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. 2007 Mar 19;204(3):497-510.
doi: 10.1084/jem.20061780. Epub 2007 Feb 26.

A Central Role for DOCK2 During Interstitial Lymphocyte Motility and sphingosine-1-phosphate-mediated Egress

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Free PMC article

A Central Role for DOCK2 During Interstitial Lymphocyte Motility and sphingosine-1-phosphate-mediated Egress

César Nombela-Arrieta et al. J Exp Med. .
Free PMC article

Abstract

Recent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyte-expressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K)gamma, signaling molecules that act downstream of G protein-coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kgamma displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kgamma alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the sphingosine-1-phosphate (S1P) receptor 1, a G(alphai) protein-coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kgamma contributed to S1P-triggered signaling events. S1P-induced cell migration was significantly reduced in T and B cells lacking DOCK2, whereas T cell-expressed PI3Kgamma contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These findings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3Kgamma, and a markedly reduced cell motility of DOCK2-deficient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell-expressed PI3Kgamma, for signal transduction during interstitial lymphocyte migration and S1P-mediated egress.

Figures

Figure 1.
Figure 1.
Paracortical T cell migration in the absence of DOCK2 and PI3Kγ. Fluorescently labeled control T cells and DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− T cells were adoptively transferred into wild-type recipients, and their migratory behavior inside popliteal lymph nodes was analyzed using MP-IVM. The grid length of each square corresponds to 20.8 μm. (A) Representative three-dimensional tracks of control T cells (+/+; left column) and DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− T cells (−/−; right column) over 30-min periods. Each colored line represents a single T cell track. (B) Velocity profiles of control T cells (green bars) and DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− T cells (red bars). Histograms show the relative frequency distribution of different populations compared with control T cells. (C) Turning angles of control T cells (+/+; top green panel) and DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− T cells (−/−; bottom red panel). The wider angle distribution of DOCK2-deficient T cells is indicative of less directed cell movement. (D) Motility coefficients of T cells in the presence or absence of DOCK2 and/or PI3Kγ. Each dot represents the combined motility coefficient of one experiment. Filled dots, control T cells; empty dots, genetically deficient T cells. The bars indicate mean values. The statistical analysis is summarized in Table I.
Figure 2.
Figure 2.
Follicular B cell migration in the absence of DOCK2 and PI3Kγ. Control B cells were adoptively transferred with DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− B cells, and their migratory behavior was analyzed inside B cell follicles using MP-IVM as in Fig. 1. (A) Representative three-dimensional tracks of control B cells (+/+; left column), DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− B cells (−/−; right column) during 30-min observation periods. Each colored line represents a single B cell track. (B) Velocity profiles of control B cells (green bars) and DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− B cells (red bars). Histograms show the relative frequency distribution of different populations compared with control B cells. (C) Turning angles of control B cells (+/+; top green panel) and DOCK2−/−, PI3Kγ−/−, or DOCK2−/− × PI3Kγ−/− mice B cells (−/−; bottom red panel). (D) Motility coefficients of B cells in the presence or absence of DOCK2 and/or PI3Kγ. Each dot represents the motility coefficient of one experiment. Filled dots, control B cells; empty dots, genetically deficient B cells. The bars indicate mean values. The statistical analysis is summarized in Table I.
Figure 3.
Figure 3.
