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but Not p110
Promotes Optimal Allergic Responses In Vivo1
* Centre for Cell Signalling, Institute of Cancer, Queen Mary University of London, London, United Kingdom;
Geneva Research Center, Merck Serono International, Geneva, Switzerland; and
Frimorfo, Fribourg, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and p110
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 (Fc
RI). 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 p110 and p110 in mast cell function, using genetic approaches and newly developed isoform-selective pharmacologic inhibitors, confirms that both PI3K isoforms play an important role in FcRI-activated mast cell degranulation in vitro. In vivo, however, only p110 was found to be required for optimal IgE/Ag-dependent hypersensitivity responses in mice. These observations identify p110 as a key therapeutic target among PI3K isoforms for allergy- and mast cell-related diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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RI) on mast cells leads to the activation of an intracellular signaling cascade that culminates in secretory granule exocytosis and allergic responses in vivo (1, 2, 3). PI3Ks, a group of signal transduction enzymes that produce intracellular lipid second messengers, have been implicated in signaling through the FcRI and various other receptors in mast cells (4). The exact role of PI3K activation downstream of the FcRI remains unclear. Most likely, PI3K action is involved in the assembly of a "signalosome" complex, which promotes, among other events, calcium mobilization and activation of protein kinase C, which together lead to mast cell exocytosis (4, 5, 6, 7, 8).
Mammals have eight isoforms of PI3K (5, 9). The subset of PI3K enzymes that are acutely activated by membrane-bound receptors are known as the class I PI3Ks. Of these, the class IA PI3Ks signal downstream of tyrosine (Tyr) kinases and consist of a p110 catalytic subunit (p110
, p110β, or p110) complexed to one of five regulatory subunits (collectively known as "p85s"). The p85s have SH2 domains, which allow the p85/p110 complex to become recruited to phospho-Tyr residues upon activation of Tyr kinase signaling.
In contrast, p110, the only class IB PI3K, signals downstream of G protein-coupled receptors (GPCRs).3 p110 forms a heterodimer either with p101 or p84/p87, highly homologous regulatory subunits which are unrelated to p85 (9, 10, 11). Whereas p110 and p110β are widely distributed, p110 and p110 are enriched in leukocytes (12, 13, 14). Combined with the fact that mice with loss-of-function of p110 or p110 are viable (15), immunological studies have initially focused on these isoforms of PI3K (16).
Cross-linking of the FcRI by multivalent Ag is known to activate a Tyr kinase signaling cascade, which provides a direct molecular link to class IA PI3K signaling (4, 8). Genetic or pharmacological inactivation of p110 has been shown to lead to a substantial, but not complete, block in the allergic responses in mice (3, 17, 18). Surprisingly, genetic inactivation of p110 in mice has been reported to lead to a complete block in passive cutaneous and systemic anaphylaxis responses in vivo (19). This is remarkable, given that the FcRI Tyr kinase signaling pathway does not appear to provide a direct molecular link to this GPCR-coupled PI3K. Evidence has been presented for p110 being part of an auto/paracrine mechanism whereby exocytosed mast cell-derived GPCR agonists, initially released by an FcRI-dependent pathway, promote hyperactivation of mast cells through GPCR signaling to overcome inhibition by the lipid phosphatases SHIP and PTEN, which antagonize PI3K signaling (11, 19).
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 p110 and p110 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 (20, 21, 22). We find that in vitro, both p110 and p110 are important for IgE/Ag-dependent mast cell activation. In vivo, however, IgE/Ag-triggered allergic responses appear to a large extent driven by p110 and are not dependent on p110. These findings have implications for the ongoing development of small molecule-PI3K inhibitors for allergy and inflammation.
