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Cytoskeletal Protein 4.1R Is a Positive Regulator of the FcεRI Signaling and Chemotaxis in Mast Cells

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Cytoskeletal Protein 4.1R Is a Positive Regulator of the FcεRI Signaling and Chemotaxis in Mast Cells

Lubica Draberova et al. Front Immunol.

Abstract

Protein 4.1R, a member of the 4.1 family, functions as a bridge between cytoskeletal and plasma membrane proteins. It is expressed in T cells, where it binds to a linker for activation of T cell (LAT) family member 1 and inhibits its phosphorylation and downstream signaling events after T cell receptor triggering. The role of the 4.1R protein in cell activation through other immunoreceptors is not known. In this study, we used 4.1R-deficient (4.1R-KO) and 4.1R wild-type (WT) mice and explored the role of the 4.1R protein in the high-affinity IgE receptor (FcεRI) signaling in mast cells. We found that bone marrow mast cells (BMMCs) derived from 4.1R-KO mice showed normal growth in vitro and expressed FcεRI and c-KIT at levels comparable to WT cells. However, 4.1R-KO cells exhibited reduced antigen-induced degranulation, calcium response, and secretion of tumor necrosis factor-α. Chemotaxis toward antigen and stem cell factor (SCF) and spreading on fibronectin were also reduced in 4.1R-KO BMMCs, whereas prostaglandin E2-mediated chemotaxis was not affected. Antibody-induced aggregation of tetraspanin CD9 inhibited chemotaxis toward antigen in WT but not 4.1R-KO BMMCs, implying a CD9-4.1R protein cross-talk. Further studies documented that in the absence of 4.1R, antigen-mediated phosphorylation of FcεRI β and γ subunits was not affected, but phosphorylation of SYK and subsequent signaling events such as phosphorylation of LAT1, phospholipase Cγ1, phosphatases (SHP1 and SHIP), MAP family kinases (p38, ERK, JNK), STAT5, CBL, and mTOR were reduced. Immunoprecipitation studies showed the presence of both LAT1 and LAT2 (LAT, family member 2) in 4.1R immunocomplexes. The positive regulatory role of 4.1R protein in FcεRI-triggered activation was supported by in vivo experiments in which 4.1R-KO mice showed the normal presence of mast cells in the ears and peritoneum, but exhibited impaired passive cutaneous anaphylaxis. The combined data indicate that the 4.1R protein functions as a positive regulator in the early activation events after FcεRI triggering in mast cells.

Keywords: 4.1R protein; chemotaxis; degranulation; mast cell; passive cutaneous anaphylaxis.

