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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.

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Determination of cellular F-actin and time lapse video microscopy of BMMCs activated by FcεRI ligation. (A) IgE-sensitized BMMCs were stimulated by cross-linking of FcεRI for the indicated time. Cells were then fixed, permeabilized, stained with phalloidin-TRITC, and analyzed by FACS®. Results represent mean ± SD of three experiments. (B) Barbed and pointed end counts of activated BMMCs determined by pyrene-labeled actin assembly assay. (C) IgE-sensitized BMMCs were allowed to attach to fibronectin-coated glass coverslips for 20 min at 37°C. Cells were then videotaped for 15 min and FcεRI cross-linking was induced after 1 min (frame 0). The indicated time frames were processed using NIH Image software. ×400.
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fig8: Determination of cellular F-actin and time lapse video microscopy of BMMCs activated by FcεRI ligation. (A) IgE-sensitized BMMCs were stimulated by cross-linking of FcεRI for the indicated time. Cells were then fixed, permeabilized, stained with phalloidin-TRITC, and analyzed by FACS®. Results represent mean ± SD of three experiments. (B) Barbed and pointed end counts of activated BMMCs determined by pyrene-labeled actin assembly assay. (C) IgE-sensitized BMMCs were allowed to attach to fibronectin-coated glass coverslips for 20 min at 37°C. Cells were then videotaped for 15 min and FcεRI cross-linking was induced after 1 min (frame 0). The indicated time frames were processed using NIH Image software. ×400.

Mentions: FcεRI signaling induces changes in cellular F-actin content in mast cells lines (21, 43). We compared F-actin content in WT and WIP−/− BMMCs after FcεRI ligation. Resting WIP−/− BMMCs had a reduced baseline F-actin content compared with WT BMMCs. The mean reduction of cellular F-actin in WIP−/− BMMCs was 12 ± 2.4% in five experiments (P < 0.01). FcεRI ligation in WT BMMCs resulted in an initial decrease in F-actin content, with the maximum decrease being observed after 2 min of stimulation. Subsequently, F-actin content slowly increased but was still lower than baseline 30 min after FcεRI ligation (Fig. 8 A). The rapid decrease of F-actin was found to be dependent on the influx of extracellular calcium as no change of F-actin content was observed in the presence of EGTA and as ionomycin also caused a decrease in F-actin content that was sustained (not depicted). WIP−/− BMMCs exhibited an initial decrease in F-actin that was similar to that observed in WT controls. However, in contrast to what was observed in WT BMMCs, the F-actin content of WIP−/− BMMCs returned almost immediately back to prestimulation level (Fig. 8 A).


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)

Determination of cellular F-actin and time lapse video microscopy of BMMCs activated by FcεRI ligation. (A) IgE-sensitized BMMCs were stimulated by cross-linking of FcεRI for the indicated time. Cells were then fixed, permeabilized, stained with phalloidin-TRITC, and analyzed by FACS®. Results represent mean ± SD of three experiments. (B) Barbed and pointed end counts of activated BMMCs determined by pyrene-labeled actin assembly assay. (C) IgE-sensitized BMMCs were allowed to attach to fibronectin-coated glass coverslips for 20 min at 37°C. Cells were then videotaped for 15 min and FcεRI cross-linking was induced after 1 min (frame 0). The indicated time frames were processed using NIH Image software. ×400.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211794&req=5

fig8: Determination of cellular F-actin and time lapse video microscopy of BMMCs activated by FcεRI ligation. (A) IgE-sensitized BMMCs were stimulated by cross-linking of FcεRI for the indicated time. Cells were then fixed, permeabilized, stained with phalloidin-TRITC, and analyzed by FACS®. Results represent mean ± SD of three experiments. (B) Barbed and pointed end counts of activated BMMCs determined by pyrene-labeled actin assembly assay. (C) IgE-sensitized BMMCs were allowed to attach to fibronectin-coated glass coverslips for 20 min at 37°C. Cells were then videotaped for 15 min and FcεRI cross-linking was induced after 1 min (frame 0). The indicated time frames were processed using NIH Image software. ×400.
Mentions: FcεRI signaling induces changes in cellular F-actin content in mast cells lines (21, 43). We compared F-actin content in WT and WIP−/− BMMCs after FcεRI ligation. Resting WIP−/− BMMCs had a reduced baseline F-actin content compared with WT BMMCs. The mean reduction of cellular F-actin in WIP−/− BMMCs was 12 ± 2.4% in five experiments (P < 0.01). FcεRI ligation in WT BMMCs resulted in an initial decrease in F-actin content, with the maximum decrease being observed after 2 min of stimulation. Subsequently, F-actin content slowly increased but was still lower than baseline 30 min after FcεRI ligation (Fig. 8 A). The rapid decrease of F-actin was found to be dependent on the influx of extracellular calcium as no change of F-actin content was observed in the presence of EGTA and as ionomycin also caused a decrease in F-actin content that was sustained (not depicted). WIP−/− BMMCs exhibited an initial decrease in F-actin that was similar to that observed in WT controls. However, in contrast to what was observed in WT BMMCs, the F-actin content of WIP−/− BMMCs returned almost immediately back to prestimulation level (Fig. 8 A).

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