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Materials and Methods |
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Mice in which p110 or p110 have been inactivated have been described previously (23, 24). Mice were backcrossed onto a C57BL/6 genetic background for >10 generations. Age-matched, 6–10-wk-old mice were used for all experiments. C57BL/6 mice (Harlan, U.K.) 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 (p110β-selective; (25, 26)), IC87114 (p110-selective; (27)), and AS-605240, AS-604850 (22) and AS-252424 (21) (p110-selective). Compound(s) or vehicle (0.5% carboxy-methyl-cellulose in 0.25% Tween 20 (Sigma-Aldrich)) were 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 (17), and maintained in RPMI 1640 medium (Invitrogen Life Technologies) containing 10% ultra-low IgG FBS (Invitrogen Life Technologies), penicillin and streptavidin, glutamine and 20 ng/ml recombinant mouse stem cell factor (SCF), and 20 ng/ml IL-3 (PeproTech) for at least 4 wk and with culture times not exceeding 8 wk. Expression of FcRI and Kit were confirmed by flow cytometry as described (17).
Assessment of Akt/protein kinase B (PKB) 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 (2.5 x 106 cells in a 1 ml volume) were then treated with compound or 0.5% DMSO for 15 min, followed by stimulation with SCF (20 ng/ml, 5 min) or adenosine (300 nM, 1 min). Cell stimulation was terminated by the addition of 2x Laemmli electrophoresis buffer followed by assessment of Akt/PKB phosphorylation by western blot using anti-phospho-Ser473 Akt/PKB Ab (Cell Signaling Technology) as described (17). For Ag stimulation, mast cells were sensitized overnight by incubation with 0.1 µg/ml IgE-DNP (Clone SPE-7; Sigma-Aldrich) at 37°C and challenged with DNP (30 ng/ml) the next day for the indicated periods of time.
In vitro cell adhesion of mast cells
A total of 80 µl of a mast cells suspension (6.25 x 105 cells/ml Tyrode’s buffer (10 mM HEPES (pH 7.4), 130 mM NaCl, 6.2 mM D-glucose, 5.0 mM KCl, 1.4 mM CaCl2, 1.0 mM MgCl2, and 0.1% BSA)) was incubated on prewarmed fibronectin-precoated 96-well plates (human fibronectin Multiwell; BD Biosciences) containing 10 µl of inhibitor solution or 0.1% DMSO per well. To stimulate cell adhesion, 10 µl of a 200 ng/ml solution of SCF in Tyrode’s buffer was added and cells were incubated at 37°C for 30 min. After washing 3 times with Tyrode’s buffer to remove nonadherent cells, the adherent cells were lysed in 100 µl of Tyrode’s buffer containing 0.5% Triton X-100, followed by quantification of β-hexosaminidase content as described below. Cell adhesion was expressed as the % of adhesion induced by stimulation with PMA (10 ng/ml, 60 min) in adjacent wells.
In vitro mast cell degranulation
Mast cells were sensitized overnight by incubation with 0.1 µg/ml IgE-DNP (Clone SPE-7; Sigma-Aldrich) at 37°C. The next day, cells were resuspended in Tyrode’s buffer at 2 x 106 cells/ml. 105 cells were plated in 96-well plates, preincubated for 20 min with inhibitor or 0.1% DMSO, followed by stimulation for 20 min with 30 ng/ml DNP-human serum albumin (HSA) (Sigma-Aldrich), in a final volume of 100 µl following. Cell supernatant and cellular pellets were harvested by 5 min centrifugation at 1500 rpm. To measure β-hexosaminidase activity, 50 µl of supernatant or cell pellet (lysed in 100 µl of Tyrode’s buffer containing 0.5% Triton X-100) were transferred to 96-well flat-bottom plates containing 50 µl of 3.7 mM p-nitrophenol-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) in 100 mM Na-acetate (pH 4.5) and further incubated for 1 h at 37°C. Reaction was stopped by addition of 100 µl of 2 M NaOH, followed by measurement of absorbance at 405 nm.
