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. 2011 Nov 17;118(20):5466-75.
doi: 10.1182/blood-2010-09-309955. Epub 2011 Sep 16.

PTEN deficiency in mast cells causes a mastocytosis-like proliferative disease that heightens allergic responses and vascular permeability

Yasuko Furumoto  1 , Nicolas Charles, Ana Olivera, Wai Hang Leung, Sandra Dillahunt, Jennifer L Sargent, Kevin Tinsley, Sandra Odom, Eric Scott, Todd M Wilson, Kamran Ghoreschi, Manfred Kneilling, Mei Chen, David M Lee, Silvia Bolland, Juan Rivera
Affiliations

PTEN deficiency in mast cells causes a mastocytosis-like proliferative disease that heightens allergic responses and vascular permeability

Yasuko Furumoto et al. Blood. .

Abstract

Kit regulation of mast cell proliferation and differentiation has been intimately linked to the activation of phosphatidylinositol 3-OH kinase (PI3K). The activating D816V mutation of Kit, seen in the majority of mastocytosis patients, causes a robust activation of PI3K signals. However, whether increased PI3K signaling in mast cells is a key element for their in vivo hyperplasia remains unknown. Here we report that dysregulation of PI3K signaling in mice by deletion of the phosphatase and tensin homolog (Pten) gene (which regulates the levels of the PI3K product, phosphatidylinositol 3,4,5-trisphosphate) caused mast cell hyperplasia and increased numbers in various organs. Selective deletion of Pten in the mast cell compartment revealed that the hyperplasia was intrinsic to the mast cell. Enhanced STAT5 phosphorylation and increased expression of survival factors, such as Bcl-XL, were observed in PTEN-deficient mast cells, and these were further enhanced by stem cell factor stimulation. Mice carrying PTEN-deficient mast cells also showed increased hypersensitivity as well as increased vascular permeability. Thus, Pten deletion in the mast cell compartment results in a mast cell proliferative phenotype in mice, demonstrating that dysregulation of PI3K signals is vital to the observed mast cell hyperplasia.

