Limits...
WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells.

Kettner A, Kumar L, Antón IM, Sasahara Y, de la Fuente M, Pivniouk VI, Falet H, Hartwig JH, Geha RS - J. Exp. Med. (2004)

Bottom Line: Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs.However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs.These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

Show MeSH

Related in: MedlinePlus

Adhesion and spreading of BMMCs on IgE-coated glass slides. (A) Quantification of BMMCs that adhered to IgE-coated glass coverslips. Cells were incubated for 1 h on IgE-coated glass coverslips. The percentage of adherent cells was then determined by β-hexosaminidase content of attached versus detached cells. (B) Quantification of BMMCs spread on IgE-coated glass coverslips. Cells were fixed at indicated time points and were counted under the microscope (×400) to determine the ratio of round versus spread cells. BMMCs preincubated with cytochalasin D were counted after 1 h of incubation. Results represent ratio of spread/round cells of >500 cells per condition counted from two independent experiments. (C) Representative frames of WT (left), WT treated with cytochalasin D (middle), and WIP−/− (right) BMMCs incubated on IgE-coated glass slides (×400). (D) Cells that adhered to IgE-coated glass coverslips after 1 h of incubation were extracted and cytoskeletons were metal coated. Bars, 1 mm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211794&req=5

fig7: Adhesion and spreading of BMMCs on IgE-coated glass slides. (A) Quantification of BMMCs that adhered to IgE-coated glass coverslips. Cells were incubated for 1 h on IgE-coated glass coverslips. The percentage of adherent cells was then determined by β-hexosaminidase content of attached versus detached cells. (B) Quantification of BMMCs spread on IgE-coated glass coverslips. Cells were fixed at indicated time points and were counted under the microscope (×400) to determine the ratio of round versus spread cells. BMMCs preincubated with cytochalasin D were counted after 1 h of incubation. Results represent ratio of spread/round cells of >500 cells per condition counted from two independent experiments. (C) Representative frames of WT (left), WT treated with cytochalasin D (middle), and WIP−/− (right) BMMCs incubated on IgE-coated glass slides (×400). (D) Cells that adhered to IgE-coated glass coverslips after 1 h of incubation were extracted and cytoskeletons were metal coated. Bars, 1 mm.

Mentions: T cells spread and extend protrusions when exposed to anti-TCR/CD3–coated surfaces. This process involves actin cytoskeleton reorganization, which is dependent on WASP and WIP (41, 42). Given the role of WIP in actin cytoskeleton organization, we tested the ability of BMMCs from WT and WIP−/− mice to attach and spread on IgE-coated glass coverslips. WT mast cells did not attach to untreated glass slides (not depicted) but adhered to IgE-coated coverslips. WIP−/− mast cells adhered to IgE-coated coverslips equally as well as WT BMMCs (Fig. 7 A). After attachment, a significant proportion of WT BMMCs spread and extended membrane protrusions (Fig. 7, B and C). Preincubation of WT mast cells with the inhibitor of actin polymerization, cytochalasin D, suppressed their spreading on IgE-coated surfaces (Fig. 7, B and C), suggesting that actin polymerization is critical in this process. WIP−/− BMMCs had reduced ability to spread and form protrusions (Fig. 7 C).


WIP regulates signaling via the high affinity receptor for immunoglobulin E in mast cells.