Passive cutaneous anaphylaxis (PCA)
Mice were lightly anesthetized with isoflourane/oxygen in an anesthesia chamber, followed by intradermal (i.d.) injection into the pinnea of the ear. For each experimental mouse, 20 µl PBS or 50 ng anti-DNP IgE (SPE-7; Sigma-Aldrich) in 20 µl PBS were injected in the right and left ear, respectively, followed 24 h later by an i.v. injection of 100 µg DNP-HSA (Sigma-Aldrich) in 100 µl 0.5% (w/v) Evans blue dye in PBS (Evans blue was coinjected to allow visualization of vascular leakage as a measure of increased vascular permeability). Thirty minutes after the i.v. injection, the mice were sacrificed in a CO2 asphyxiation chamber. Tissue sections around the i.d. injection site were excised with a sample corer, followed by weighing and extraction of the extravasated Evans blue by incubation in 200 µl formamide at 55°C for 24 h and measurement of absorbance at 620 nm (OD620 nm). Data are expressed as OD620 nm = absorbance (at 620 nm) of IgE-injected skin biopsy minus absorbance (at 620 nm) of PBS-injected skin biopsy.
Vascular permeability assay
The procedure to determine vascular permeability was similar to that of the PCA assay. Following i.v. injection of 100 µl 0.5% Evans blue in saline, the ears were injected i.d. 1 hr later either with 20 µl volume of PBS, adenosine (5 x 10–3 M; Sigma-Aldrich), histamine (5 ng/ml; Sigma-Aldrich), or mast cell extract (obtained by sonicating 2 x 107 4-wk-old bone marrow-derived mast cells (BMMCs) (cultured as in Ref. 17) in 2 ml of ice-cold PBS). Thirty minutes later, animals were sacrificed in a CO2 asphyxiation chamber and tissue biopsies taken and processed as described above. Data are expressed as OD620 nm = absorbance (at 620 nm) of histamine/mast cell extract skin biopsy minus absorbance (at 620 nm) of PBS-injected skin biopsy.
Statistical analysis
Results from in vivo experiments (mean) were assessed using a nonparametric Mann-Whitney U test with results of analysis and animal numbers presented in the relevant figure legends. The differences between wild-type (WT) and mutant animals or untreated and treated groups were statistically not significant if p > 0.05 (labeled as n.s.), significant if p < 0.05 (*), very significant if 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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as a consequence of gene deletion/knockout (KO) are referred to as KO (23). Mice expressing a germline mutation encoding a kinase-dead version of p110 (p110D910A) are referred to as D910A (24). Both mouse lines were backcrossed onto the C57BL/6 genetic background for >10 generations. For genetic studies, the WT control mice were derived from intercrosses 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 (data not shown).
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activity is important for the development or maintenance of tissue site-specific mast cell population(s)
We previously reported that genetic inactivation of p110 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 (17). Mast cell numbers in other tissues, such as the dermis of the back and the mucosa layer of the stomach, were unaffected ((17); Fig. 1A). We have now also assessed the impact of p110 deletion on mast cell numbers and found comparable mast cell numbers in KO and WT mice at all anatomical sites assessed, in line with previously published data on a more limited set of tissues (19). Only the dermis of the back skin showed a minor reduction (
22%) of toluidine blue-positive mast cells in p110KO mice (Fig. 1A). These data show that p110, unlike p110, has an impact on mast cell differentiation, which should be taken into account when interpreting studies using D910A mice.
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or p110 does not affect vascular responsiveness to proinflammatory stimuli
Recently, evidence has been presented for the presence of p110 and p110 in endothelial cells and vascular smooth muscle cells (28, 29, 30, 31). Given that allergic responses in p110 and p110 mutant mice have been assessed by leakage of Evans blue out of the vessels (17, 19), 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 (Fig. 1B; note that the tendency for increased responsiveness of KO mice is statistically not significant). Vascular permeability responses to mast cell extracts (prepared by sonication of BMMCs, as a model for a more complex agonist) were also similar in WT, KO, and D910A mice (Fig. 1B). Taken together, these data show an intact responsiveness of the vasculature to inflammatory stimuli upon systemic inactivation of p110 or p110.
Distinct roles for p110 and p110 in adenosine signaling in mast cells
In line with a previous report (19), we find that adenosine-stimulated phosphorylation of Akt, a surrogate marker of PI3K activity, is abrogated in KO BMMCs (Fig. 2A). In agreement with this observation, adenosine-induced Akt/PKB phosphorylation was very sensitive to pharmacological inhibition of p110, with an IC50 for AS-252424 of 85 nM, as compared with 3.6 µM for the p110 inhibitor IC87114 (Fig. 2A).
