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, 92 (5), 1029-41

Molecular Mechanisms of Spontaneous and Directed Mast Cell Motility

Affiliations

Molecular Mechanisms of Spontaneous and Directed Mast Cell Motility

Jinmin Lee et al. J Leukoc Biol.

Abstract

Migration is a fundamental function of immune cells, and a role for Ca(2+) in immune cell migration has been an interest of scientific investigations for many decades. Mast cells are the major effector cells in IgE-mediated immune responses, and cross-linking of IgE-FcεRI complexes at the mast cell surface by antigen activates a signaling cascade that causes mast cell activation, resulting in Ca(2+) mobilization and granule exocytosis. These cells are known to accumulate at sites of inflammation in response to parasite and bacterial infections. Using real-time imaging, we monitored chemotactic migration of RBL and rat BMMCs in response to a gradient of soluble multivalent antigen. Here, we show that Ca(2+) influx via Orai1 plays an important role in regulating spontaneous motility and directional migration of mast cells toward antigen via IgER complexes. Inhibition of Ca(2+) influx or knockdown of the Ca(2+) entry channel protein Orai1 by shRNA causes inhibition of both of these processes. In addition, a mutant Syk- shows impaired spontaneous motility and chemotaxis toward antigen that is rescued by expression of Syk. Our findings identify a novel Ca(2+) influx-mediated, Orai1-dependent mechanism for mast cell migration.