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Figures

Figure 1
Figure 1
PTEN deficiency enhances BMMC proliferation and differentiation. (A) Bone marrow cells from WT (red) or PTEN-null mice (blue) were stained with CFSE and analyzed for cell division on day 7, day 13, and day 23 of culture. Fluorescence intensity was measured by flow cytometry. One representative of 4 experiments is shown. (B) Percentage of FcϵRI and Kit double-positive cells in bone marrow cell cultures of the indicated genotype over time. Cells (1 × 105 cells) were labeled with PE-MAR-1 (anti-FcϵRI) and APC–anti-Kit. Mean fluorescence intensity was determined by flow cytometry. (C) BMMCs (1 × 104) of the indicated genotypes were incubated with 3H-thymidine (1 mCi) and IgE, SCF, or IgE plus SCF. After 12 hours, the amount of incorporated thymidine was determined. (B-C) A minimum of 4 experiments were conducted.
Figure 2
Figure 2
Pten−/− mice have circulating MCs and increased MC numbers in tissues. (A) Tissue samples of the indicated tissues from 8-week-old mice were fixed with 4% formalin, paraffin-embedded, and sections were stained with toluidine blue. MCs from Pten−/− mice showed spindle-shaped and partially degranulated phenotypes (inset). MC numbers per millimeter squared (graphs) were determined by counting blinded samples from individual mice. Images were visualized on a Leica DM LB2 microscope using a 20× (0.40 NA) objective for dry mounted slides. Image capture was with a QImacong Micro Publisher 3.3 RTV camera using QCapture 2.7.3 software. Image analysis was with ImageJ 1.45 (National Institutes of Health). Statistical analysis was by an unpaired 2-tailed Student t test: *P < .05; **P < .01. Scale bar represents 100 μm. (B-C) Percentage of FcϵRI and Kit-positive cells was analyzed in peripheral blood (B) and in the bone marrow (C) of individual mice with the indicated genotype. Cells (1 × 105) were labeled with PE-MAR-1 (anti-FcϵRI) and APC–anti-Kit. Mean fluorescence intensity was measured by flow cytometry (n = 15 for peripheral blood cells; 6 of 15 mice had circulating MCs; n = 11 for bone marrow cells). For peripheral blood cell counts, 0.025% of double = positive cells was set as a threshold for background. (B-C) A minimum of 4 individual experiments were conducted.
Figure 3
Figure 3
Tissue-specific deletion of Pten demonstrates altered proliferation and increased MC numbers in vivo. (A) PDMCs (1 × 105 cells) from the indicated genotypes (age of mice, 16-24 weeks) were stained with toluidine blue. The controls, Ptenwt/wtFcϵRIβCre+/wt, and Ptenwt/wtFcϵRIβCre+/+ mice were heterozygotes or homozygotes, respectively, for Cre expression at the FcϵRIβ locus and did not carry floxed Pten alleles. Cell surface expression of FcϵRI and Kit in PDMCs of indicated genotypes was determined. Cells (1 × 105 cells) were incubated with PE-MAR-1 (anti-FcϵRI) and APC–anti-Kit. The mean fluorescence intensity was measured by flow cytometry. One representative of 3 individual experiments is shown. (B) PDMCs (5 × 105 cells) were lysed in RIPA buffer. PTEN expression was determined by immunoblot with a monoclonal antibody to PTEN. β-actin was used as loading control. (C) Cell proliferation was measured by CFSE staining. Mean fluorescence intensity was measured by flow cytometry. (B-C) A minimum of 3 individual experiments were conducted. (D) Tissue samples were fixed with 4% formalin, paraffin-embedded, and sections stained with toluidine blue. MCs per millimeter squared was determined by direct counts of blinded samples. Statistical analysis was by an unpaired 2-tailed Student t test: *P < .05; **P < .01. Scale bar represents 100 μm.
Figure 4
Figure 4
PTEN deficiency enhances the proliferative and survival signals of MCs. (A) Cells (5 × 105 cells) were stimulated with IgE/Ag, SCF, or IL-3 and lysed in RIPA buffer. Phosphorylated Akt was probed with anti–phosho-Akt (T308). Total Akt was used as the loading control. (B) IL-3 or SCF-mediated phosphorylation of STAT5 was determined by immunoblot. Stimulated cells (5 × 105 cells) were lysed in RIPA buffer, and phosphorylated STAT5 was detected with an antibody recognizing phosphorylated STAT5. One representative of 4 experiments is shown. (C) BMMCs (5 × 105 cells) incubated in the absence of cytokines for 3 hours and restimulated with SCF, IL-3, or SCF and IL-3 for 24 hours. Expression of antiapoptotic factors (Bcl-2 and Bcl-XL) was measured after 24 hours by immunoblotting with antibodies to Bcl-2 or Bcl-XL. β-actin was used as loading control. (A-C) One representative of a minimum of 4 experiments is shown. Fold induction reflects the mean of all experiments normalized to WT control (0 time; A-B) or to WT control with no treatment (C).
Figure 5
Figure 5
PTEN deficiency enhances MC degranulation and cytokine production via FcϵRI or in combination with Kit. (A) FcϵRI-mediated MC degranulation as measured by β-hexosaminidase release. SCF (10 ng/mL) was used in combination with IgE/Ag. The percentage of β-hexosaminidase released after stimulation is expressed as a percentage of total cellular β-hexosaminidase. (Inset) β-hexosaminidase release with SCF stimulation in the absence of IgE/Ag. One representative of 3 experiments is shown. (B-D) IL-3, IL-6, and TNF production was measured by ELISA. (Inset) Cytokine release with SCF stimulation in the absence of IgE/Ag. Statistical analysis by an unpaired 2-tailed Student t test compared with control cells stimulated with SCF: *P < .05; **P < .01; ***P < .001. One representative of 4 experiments is shown.
Figure 6
Figure 6
Deletion of Pten in MCs causes increased anaphylactic responses and enhanced vascular permeability in vivo. (A) BMMCs and peritoneal MCs (PMCs; 5 × 105 cells) were lysed in RIPA buffer. PTEN expression was determined by immunoblot with an antibody to PTEN. β-actin was used as loading control. One representative of 4 experiments is shown. (B) Ptenwt/wtFcϵRIβCre+/wt mice and Ptenfl/flFcϵRIβCre+/wt mice (age, 24 weeks) were passively sensitized with DNP-specific IgE and challenged 24 hours later with Ag (DNP-HSA) or pseudo-challenged with an equal volume of PBS. Anaphylactic reaction was measured by temperature change. Data are mean ± SEM of a minimum of 5 independent experiments. Statistical analysis was by 2-way ANOVA: ***P < .0001. Significant differences were observed between 25 and 45 minutes after challenge. (C) Ptenwt/wtFcϵRIβCre+/+ mice and Ptenfl/flFcϵRIβCre+/+ mice (age, 16 weeks) were inoculated intraperitoneally with K/BxN serum containing autoantibodies to glucose-6-phosphate isomerase (GPI) or with control serum (Ctrl). Joint swelling and clinical score were determined as described in “Anaphyloxis and arthritis models.” Data are mean ± SEM of a minimum of 8 mice. Statistical analysis was by a 2-way ANOVA: ***P < .0001. Significant differences were observed between 6 and 9 days after challenge. (D) Evans blue dye (200 μL) was injected intravenously in Ptenwt/wtFcϵRIβCre+/+ mice and Ptenfl/flFcϵRIβCre+/+ mice. Subsequently, they were challenged intradermally with 20 μL compound 48/80 (C48/80, 1.75 mg/mL) in PBS or as control with an equal volume of PBS. Vascular permeability was measured by extravasation of the dye into the ear tissue. Data are mean ± SEM from 10 mice. Statistical analysis was by unpaired 2-tailed Student t test: **P < .001.
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References

    1. Wedemeyer J, Tsai M, Galli SJ. Role of mast cells and basophils in innate and acquired immunity. Curr Opin Immunol. 2000;12(6):624–631. - PubMed
    1. Williams CM, Galli SJ. The diverse potential effector and immunoregulatory roles of mast cells in allergic disease. J Allergy Clin Immunol. 2000;105(5):847–859. - PubMed
    1. Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CM, Tsai M. Mast cells as “tunable” effector and immunoregulatory cells: recent advances. Annu Rev Immunol. 2005;23:749–786. - PubMed
    1. Valent P, Akin C, Escribano L, et al. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Invest. 2007;37(6):435–453. - PubMed
    1. Akin C, Jaffe ES, Raffeld M, et al. An immunohistochemical study of the bone marrow lesions of systemic mastocytosis: expression of stem cell factor by lesional mast cells. Am J Clin Pathol. 2002;118(2):242–247. - PubMed

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