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KO mice have been reported to be completely resistant to adenosine-stimulated increases in vascular permeability (19). Using a similar protocol as was used in Ref. 19 , we found a severe, but not complete, reduction in adenosine-stimulated vascular permeability upon genetic or pharmacological inactivation of p110 (Fig. 2B). D910A mice (Fig. 2A) and WT mice treated with the p110-selective inhibitor IC87114 (Fig. 2C) remained sensitive to this type of stimulation. The observation that IC87114, at the doses tested in these experiments, did not affect the adenosine response suggests that IC87114 has no off-target effects on p110 under these conditions in vivo. Together with the in vitro data described above, these data confirm that p110 plays an important role in adenosine-stimulated vascular permeability.
Distinct roles for p110 and p110 in Kit receptor signaling in mast cells
We have previously shown that p110 is the main source of PI3K activity downstream of the activated Kit Tyr kinase receptor for SCF and largely controls SCF-stimulated proliferation, migration, and adhesion (17). SCF can also potentiate FcRI-activated mast cell degranulation, a response which can be attenuated by the p110-selective inhibitor IC87114 (17). Indeed, SCF-stimulated Akt/PKB phosphorylation is very sensitive to IC87114 (IC50 of 0.27 µM) compared with the p110-selective compound AS-252424 (IC50 of 4.29 µM) (Fig. 3A). These data confirm and extend our previous data on the critical role of p110 in SCF/Kit signaling in BMMCs (17). This is further corroborated by the blockade of SCF-induced mast cell adhesion upon genetic (Fig. 3B) or pharmacological (Fig. 3C) inactivation of p110. This biological response is refractory to genetic or pharmacological blockade of p110. These data further demonstrate the functional distinction which can exist between different PI3K isoforms in a specific biological response.
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and p110 play important roles in FcRI-driven mast cell degranulation in vitro
Reduced IgE/Ag-induced degranulation upon genetic or pharmacological inactivation of p110, or genetic inactivation of p110, has been reported in separate studies (17, 19). We have now tested BMMCs under the same experimental conditions and also used newly developed inhibitors against p110 (21, 22) (Table I). We confirm that genetic inactivation of p110 or p110 impairs in vitro degranulation (Fig. 4A) and show that acute PI3K inactivation using isoform-selective inhibitors mirrors this response (Fig. 4B).
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KO mast cells activated through FcRI in the absence of any costimulation but is strongly reduced upon costimulation of FcRI with adenosine (19). Using Akt/PKB phosphorylation as a surrogate marker of PI3K activation, we found that the early phase of PI3K activity (30 s) downstream of activated FcRI was, surprisingly, refractory to IC87114 inhibition and dependent on p110 (Fig. 4C, top panel), with an IC50 of 327 nM (data not shown). The later phase (5 min), which remained equally sensitive to AS-252424, became more sensitive to IC87114 (IC50 of 86 nM; Fig. 4C and data not shown). Our findings suggest that PI3K activation downstream of the activated FcRI in vitro is biphasic, with p110 being activated before p110 upon FcRI engagement.
p110, but not p110, is dispensable for allergic responsiveness in vivo
Mast cells in vivo are exposed to stimuli from the microenvironment other than Ag which can modulate the FcRI response, and it is therefore not always possible to extrapolate in vitro observations such as those shown in Fig. 4, A and B, to the organismal context. We therefore tested the in vivo allergic response of KO and D910A mice, side-by-side in the same experiment and using mice on the same genetic background (C57BL/6). Mice were sensitized locally (either in the dermis of the ear or the back) by injection of Ag-specific IgE (directed against the hapten DNP) and challenged systemically 24 h later with DNP-HSA (administered together with Evans blue). Thirty minutes later, the mast cell response was quantified by measuring extravasated Evans blue.
In line with our previously published results in D910A mice on the BALB/c genetic background (17), inactivation of p110 on the C57BL/6 background led to a significant (70%) reduction in IgE/Ag-dependent vascular permeability in the ears of sensitized mice (Fig. 4D). Similar results (69% reduction) were observed in the back dermis (data not shown). Surprisingly, KO mice did not show reduced in vivo allergic responses (Fig. 4D).