Figures

Figure 1
Figure 1
4.1R-deficient BMMCs exhibit normal surface expression of FcεRI and c-KIT, but reduced antigen- and thapsigargin-induced degranulation. (A–D) BMMCs derived from 4.1R-WT and 4.1R-KO mice were stained for FcεRI and c-KIT with Alexa Fluor 647-conjugated anti-FcεRI and PE/Cy7-conjugated anti-c-KIT. Unstained 4.1R-WT and 4.1R-KO cells were used as negative controls (Ctrls). Samples were analyzed by flow cytometry. Representative histograms of the expression of FcεRI (A) and c-KIT (B) gated for live cells after six weeks of culture are shown. (C,D) Quantification of representative flow cytometry histograms showing the median fluorescence intensity (MFI) of FcεRI (C) and c-KIT (D) expressed as means and SEM from three independent experiments. (E) 4.1R protein in lysates from 4.1R-WT and 4.1R-KO BMMCs was determined by immunoblotting with the 4.1R-specific antibody. The same membrane was developed for β-actin used as a loading control. Numbers on the right indicate positions of molecular weight markers in kDa. A typical result of four experiments is shown. (F,G) Degranulation response of 4.1R-WT and 4.1R-KO BMMCs. The cells were sensitized (F) or not (G) with TNP-specific IgE (1 μg/ml) and then stimulated for 30 min with various concentrations of antigen (Ag, TNP-BSA; F) or thapsigargin (Th; G). Data represent means ± SEM calculated from five independent experiments. Statistical significance of differences between 4.1R-WT and 4.1R-KO cells was evaluated with Student's unpaired, two-tailed t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 2
Figure 2
Positive regulatory role of 4.1R protein on antigen-induced calcium response. (A–C) Measurement of free intracellular Ca2+. 4.1R-WT and 4.1R-KO BMMCs were sensitized with IgE (A,B) or not (C), loaded with Fura-2 AM, and then stimulated with antigen (Ag) at a concentration 0.1 μg/ml (A) or 0.5 μg/ml (B), or 0.5 μM thapsigargin (Th; C), added as indicated by arrows. Free intracellular Ca2+ was measured at different time intervals as Fura-2 emission at 510 nm after excitation with 340 and 380 nm. (D,E) Measurement of 45Ca2+ uptake. The cells were sensitized with IgE (D) or not (E) and then stimulated for various time intervals with antigen at a concentration 0.5 μg/ml (D) or 1 μM thapsigargin (E) in the presence of 1 mM extracellular 45Ca2+. The reactions were terminated by centrifugation of the cells through BSA gradient, and cell-bound radioactivity (in cell pellet) was determined. Data represent means ± SEM from 4 to 6 independent experiments performed in duplicates or triplicates. Statistical significance of differences between 4.1R-WT and 4.1R-KO cells was determined using two-way ANOVA with Bonferroni post-test (A–C) or unpaired two-tailed Student's t-test (D,E). *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 3
Figure 3
Positive regulatory role of 4.1R protein on TNF-α cytokine gene expression and TNF-α production in FcεRI-activated cells. 4.1R-WT and 4.1R-KO BMMCs were sensitized with IgE and then activated or not with antigen. After 1 h, TNF-α mRNA was quantified by RT-qPCR (A), and after 6 h, TNF-α cytokine secreted into the media was quantified by the immuno-PCR method (B). Means ± SEM were calculated from three independent experiments performed in duplicates or triplicates. Statistical significance of intergroup differences was determined by unpaired two-tailed Student's t-test. **P < 0.01.
Figure 4
Figure 4
Positive regulatory role of 4.1R on antigen- and SCF-mediated chemotaxis and spreading and cross-talk of 4.1R with tetraspanin CD9 and PKA in chemotaxis toward antigen. (A) 4.1R-WT and 4.1R-KO BMMCs were sensitized with IgE and their migration toward the antigen (Ag; TNP-BSA, 0.1 or 0.25 μg/ml in the bottom well) was determined. Migration of IgE non-sensitized cells toward SCF (0.05 μg/ml) and PGE2 (0.1 μM) was also determined. Means ± SEMs were calculated from 12 independent experiments performed in duplicates. (B) IgE-sensitized 4.1R-WT and 4.1R-KO BMMCs were treated for 15 min at 37°C with anti (α)-CD9 (2 μg/ml), PKA inhibitor H-89 (5 μM), PKA enhancer caffeine (0.1 mM), alone or in combinations, and their effect on chemotaxis toward antigen (0.25 μg/ml in the bottom well) was determined. Cells incubated in chemotaxis medium alone were used as positive controls (Ctrl). (C–F) Phosphorylation of c-KIT in cells activated with SCF (0.05 μg/ml; C,D) or phosphorylation of PKA in IgE-sensitized cells activated with antigen (0.25 μg/ml, E,F). Cells activated for the indicated time intervals were lysed in 2-mercaptoethanol-containing sample buffer, size-fractionated by SDS-PAGE, and analyzed by immunoblotting with antibodies specific for p-c-KIT and loading control c-KIT (C,D), and with p-PKA- and loading control PKA-specific antibodies (E,F). (C,E) Show representative immunoblots. (D,F) Show the results of densitometry analysis of the corresponding immunoblots in which signals from tyrosine-phosphorylated proteins in activated cells were normalized to the tyrosine-phosphorylated proteins in non-activated 4.1R-WT cells and the amounts of corresponding loading control proteins. Means ± SEM were calculated from a minimum of five independent experiments in each group. (G) IgE-sensitized 4.1R-WT and 4.1R-KO BMMCs were attached to the fibronectin-coated glass surface and then non-stimulated (Ctrl) or stimulated with antigen (Ag; 0.25 μg/ml), or SCF (0.05 μg/ml). After 30 min, the cells were fixed and stained for actin with Alexa Fluor 488-phalloidin conjugate. The images are confocal micrographs taken at equatorial planes. Scale bar, 10 μm. (H) Cell areas from micrographs as in (G) were determined using automated CellProfiller software and the data were normalized to non-activated 4.1R-WT cells. At least 200 cells were evaluated in each experiment. Means + SEM were calculated from three independent experiments. Statistical significance of intergroup differences in (A,D,F,H) was determined by unpaired two-tailed Student's t-test, and in (B) by one-way ANOVA with Tukey's post-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 5
Figure 5