DOCK2 and PI3Kγ transmit signals downstream of S1P receptors. (A) Flow cytometric analysis of PKB phosphorylation 30 s after the addition of S1P (1 μM final concentration; red line) or 1 min after the addition of CXCL12 (100 nM final concentration; green line) in control, DOCK2−/−, or PI3Kγ−/− T and B cells using phosphorylated PKB-specific antibody. The black line represents background staining in the absence of stimuli. (B) Flow cytometric analysis of F-actin polymerization after S1P addition (500 nM final concentration) in control, DOCK2−/−, PI3Kγ−/−, and DOCK2−/− × PI3Kγ−/− T and B cells. Data are presented as normalized mean fluorescence intensity of FITC-Phalloidin binding, with the “0” time point = 100. Each value corresponds to the mean ± SD of at least three independent experiments. au, arbitrary units. (C) Chemotaxis of control, DOCK2−/−, PI3Kγ−/−, and DOCK2−/− × PI3Kγ−/− T and B cells to 25 nM S1P for 4 h at 37°C. Significant migration to S1P over medium is marked with an asterisk (P < 0.05). Interconnected bars indicate significant difference (P < 0.05; ANOVA). Data represent mean ± SD of three independent experiments.
Figure 4.
Figure 4.
DOCK2 deficiency delays lymphocyte egress from PLNs. (A) Absolute numbers of control and DOCK2−/− lymphocytes recovered from PLNs at the indicated times after Mel-14 treatment (20 h after adoptive transfer). Fluorescently labeled control and DOCK2−/− lymphocytes were adoptively transferred into recipient mice, and after 20 h, further homing was blocked by the injection of Mel-14 mAb. n = 4 mice per time point from two independent experiments. (B) Kinetic of control and DOCK2−/− lymphocyte egress expressed as percent of normalized initial population. Asterisks indicate significant difference compared with control lymphocytes (P < 0.05). (C) Retention ratio of adoptively transferred total lymphocytes (open bars), T cells (gray bars), and B cells (filled bars) at the indicated times after Mel-14 treatment (20 h after adoptive transfer). Individual mice are represented by open circles, and bars correspond to mean values. The relative frequency of DOCK2−/− T and B cells increases during Mel-14 treatment, indicating slower egress kinetics. One-way ANOVA was used to determine significance between different time points. Interconnected columns are significantly different (P < 0.05). (D) Cryosections of PLNs isolated 2 and 12 h after Mel-14 treatment (20 h after adoptive transfer) showing distribution of CMFDA-labeled DOCK2+/+ lymphocytes and CMTMR-labeled DOCK2−/− lymphocytes. HEVs are stained with anti-PNAd mAb MECA-79 in the top panel and lymphatic endothelium with LYVE-1 antibody in the bottom panel (blue). For clarity, a rectangular segment of each section has been magnified as indicated. Bar, 100 μm.
Figure 5.
Figure 5.
PI3Kγ deficiency does not affect lymphocyte egress from PLNs. (A) Total numbers of PI3Kγ+/+ and PI3Kγ−/− cells present in PLNs at 2, 12, and 24 h after Mel-14 mAb treatment (4 h after adoptive transfer). Data represent mean ± SD from at least three mice from two independent experiments. (B) Retention ratio of PI3Kγ+/+ and PI3Kγ−/− T and B cells at 2, 12, and 24 h after cell transfer. Each circle represents individual mice, and bars depict mean values. Shown are transferred total lymphocytes (open bars), T cells (gray bars), and B cells (filled bars) at the indicated times after Mel-14 mAb treatment. No significant difference in ratio became apparent. (C) Retention ratio of DOCK2−/− and DOCK2−/− × PI3Kγ−/− T and B cells. 20 h after cell transfer, further homing was blocked by Mel-14 mAb administration, and mice were killed at 2 and 20 h later. Each circle represents individual mice, and bars depict mean values. Shown are transferred total lymphocytes (open bars), T cells (gray bars), and B cells (filled bars) at the indicated times after Mel-14 mAb treatment. Data are from two independent experiments with four mice per time point. No significant difference in ratio became apparent.
Figure 6.
Figure 6.
Medullar T cell migration in the absence of DOCK2. (A) Image sequence of control and DOCK2-deficient T cells proximal to efferent WGA+ lymphatic vessels (green, outline labeled white for greater clarity). A control T cell entering WGA-labeled lymphatic vessel is shown, whereas DOCK2-deficient T cells remained stationary during the observation period. The asterisks mark the initial tracking spot. Time is in minutes and seconds. Bar, 10 μm. (B) Representative three-dimensional tracks of control and DOCK2−/− T cells tracked for 30 min. 10 tracks are shown for each genotype. Scale is in micrometers. (C) Velocity profile of control and DOCK2-deficient medullar T cells. 886 control T cell and 138 DOCK2−/− T cell tracks were analyzed from four to five independent videos.

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