Nf1-/- Schwann cell-conditioned medium modulates mast cell degranulation by c-Kit-mediated hyperactivation of phosphatidylinositol 3-kinase

Abstract

Neurofibromatosis type 1 (NF1) is a common genetic disorder and is characterized by both malignant and nonmalignant neurofibromas, which are composed of Schwann cells, degranulating mast cells, fibroblasts, and extracellular matrix. We and others have previously shown that hyperactivation of the c-Kit pathway in an Nf1 haploinsufficient microenvironment is required for both tumor formation and progression. Mast cells play a key role in both tumorigenesis and neoangiogenesis via the production of matrix metalloproteinases, heparin, and a range of different growth factors. In the present study, we show that tumorigenic Schwann cells derived from Nf1(-/-) embryos promote increased degranulation of Nf1(+/-) mast cells compared with wild-type mast cells via the secretion of the Kit ligand. Furthermore, we used genetic intercrosses as well as pharmacological agents to link the hyperactivation of the p21(Ras)-phosphatidylinositol 3-kinase (PI3K) pathway to the increased degranulation of Nf1(+/-) mast cells both in vitro and in vivo. These studies identify the p21(Ras)-PI3K pathway as a major regulator of the gain in Nf1(+/-) mast cell degranulation in neurofibromas. Collectively, these studies identify both c-Kit and PI3K as molecular targets that modulate mast cell functions in cases of NF1.

Figure 1

Nf1^−/− SCCM significantly promotes Nf1^+/− mast cell degranulation. Degranulation of mast cells was assessed by the release of β-hexosaminidase. β-Hexosaminidase activity is measured in the supernatant and the extent of degranulation is reported as a percentage of total cellular β-hexosaminidase activity. A: Degranulation of WT and Nf1^+/− BMMCs was assessed by the release of β-hexosaminidase after SCCM stimulation. *P < 0.01 for WT versus Nf1^−/− SCCM. **P < 0.01 for Nf1^+/− versus WT mast cells stimulated with Nf1^−/− SCCM. Data are means ± SEM from triplicate samples in four independent experiments. B: β-Hexosaminidase release was measured after stimulation with either Kit-L plus DNP or SCCM (WT or Nf1^−/−) with or without the addition of Ack. *P < 0.01 for WT versus Nf1^+/− mast cells stimulated with Kit-L/DNP. **P < 0.01 for Nf1^+/− versus WT mast cells stimulated with Nf1^−/− SCCM and Nf1^+/− treated with Ack versus Nf1^+/− treated with vehicle. Data are means ± SEM from triplicate samples in four independent experiments. C: The Nf1^−/− SCCM promotes mast cell degranulation via the c-Kit receptor. Degranulation of BMMCs derived from WT, Nf1^+/−, W41/W41, and Nf1^+/−;W41/W41 mice was assessed by the release of β-hexosaminidase after stimulation with Nf1^−/− Schwann cell-conditioned medium. *P < 0.01 for WT versus Nf1^+/− mast cells stimulated with Nf1^−/− SCCM. **P < 0.01 for WT versus W41/W41 mast cells stimulated with Nf1^−/− SCCM. ***P < 0.01 for Nf1^+/− versus Nf1^+/−;W41/W41 mast cells stimulated with Nf1^−/− SCCM. Data are means ± SEM from triplicate samples in four independent experiments.

Figure 2

Effect of a pharmacological PI3K inhibitor on Nf1^+/− mast cell degranulation. A: WT or Nf1^+/− mast cells were incubated with or without Ly294002 for the time indicated and then were stimulated with Kit-L. AKT phosphorylation at Ser-473 was examined by Western blot. Data are representative of one of three independent experiments using different primary cell lines. B: WT and Nf1^+/− mast cells were incubated in the absence or presence of Ly294002 and stimulated with Kit-L/DNP for 15 minutes. β-Hexosaminidase release was examined to determine degranulation. Results are representative of one of four independent experiments performed in triplicate. *P < 0.01 for Kit-L-induced WT mast cell degranulation versus basal level. **P < 0.01 for Nf1^+/− versus WT mast cell degranulation induced by Kit-L. ***P < 0.01 for WT or Nf1^+/− mast cell degranulation mediated by Kit-L with versus without Ly294002 inhibition.

Figure 3

Genetic disruption of p85α restores Kit-L-mediated gain of function in Nf1^+/− mast cells to WT levels. A: Mast cells derived from WT, Nf1^+/−, p85α^−/−, and Nf1^+/−;p85α^−/− mice were stimulated with Kit-L, and Akt phosphorylation was examined by Western blot. Data are representative of one of three independent experiments using different primary cell lines. B: Mast cells derived from WT, Nf1^+/−, p85α^−/−, and Nf1^+/−;p85α^−/− mice were stimulated with Kit-L/DNP for 15 minutes, and mast cell degranulation was examined. Results are representative of one of four independent experiments performed in triplicate. *P < 0.01 for Nf1^+/− versus WT mast cell degranulation induced by Kit-L. **P < 0.01 for p85α^−/− versus WT mast cell degranulation induced by Kit-L. ***P < 0.01 for Nf1^+/− versus Nf1^+/−;p85α^−/− mast cell degranulation induced by Kit-L.

Figure 4

Effect of genetic inactivation of p85α on mast cell numbers in vivo. A and B: Ear sections were stained with Alcian Blue, and mast cells were quantitated in a blinded fashion by counting 1-mm² sections. Mast cells are identified by arrowheads. *P < 0.01 comparing Nf1^+/− versus WT mice. **P < 0.01 for p85α^−/− versus WT mice. ***P < 0.01 for Nf1^+/− versus Nf1^+/−;p85α^−/− mice. C and D: Representative cytospins from peritoneal lavages stained for mast cells from individual mice of the four Nf1 and p85α genotypes. Peritoneal cells were stained with toluidine blue to quantify the total number of mast cells per peritoneal lavage. *P < 0.01 for Nf1^+/− versus WT mice. **P < 0.01 for p85α^−/− versus WT mice. ***P < 0.01 for Nf1^+/− versus Nf1^+/−;p85α^−/− mice. E and F: Genetic disruption of p85α diminishes PCA in vivo. WT, Nf1^+/−, p85α^−/−, and Nf1^+/−;p85α^−/− mice were sensitized by intradermal injection of anti-DNP IgE (1:44 dilution, 1 μg/ml) into the right ear (20 μl/injection) and PBS (20 μl/injection) into the left ear. After 20 hours, mice were challenged by intravenous injection of antigen (DNP-HSA) and Kit-L along with Evans blue injection. Photographs of representative IgE-primed (left) and control (right) ears 20 minutes after antigen/Kit-L challenge are shown qualitatively. From each ear, Evans blue was extracted, and the intensity of the dye was measured by absorption at 620 nm. *P < 0.01 comparing Nf1^+/− versus WT mice. **P < 0.01 for Nf1^+/− versus Nf1^+/−;p85α^−/− mice.