To exclude that altered PCA responses in gene-targeted mice are related to developmental defects, we next pharmacologically intervened with PI3K function using isoform-selective PI3K inhibitors. Treatment of WT mice with the p110-selective inhibitor IC87114 at doses which do not affect p110 (as implied by the findings shown in Fig. 2C) consistently diminished the allergic immune response by 40% (Fig. 4E). This milder reduction upon pharmacological, compared with genetic, inactivation of p110 (Fig. 4D) most likely relates to the reduced number of mast cells in the ears of D910A mice (Fig. 1A), as previously discussed (17), and the notion that IC87114, in contrast to genetic inactivation, is not expected to provide full inhibition of p110 as is the case in homozygous D910A mice. In contrast to IC87114, the p110-selective compounds AS-604850 and AS-252424 had no significant impact on the allergic response (Fig. 4E), in line with our observations in KO mice (Fig. 4D). Administration of the p110β-selective compound TGX-155 also did not impact on the acute allergic response (Fig. 4E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, in isolation, plays a significant role in the in vivo allergic cascade. This appears to be in contradiction with previous work, which suggested that p110 is essential for and is the only PI3K subunit which drives the in vivo IgE/Ag-triggered allergic response (19). It is possible that the proposed GPCR-driven auto/paracrine signaling amplification mechanism, largely based on in vitro observations on cultured mast cells (19), may not be operational in vivo. This conclusion is in line with the observation that KO mice for A(3), the main adenosine receptor, retain normal IgE/Ag-dependent PCA responses, despite a complete abrogation of adenosine responsiveness (32). Differences in genetic backgrounds of mice could also contribute to the discrepancies between our studies and earlier work (19). Indeed, previous studies in which p110 function was assessed used mice bred onto the 129sv background, in contrast to our studies in which we used C57BL/6 mice (for p110 and p110; this work) and BALB/c (for p110; data shown in Ref. 17). However, why a reduced sensitivity of KO mice to adenosine would be retained across genetic backgrounds, in contrast to responsiveness to allergic responses, is difficult to explain. For a molecule to have an "essential" role in a process such as allergy, we believe it must have a function across genetic backgrounds, similar as what is observed for p110. Other experimental differences to measure the allergic response may also contribute to the observed discrepancies. Indeed, whereas both studies used vascular permeability as a measure of mast cell activation, a different sensitization protocol was applied, namely intradermal local sensitization (PCA; this study) vs i.v. systemic sensitization (passive systemic anaphylaxis; (19)). We have found the i.v. sensitization procedure in passive systemic anaphylaxis experiments to give extremely variable results in WT mice, for reasons unclear to us, but apparently unrelated to age or sex of the mice (data not shown). Other than being more robust, we also believe that the PCA protocol is a more accurate measure of mast cell contribution in allergy, given that it assesses the function of tissue-resident mast cells as the primary targets of the intradermal sensitization step, unlike in systemic sensitization protocols which also sensitize (and in the Ag challenge phase activate) other FcRI-expressing cells, including basophils and eosinophils.
In this study we show that specific signaling and biological responses are, to a large extent, selectively driven by a single PI3K isoform. This is the case for SCF and adenosine, which are controlled by p110 and p110, respectively. In constrast, the FcRI enlists both p110 and p110. Kinetic studies measuring FcRI-associated PI3K activation show that p110 and p110 PI3Ks are activated sequentially downstream of the activated FcRI with p110 being activated before p110. It is puzzling how the FcRI, which is considered to signal intracellularly mainly through tyrosine kinases (7), activates the GPCR-coupled p110 so early, even before p110. However, despite the apparent importance of p110 in FcRI-activated mast cell exocytosis in vitro, our work indicates that this need for p110 activity does not translate to the in vivo situation, where p110 appears to be dispensable. It is also possible that the density of mast cells in an in vitro Ag-activated exocytosis experiment may produce a substantially greater concentration of adenosine (or other factor(s)) in the immediate environment than may be seen in vivo where mast cells are more diffusely distributed in the tissues. Furthermore, unlike in tissue culture, adenosine would be rapidly metabolized in vivo. It is also possible that in tissues, agonists other than adenosine may override the necessity for p110.