Kettner A, Kumar L, Antón IM, Sasahara Y, de la Fuente M, Pivniouk VI, Falet H, Hartwig JH, Geha RS - J. Exp. Med. (2004)

Adhesion and spreading of BMMCs on IgE-coated glass slides. (A) Quantification of BMMCs that adhered to IgE-coated glass coverslips. Cells were incubated for 1 h on IgE-coated glass coverslips. The percentage of adherent cells was then determined by β-hexosaminidase content of attached versus detached cells. (B) Quantification of BMMCs spread on IgE-coated glass coverslips. Cells were fixed at indicated time points and were counted under the microscope (×400) to determine the ratio of round versus spread cells. BMMCs preincubated with cytochalasin D were counted after 1 h of incubation. Results represent ratio of spread/round cells of >500 cells per condition counted from two independent experiments. (C) Representative frames of WT (left), WT treated with cytochalasin D (middle), and WIP−/− (right) BMMCs incubated on IgE-coated glass slides (×400). (D) Cells that adhered to IgE-coated glass coverslips after 1 h of incubation were extracted and cytoskeletons were metal coated. Bars, 1 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211794&req=5

fig7: Adhesion and spreading of BMMCs on IgE-coated glass slides. (A) Quantification of BMMCs that adhered to IgE-coated glass coverslips. Cells were incubated for 1 h on IgE-coated glass coverslips. The percentage of adherent cells was then determined by β-hexosaminidase content of attached versus detached cells. (B) Quantification of BMMCs spread on IgE-coated glass coverslips. Cells were fixed at indicated time points and were counted under the microscope (×400) to determine the ratio of round versus spread cells. BMMCs preincubated with cytochalasin D were counted after 1 h of incubation. Results represent ratio of spread/round cells of >500 cells per condition counted from two independent experiments. (C) Representative frames of WT (left), WT treated with cytochalasin D (middle), and WIP−/− (right) BMMCs incubated on IgE-coated glass slides (×400). (D) Cells that adhered to IgE-coated glass coverslips after 1 h of incubation were extracted and cytoskeletons were metal coated. Bars, 1 mm.
Mentions: T cells spread and extend protrusions when exposed to anti-TCR/CD3–coated surfaces. This process involves actin cytoskeleton reorganization, which is dependent on WASP and WIP (41, 42). Given the role of WIP in actin cytoskeleton organization, we tested the ability of BMMCs from WT and WIP−/− mice to attach and spread on IgE-coated glass coverslips. WT mast cells did not attach to untreated glass slides (not depicted) but adhered to IgE-coated coverslips. WIP−/− mast cells adhered to IgE-coated coverslips equally as well as WT BMMCs (Fig. 7 A). After attachment, a significant proportion of WT BMMCs spread and extended membrane protrusions (Fig. 7, B and C). Preincubation of WT mast cells with the inhibitor of actin polymerization, cytochalasin D, suppressed their spreading on IgE-coated surfaces (Fig. 7, B and C), suggesting that actin polymerization is critical in this process. WIP−/− BMMCs had reduced ability to spread and form protrusions (Fig. 7 C).

Bottom Line: Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs.However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs.These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Wiskott-Aldrich syndrome protein-interacting protein (WIP) stabilizes actin filaments and is important for immunoreceptor-mediated signal transduction leading to actin cytoskeleton rearrangement in T and B cells. Here we report a role for WIP in signaling pathways downstream of the high affinity receptor for immunoglobulin (Ig)E (FcepsilonRI) in mast cells. WIP-deficient bone marrow-derived mast cells (BMMCs) were impaired in their capacity to degranulate and secrete interleukin 6 after FcepsilonRI ligation. Calcium mobilization, phosphorylation of Syk, phospholipase C-g2, and c-Jun NH2-terminal kinase were markedly decreased in WIP-deficient BMMCs. WIP was found to associate with Syk after FcepsilonRI ligation and to inhibit Syk degradation as evidenced by markedly diminished Syk levels in WIP-deficient BMMCs. WIP-deficient BMMCs exhibited no apparent defect in their subcortical actin network and were normal in their ability to form protrusions when exposed to an IgE-coated surface. However, the kinetics of actin changes and the cell shape changes that follow FcepsilonRI signaling were altered in WIP-deficient BMMCs. These results suggest that WIP regulates FcepsilonRI-mediated mast cell activation by regulating Syk levels and actin cytoskeleton rearrangement.

Show MeSH
Related in: MedlinePlus