Figures

Figure 1.
Figure 1.. Morphology and motility properties of RBL-2H3 mast cells and rat BMMCs.
(A) Phase contrast images of RBL-2H3 cells (left) and rat BMMCs (right) in culture medium. Note polarized morphologies with extended protrusions that are common for these cells after several hours on glass surfaces (arrows). (B) Representative images of first (left) and last (right) snapshots of time-lapse images of RBL-2H3 cells that were automatically tracked for 3 h, as described in Materials and Methods. Numbers identify identical cells in both images, and colored lines in the right panel represent the cell migration tracks. (C and D) Average motility coefficients of RBL-2H3 cells, as determined by Equation 2. Error bars show sem for n = 15–97 cells for each sample. Cell motility was monitored for 1.5–3 h in culture medium (C) or in BSS with 1 mg/ml BSA (D). Inhibitors (1 μM cytochalasin D, 1 μM BiM hydrochloride, or 200 nM wortmannin) were added just prior to motility measurements. For samples with wortmannin, cell motility was monitored in buffer without BSA. C1 is a mutant RBL cell line, RBL-C1, defective in Rho GTPase activation. (E) Average motility coefficients of rat BMMCs ± sem for n = 30–67/each sample. Cytochalasin D (1 μM) was added just prior to motility measurements. *P < 0.05; **P < 0.01; ****P < 0.0001 compared with untreated control.
Figure 2.
Figure 2.. Extracellular Ca2+ is important for mast cell motility.
Motilities of individual RBL-2H3 cells (A) and rat BMMCs (B) were monitored for 1.5 h in BSS, and average motility coefficients ± sem (n=14–88/each sample) calculated from Equation 2 are shown. No Ca2+ refers to BSS without CaCl2 and with 1 mM EGTA and 2 mM MgCl2. SOCE inhibitor 2-APB (10 μM), Orai1 channel inhibitor GdCl3 (2 μM), and PLC inhibitor U-73122 (2 μM) were added prior to motility measurements. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 compared with control.
Figure 3.
Figure 3.. Orai1 is involved in RBL mast cell motility.
RBL-2H3 cells were transiently transfected with shRNA specific (KD) for Orai1, STIM1, or parallel control vectors (TRPC1), respectively. TRPC1 shRNA was cotransfected with mRFP, and in control experiments, RBL cells were transiently transfected with mRFP only. Cell tracks were monitored for 1.5 h in media, and relative average velocities are shown ± sem (n=49–76 cells/each sample). *P < 0.05; ****P < 0.0001 compared with controls. Averages of absolute velocity values are shown in Supplemental Fig. 1.
Figure 4.
Figure 4.. RBL-2H3 mast cells exhibit spontaneous Ca2+ transients with influx dependence that correlates with motility.
(A) Confocal images of representative RBL-2H3 mast cells expressing GCaMP3. Time-lapse images were taken every 2 s for 20 min. Warmer colors indicate higher Ca2+ levels. Note local Ca2+ transients (red or white) frequently occurring in protrusions (arrows). No Ca2+ refers to BSS without CaCl2 and with 1 mM EGTA and 2 mM MgCl2. Inhibitors 2-APB (10 μM) or GdCl3 (2 μM) were added just prior to collecting time-lapse images. (B) Summary of average percentages of cells with Ca2+ transients compared with total GCaMP3-expressing cells ± sem (n=27–42/each condition; monitored in three experiments). **P < 0.01; ***P < 0.001 compared with control.
Figure 5.
Figure 5.. Monitoring and analyzing mast cell chemotaxis in real time.
(A) Representative image of RBL-2H3 cells in an ibidi chemotaxis μ-slide after 16 h tracking. Colored lines show migration tracks derived from ImageJ Manual Tracking plugin program. (B) Representative plots from a single experiment showing migration tracks of RBL-2H3 cells with (right) and without (left) 10 ng/mL DNP-BSA in the lower reservoir buffer. (C) Representative plots for rat BMMCs with (right) and without (left) 100 nM SCF in the lower reservoir buffer. The migration tracks were plotted after setting the starting point for each cell at x = 0, and y = 0, using ImageJ Chemotaxis and Migration Tool plugin. Red tracks indicate individual cells with net migration toward the lower chamber that contained chemoattractant (or control). Blue crosses represent the average endpoints.
Figure 6.
Figure 6.. RBL-2H3 mast cells show chemotaxis toward antigen.
Mast cell chemotaxis is represented as the average yFMI ± sem (n=36–137 cells/each condition). yFMI is determined by dividing the net y value of a given track by accumulated distance (Equation 3). RBL-2H3 cells (black bars) or rat BMMCs (open bars) were sensitized with anti-DNP IgE, plated onto ibidi chemotaxis μ-slide chambers overnight, and then monitored for 16 h in the absence (Control) or presence of varying doses of DNP-BSA or SCF, as indicated, in the lower reservoir buffer. **P < 0.01; ***P < 0.001; ****P < 0.0001 compared with respective control sample.
Figure 7.
Figure 7.. Directed migration of RBL-2H3 cells toward antigen depends on Syk kinase.
Syk− cells were sensitized with anti-DNP IgE, plated onto ibidi chemotaxis μ-slide chambers overnight, and then monitored for 16 h in the absence (Control) or presence of 10 ng/mL DNP-BSA (+Ag) in the lower reservoir buffer. +Syk refers to Syk− cells transiently expressing Syk-CFP. +SH2 refers to Syk− cells transiently expressing PLCγ-(SH2)2-GFP. Average yFMI ± sem (n=27–108 cells/each condition) is shown. *P < 0.05; ****P < 0.0001 compared with respective control sample.
Figure 8.
Figure 8.. Orai1 contributes to RBL-2H3 mast cell chemotaxis toward antigen.
RBL-2H3 cells were sensitized with anti-DNP IgE, plated onto ibidi chemotaxis μ-slide chambers overnight, and then monitored for 16 h in the absence (Control) or presence of 10 ng/mL DNP-BSA (+Ag). +S1P refers to chemotaxis of RBL cells in the presence of 1 μM S1P. No Ca2+ refers to RBL-2H3 cells in medium with 4 mM EGTA and 3 mM MgCl2. RBL-2H3 cells were transiently transfected with shRNA specific for Orai1 (Orai1 KD) or with corresponding control vector (Vector), sensitized with anti-DNP IgE, plated onto ibidi chemotaxis μ-slide chambers overnight, and then monitored for 16 h. Average yFMI ± sem (n=11–108 cells/each condition) is shown. *P < 0.05; **P < 0.01; ****P < 0.0001 compared with respective control sample.

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