In contrast to p110, disruption of p110 signaling has an inhibitory effect on the allergic response across different genetic backgrounds and in WT mice treated with a p110-selective inhibitor. This most likely relates to the fact that blockade of p110 has effects beyond the inhibition of activated FcRI. Indeed, p110 function is critical for signaling through the Kit receptor (Fig. 3), known to potentiate allergic responses in vitro and in vivo (17). Mast cells actively participate in allergy and allergic airway inflammation, and our data provide a partial mechanism for the observation that genetic or pharmacological inactivation of p110 impairs airway hyperresponsiveness in murine models (3, 18, 33). Unfortunately, despite the availability of several strains of p110-deficient mice and small molecule inhibitors to p110, there are as yet no published reports to suggest a role for p110 in allergic airway inflammation.
Intracellularly, class IA PI3Ks couple to the FcRI via the adaptor protein Gab2, which recruits class IA PI3Ks to the activated FcRI signaling complex. Deletion of Gab2 in BMMCs has a severe negative impact on both PI3K activation downstream of Kit and FcRI, and Gab2-deficient mice have an almost complete block in the allergic response (34). This reduction is more severe than that observed in p110-deficient mice (17), possibly because Gab2 also binds other class IA PI3Ks, including p110 and p110β. We have previously reported that a high dose of IC87114 (40 µM serum dose) could completely wipe out the PCA response (17). We presumed at the time that this was due to possible off-target effects of this compound on p110 (17). Our current data show that this is not the case and that other PI3K isoforms, either on their own or in combination, account for the PI3K-dependent fraction of the IgE/Ag-dependent allergic response. Taken together, it is therefore possible that the p110 and p110β isoforms of PI3K together contribute to the residual PI3K-dependent PCA response observed upon p110 inactivation (Fig. 4, D and E). However, on its own, p110β does not significantly contribute to the PCA response (Fig. 4E). Unfortunately, selectivity of inhibitors for p110 cannot be achieved at present without resulting in many off-target effects, so that the currently available p110 inhibitors also inhibit other relevant kinases including isoforms of protein kinase C (35). Genetic investigation of the role of p110 (and p110β) PI3K isoforms has thus far also been precluded due to the embryonic lethality of homozygous p110 and p110β gene-targeted mice and the incapacity to derive cell lines from these mice (36, 37, 38). The creation of mice with conditional p110 and p110β alleles and the development of small molecule inhibitors with higher p110 isoform-selectivity will be critical to gain insight into which other PI3K isoforms may complement p110 in controlling the IgE/Ag-dependent allergic response.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by the Biotechnology and Biological Science Research Council U.K. (Grant BB/C505659/1), the European Union FP6-502935, Barts and the London Charity, and the Ludwig Institute for Cancer Research. 
2 Current address: Intellikine, La Jolla, CA 92037.
3 Address correspondence and reprint requests to Dr. Bart Vanhaesebroeck, Centre for Cell Signaling, Institute of Cancer, Queen Mary, University of London, Sir John Vane Research Centre, Charterhouse Square, London EC1M 6BQ, United Kingdom. E-mail address: bart.vanh{at}qmul.ac.uk
4 Abbreviations used in this paper: GPCR, G protein-coupled receptor; BMMC, bone marrow-derived mast cell; HSA, human serum albumin; i.d., intradermal; KO, knock-out; PCA, passive cutaneous anaphylaxis; SCF, stem cell factor; WT, wild type; Tyr, tyrosine; PKB, protein kinase B.
Received for publication May 7, 2007. Accepted for publication December 5, 2007.
