vessels , it is not clear to what extent altered vascular responsiveness of PI3K mutant mice may have contributed to the observed reduced allergic responses in these mice. To gain insight into this question, we tested the direct effect of vasoactive compounds on vascular permeability in mutant mice, again using leakage of Evans blue dye into the surrounding tissue as a read-out. Injection of histamine led to a robust increase in vascular permeability that was similar in all genotypes.
Vascular permeability responses to mast cell extracts were also similar in WT, γKO, and δD910A mice. Taken together, these data show an intact responsiveness of the vasculature to inflammatory stimuli upon systemic inactivation of p110γ or p110δ.Distinct roles for p110γ and p110δ in adenosine signaling in mast cells In line with a previous report , we find that adenosine-stimulated phosphorylation of Akt, a surrogate marker of PI3K activity, is abrogated in γKO BMMCs. In agreement with this observation, adenosine-induced Akt/