Positive regulatory role of 4.1R on tyrosine phosphorylation of SYK, LAT1, and CBL, but not FcεRI and LAT2, in antigen-activated BMMCs. 4.1R-WT and 4.1R-KO BMMCs were sensitized with IgE and activated for the indicated time intervals with antigen (TNP-BSA; 0.25 μg/ml). The cells were lysed and FcεRI (A,B), LAT1 (E,F), and LAT2 (G,H) were immunoprecipitated with the corresponding protein-specific antibodies, followed by size fractionation and examination by immunoblotting with tyrosine-specific mAb PY20-HRP conjugate (PY). For SYK (C,D) and CBL (I,J), size-fractionated proteins in cell lysates were directly analyzed by immunoblotting with the antibodies specific for phospho-SYKY519/Y520 and phospho-CBLY700, respectively. For loading controls, the membranes were analyzed by immunoblotting with the corresponding protein-specific antibodies. (A,C,E,G,I) Show representative immunoblots from at least five experiments. (B,D,F,H,J) Show the results of densitometry analysis of the corresponding immunoblots in which signals from tyrosine-phosphorylated proteins in activated cells are normalized to the tyrosine-phosphorylated proteins in non-activated cells and the amounts of corresponding loading control proteins. Means ± SEM were calculated from a minimum of five independent experiments (three BMMC isolates) in each group. The statistical significance of the difference between 4.1R-WT and 4.1R-KO BMMCs was determined by two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 6
Figure 6
Positive regulatory role of 4.1R in phosphorylation of PLCγ, phosphatases SHP1 and SHIP, and mTOR in antigen-activated BMMCs. 4.1R-WT and 4.1R-KO BMMCs were sensitized with IgE and activated for the indicated time intervals with antigen (TNP-BSA; 0.25 μg/ml). The cells were lysed in 2-mercaptoethanol containing sample buffer, sonicated, size-fractionated by SDS-PAGE, and analyzed by immunoblotting with the antibodies specific for phospho-PLCγ1Y783, phospho-SHP1Y564, phospho-SHIP1Y1020, and phospho-mTORS2448. For loading controls, the membranes were analyzed by immunoblotting with the corresponding protein-specific antibodies. (A,C,E,G) Show representative immunoblots. (B,D,F,H) Show the results of densitometry analysis of the corresponding immunoblots in which signals from tyrosine-phosphorylated proteins in activated cells were normalized to the tyrosine-phosphorylated proteins in non-activated cells and the amounts of corresponding loading control proteins. Means ± SEM were calculated from a minimum of five independent experiments in each group. The statistical significance of the difference between 4.1R-WT and 4.1R-KO BMMCs was determined by two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 7
Figure 7
Positive regulatory role of 4.1R in phosphorylation of transcriptional regulators p-38, ERK, JNK, and STAT5 in antigen-activated BMMCs. 4.1R-WT and 4.1R-KO BMMCs were sensitized with IgE and activated for the indicated time intervals with antigen (TNP-BSA; 0.25 μg/ml). The cells were sonicated, size fractionated, and analyzed by immunoblotting with the antibodies specific for phosho-p38Y182, phosho-ERKY204, phospho-JNKT183/Y185, and phospho-STAT5Y694. For loading controls, the membranes were analyzed by immunoblotting with the corresponding protein-specific antibodies. (A,C,E,G) Show representative immunoblots. (B,D,F,H) Show the results of densitometry analysis of the corresponding immunoblots in which signals from tyrosine-phosphorylated proteins in activated cells were normalized to the tyrosine-phosphorylated proteins in non-activated cells and the amounts of corresponding loading control proteins. Means ± SEM were calculated from a minimum of four independent experiments in each group. The statistical significance of the difference between 4.1R-WT and 4.1R-KO BMMCs was determined by two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Figure 8
Figure 8
4.1R forms complexes with LAT1 and LAT2 adaptor proteins. BMMCs were sensitized with IgE and activated (+) or not (–) with antigen (TNP-BSA; 0.25 μg/ml) for 5 min. The cells were lysed, and LAT1 (A) and LAT2 (B) complexes were immunoprecipitated with the LAT1 (αL1) and LAT2 (αL2) specific antibodies immobilized on beads, followed by size fractionation and immunoblotting with 4.1R-specific antibody and anti-LAT1 (A) and anti-LAT2 (B). Lysates incubated with the beads armed with normal rabbit serum (A, Co, line 3) or beads without antibodies were used as a negative controls (C). As a positive control, whole lysates were directly analyzed by immunoblotting for the presence of 4.1R and LAT1 (A, line 4) and LAT2 (B, line 3). A typical result from three independent experiments is shown.
Figure 9
Figure 9
4.1R-KO mice possess normal numbers of mast cells in the ear tissue and peritoneal cavity but exhibit impaired PCA. (A–C) The ear tissue from 4.1R-WT mice (A) and 4.1R-KO mice (B) was fixed with formaldehyde, embedded in paraffin, sectioned, and stained with toluidine blue for histological quantification of mast cells. The representative images were taken at magnification 100x. Arrows and asterisks indicate toluidine blue-positive mast cells. The numbers of mast cells in histological preparations were counted and the data are presented as the means ± SEM of mast cells per microscopy field calculated from 50 fields in each mouse and six mice for each genotype (C). (D–F) Peritoneal mast cells from 4.1R-WT mice (D) and 4.1R-KO mice (E) were stained with FcεRI-specific antibody-Alexa Fluor 647 conjugate and c-KIT-specific antibody-PE/Cy7 conjugate and analyzed by flow cytometry. Means ± SEM of the percentage of mast cells was calculated from the peritoneal cavity of six mice of each genotype (F). (G–I) PCA assay was performed as described in Materials and Methods. Examples of the left ear of 4.1R-WT mouse (G) and 4.1R-KO mouse (H) are shown. (I) Quantitative data for ear tissue-extracted Evans blue from IgE-sensitized and antigen-exposed left ear from 4.1R-WT and 4.1R-KO mice. Means ± SEM were calculated from 12 4.1R-WT mice and 11 4.1R-KO mice. Statistical significance of intergroup differences was evaluated by unpaired two-tailed Student's t-test (C,F,G). ***P < 0.001.

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