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References |
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in generation of type 2 cytokine responses and allergic airway inflammation. Eur. J. Immunol. 37: 416-424. [Medline] isoform of PI3K. Trends Immunol. 28: 80-87. [Medline], a novel phosphoinositide 3-kinase in leukocytes. Proc. Natl. Acad. Sci. USA 94: 4330-4335. , a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes. J. Biol. Chem. 272: 19236-19241. and PI3K : partners in crime in inflammation in rheumatoid arthritis and beyond?. Nat. Rev. Immunol. 7: 191-201. [Medline] phosphoinositide 3-kinase in the allergic response. Nature 431: 1007-1011. [Medline] attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model. FASEB J. 20: 455-465. is an essential amplifier of mast cell function. Immunity 16: 441-451. [Medline] in neutrophil directional movement. J. Immunol. 170: 2647-2654. . J. Med. Chem. 49: 3857-3871. [Medline] suppresses joint inflammation and damage in mouse models of rheumatoid arthritis. Nat. Med. 11: 936-943. [Medline] in inflammation. Science 287: 1049-1053. PI 3-kinase mutant mice. Science 297: 1031-1034. in neutrophil inflammatory responses. Biochem. Biophys. Res. Commun. 308: 764-769. [Medline] in promoting neutrophil trafficking into inflamed tissue. Blood 103: 3448-3456. activity in neutrophil trafficking. Blood 106: 150-157. the culprit?. Hypertension 43: 885-890. . J. Exp. Med. 201: 1217-1228. inhibitor reduces vascular permeability in a murine model of asthma. J. Allergy Clin. Immunol. 118: 403-409. [Medline] in insulin signaling. Cell 125: 733-747. [Medline] subunit of phosphoinositide 3-kinase. J. Biol. Chem. 274: 10963-10968. phosphoinositide-3-OH kinase in growth and metabolic regulation. Nature 441: 366-370. [Medline] isoform of PI3K in B-cell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110 function in B cells. Blood 107: 642-650. This article has been cited by other articles:
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|
 |
|
  J. Doukas, L. Eide, K. Stebbins, A. Racanelli-Layton, L. Dellamary, M. Martin, E. Dneprovskaia, G. Noronha, R. Soll, W. Wrasidlo, et al. Aerosolized Phosphoinositide 3-Kinase {gamma}/{delta} Inhibitor TG100-115 [3-[2,4-Diamino-6-(3-hydroxyphenyl)pteridin-7-yl]phenol] as a Therapeutic Candidate for Asthma and Chronic Obstructive Pulmonary Disease J. Pharmacol. Exp. Ther., March 1, 2009; 328(3): 758 - 765. [Abstract] [Full Text] [PDF]
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 C. Billottet, L. Banerjee, B. Vanhaesebroeck, and A. Khwaja Inhibition of Class I Phosphoinositide 3-Kinase Activity Impairs Proliferation and Triggers Apoptosis in Acute Promyelocytic Leukemia without Affecting Atra-Induced Differentiation Cancer Res., February 1, 2009; 69(3): 1027 - 1036. [Abstract] [Full Text] [PDF]
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 E. A. Papakonstanti, O. Zwaenepoel, A. Bilancio, E. Burns, G. E. Nock, B. Houseman, K. Shokat, A. J. Ridley, and B. Vanhaesebroeck Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages J. Cell Sci., December 15, 2008; 121(24): 4124 - 4133. [Abstract] [Full Text] [PDF]
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|
 E. Zebedin, O. Simma, C. Schuster, E. M. Putz, S. Fajmann, W. Warsch, E. Eckelhart, D. Stoiber, E. Weisz, J. A. Schmid, et al. Leukemic challenge unmasks a requirement for PI3K{delta} in NK cell-mediated tumor surveillance Blood, December 1, 2008; 112(12): 4655 - 4664. [Abstract] [Full Text] [PDF]
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 T Weichhart and M D Saemann The PI3K/Akt/mTOR pathway in innate immune cells: emerging therapeutic applications Ann Rheum Dis, December 1, 2008; 67(Suppl_3): iii70 - iii74. [Abstract] [Full Text] [PDF]
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 M. S. Thomas, J. S. Mitchell, C. C. DeNucci, A. L. Martin, and Y. Shimizu The p110{gamma} isoform of phosphatidylinositol 3-kinase regulates migration of effector CD4 T lymphocytes into peripheral inflammatory sites J. Leukoc. Biol., September 1, 2008; 84(3): 814 - 823. [Abstract] [Full Text] [PDF]
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