Evidence that formation of vimentin mitogen-activated protein kinase (MAPK) complex mediates mast cell activation following FcεRI/CC chemokine receptor 1 cross-talk

Abstract

Accumulating evidence points to cross-talk between FcεRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcεRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using β,β'-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcεRI and CCR1 activation.

FIGURE 1.

Synergistic CCL2 production in RBL-CCR1 cells co-stimulated via FcϵRI and CCR1. A, after sensitization with anti-DNP-IgE mAb, RBL-CCR1 cells were stimulated with 10 ng/ml DNP-HSA and/or various concentrations of rCCL3 for 2, 4, or 6 h before determining CCL2 production by ELISA. B, to inhibit p38 MAPK activation, the cells were incubated with 10 or 50 μm SB203580 for the last 1 h of sensitization. C, to inhibit ERK1/2 activation, the cells were incubated with 10 or 50 μm PD98058 for the last 1 h of sensitization. The cells were then stimulated with 10 ng/ml DNP-HSA and/or 100 ng/ml rCCL3 for 6 h. The plotted data of mean CCL2 concentrations in culture supernatants measured by ELISA are representative of three independent experiments. Error bars, S.D. *, p < 0.01.

FIGURE 2.

Increased phosphorylation of p38 MAPK and ERK1/2 in RBL-CCR1 cells stimulated via FcϵRI and CCR1. After sensitization with anti-DNP-IgE mAb, RBL-CCR1 cells were stimulated with 10 ng/ml DNP-HSA and/or 100 ng/ml rCCL3 for 5 min. To inhibit p38 kinase and ERK1/2 activation, the cells were incubated with 50 μm SB203580 or 50 μm PD98058 for the last 1 h of sensitization. Levels of phosphorylated and total p38 kinase (A) and ERK1/2 (B) protein in total cell lysates were measured by ELISA. The data are representative for three independent experiments. nd, not detectable (<1.0 unit (U)/ml). Error bars, S.D. *, p < 0.01; **, p < 0.05.

FIGURE 3.

Differential protein expression analysis in RBL-CCR1 cells stimulated via FcϵRI and CCR1. After sensitization with anti-DNP-IgE, RBL-CCR1 cells were stimulated with or without 10 ng/ml DNP-HSA and 100 ng/ml rCCL3 for 5 min. Total cell lysates of the unstimulated cells (A) or total cell lysates of the stimulated cells (B) were subjected to two-dimensional PAGE and stained with Coomassie Brilliant Blue. Protein spots up-regulated in a gel are shown with the red arrows. MW, molecular weight.

FIGURE 4.

Phosphorylation of vimentin in RBL-CCR1 cells stimulated via FcϵRI and CCR1. After sensitization with anti-DNP-IgE mAb, RBL-CCR1 cells were stimulated with 10 ng/ml DNP-HSA and/or various concentrations of rCCL3 for 5 min. Total cell lysates of non-stimulated or the stimulated cells were subjected to immunoprecipitation with anti-vimentin mAb. The immunoprecipitated proteins were analyzed by immunoblotting using antibodies against phosphotyrosine; phosphothreonine; phosphoserine; phosphorylated vimentin at Ser-6, -55, -71, or -82; or vimentin. The data are representative of two independent experiments.

FIGURE 5.

Interaction of vimentin and phosphorylated MAPK in RBL-CCR1 cells stimulated via FcϵRI and CCR1. After sensitization with anti-DNP-IgE, RBL-CCR1 cells were stimulated with 10 ng/ml DNP-HSA and/or various concentrations of rCCL3 for 5 min. A, after the stimulation, soluble forms of vimentin were extracted and analyzed by immunoblotting using anti-vimentin Abs. B, the cells were incubated with β,β′-iminodipropionitrile (IDPN (+)) to promote aggregation of vimentin intermediate filaments for the last 1 h of sensitization. Soluble forms of vimentin in the cells were extracted and subjected to immunoprecipitation using anti-vimentin mAb and analyzed by immunoblotting using anti-phosphorylated ERK1/2 mAb, anti-phosphorylated p38 kinase mAb, or anti-vimentin Abs. The data are representative of three independent experiments.

FIGURE 6.

Reduced CCL2 production by β,β′-iminodipropionitrile treatment in RBL-CCR1 cells and BMMCs stimulated via FcϵRI and CCR1. RBL-CCR1 cells (A) and BMMCs (B) were sensitized with anti-DNP-IgE and were incubated with IDPN for the last 1 h of sensitization. The cells were then stimulated with 10 ng/ml DNP-HSA and/or 10 or 100 ng/ml rCCL3. Culture supernatants were harvested after stimulation. Concentrations of CCL2 in the supernatant were measured by ELISA. The plotted CCL2 concentration data are representative of three independent experiments. Error bars, S.D. *, p < 0.01.

FIGURE 7.

Hypothetical model for FcϵRI and CCR1 mediated signaling pathways in mast cells. FcϵRI and CCR1 activation synergistically induces phosphorylation of ERK1/2, p38 MAPK and vimentin in mast cells. Phosphorylation of vimentin induces disassembly of the filament protein. The disassembled vimentin interacts with phosphorylated ERK1/2 directly or with phosphorylated p38 MAPK indirectly via other vimentin-binding protein(s) (protein X). Vimentin may then act as a shuttle protein for the activated MAPKs to translocate them into the nucleus.