1. Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells
by Kazuhito Gotoh, Yoshihiko Tanaka, Akihiko Nishikimi, Ayumi Inayoshi, Munechika Enjoji, Ryoichi Takayanagi, Takehiko Sasazuki, and Yoshinori Fukui
Blood
Volume 111(6):
March 15, 2008
©2008 by American Society of Hematology

2. DOCK2−/− plasmacytoid dendritic cells (pDCs) are impaired in their homing to and localization within secondary lymphoid organs.
DOCK2−/− plasmacytoid dendritic cells (pDCs) are impaired in their homing to and localization within secondary lymphoid organs. (A,B) Bone marrow (BM), spleen, and lymph node (LN) cells from B6 or DOCK2−/− mice were stained with fluorescein isothiocyanate (FITC)-labeled anti-B220, phycoerythrin (PE)-labeled anti-CD11c and biotinylated anti-mPDCA-1 antibodies followed by allophycocyanin (APC)-conjugated streptavidin. Before staining, BM and splenic dendritic cells (DCs) were enriched with anti-CD11c microbeads. (A) Expression of B220 and mPDCA-1 on CD11c+ BM or splenic DCs are shown. Numbers in quadrants indicate the percentage of cells in each after gating on CD11c+ cells. Data are representative of 4 independent experiments. (B) The number of myeloid dendritic cells (mDCs; CD11c+B220−mPDCA-1−) and pDCs (CD11c+B220+mPDCA-1+) in the BM, spleen, peripheral LN (PLN) and mesenteric LN (MLN) were compared between B6 (□, n = 4) and DOCK2−/− (■, n = 4) mice. Data are mean plus or minus SD; *P < .01. (C) Spleen and PLN tissue sections from B6 and DOCK2−/− mice were stained for B220 (Alexa Fluor 546; red) and mPDCA-1 (FITC; green). Scale bars, 100 μm. Data are representative of 3 independent experiments with different mice. (D-F) BM-derived pDCs from B6 and DOCK2−/− mice were labeled with PKH-26 (green) and PKH-67 (red) dyes, respectively, and were mixed in equal numbers and intravenously injected into B6 mice. (D) The ratios of DOCK2−/− pDCs (■) to B6 pDCs (□; set as an arbitrary value of 1) in the spleen, PLN and MLN of the recipient mice were analyzed at 24 hours after transfer (n = 3, *P < .05). (E) Spleen sections were prepared at 24 hours after transfer and stained for metalophilic macrophages with anti-MOMA1 antibody (Alexa Fluor 647; blue). Scale bar, 100 μm. Data are representative of 2 independent experiments with different mice. (F) The ratios of DOCK2−/− pDCs to B6 pDCs in the spleen were analyzed at indicated time points after transfer. Data are means plus or minus SD (n = 3). Images in panels C and E were acquired using an LSM 510 META confocal microscope (Carl Zeiss, Gottingen, Germany) equipped with a 20×/0.75 (C) or 10×/0.45 (E) NA Plan-Apochromat objective lens (Carl Zeiss).
Kazuhito Gotoh et al. Blood 2008;111:
©2008 by American Society of Hematology

3. DOCK2 is a Rac activator indispensable for migration of pDCs, but not mDCs.
DOCK2 is a Rac activator indispensable for migration of pDCs, but not mDCs. (A,B) Primary BM cells were used in a transwell chemotaxis assay. Before assay, BM cells were treated with either biotinylated anti-CD3, anti-CD11b, anti-CD19 and anti-CD49b antibodies plus anti-biotin microbeads or anti-B220 microbeads, anti-CD90 microbeads and biotinylated anti-Gr1 antibody plus anti-biotin microbeads to enrich pDCs or mDCs, respectively. The input cells and the cells migrating to the lower chamber were stained for B220 and CD11c. The results are expressed as the percentage of the input cells (mean ± SD of triplicate wells). (A) Chemotactic responses of BM mDCs and pDCs to CXCL12 were compared between B6 (□) and DOCK2−/− (■) mice. (B) Chemotactic responses of BM pDCs to CXCL12 plus CXCL9 were compared between B6 (□) and DOCK2−/− (■) mice. (C,D) Primary or Flt3 ligand-stimulated BM cells were stained with anti-CXCR4, anti-CCR7 or PE-labeled anti-CXCR3 antibody in combination with FITC-labeled anti-B220 and APC-labeled anti-CD11c antibodies, and B220+CD11c+ pDCs were analyzed for receptor expression. Anti-CXCR4 or anti-CCR7 antibody was detected with biotinylated anti-rabbit antibody and PE-conjugated streptavidin. Dotted lines indicate the profiles stained without the primary antibody (for CXCR4 and CCR7) or those stained with control antibody (for CXCR3). (C) The expression of CXCR4 or CXCR3 on BM pDCs is shown. (D) The expression of CCR7 on BM-derived pDCs is shown. (E,F) BM-derived pDCs chemotaxing under the CCL21 gradient were analyzed with an EZ-Taxiscan. Data were collected at 30-second intervals for 30 minutes, starting at 10 minutes after addition of 1 μl of CCL21 (250 μg/mL) to the upper side of the chamber. Scale bar, 20 μm. (F) BM-derived pDCs from B6 (□) and DOCK2−/− mice (■) were compared in terms of the speed, directional change, and straightness. The directional change is a measure of the frequency of turns that a cell makes to move 100 μm in a given direction. The straightness was estimated by dividing the distance from the initial position to the final location by the total path length. The results are expressed as the means plus or minus SEM of 3 independent experiments. *P less than .05; **P less than .01. (G) BM-derived pDCs or mDCs were stimulated in suspension with CCL21 (2 μg/mL) for the indicated times and analyzed for Rac activation. Data are representative of 3 independent experiments. (H,I) BM-derived pDCs were stimulated in suspension with CCL21 (2 μg/mL) for the indicated times and analyzed for the content (H) and localization (I) of F-actin by staining the cells with Alexa Fluor 488–conjugated phalloidin. (H) The results are expressed as the mean channel fluorescence (mean ± SD of triplicate wells). B6, [—○—]; DOCK2−/−, [—●—]. (I) Data are representative of 3 independent experiments. Scale bar, 20 μm. Images were acquired using an LSM 510 META confocal microscope equipped with a 63×/1.4 oil Plan-Apochromat objective lens (Carl Zeiss). (J) BM pDCs and mDCs were sorted and analyzed for the expression of DOCK180, DOCK2, DOCK10 or DOCK11 by reverse transcriptase–polymerase chain reaction. DOCK11 is a Cdc42-specific activator, but the specificity of DOCK10 remains unclear.21,22 The gene encoding hypoxanthine guanine phosphoribosyl transferase (Hprt1) was used as a control.
Kazuhito Gotoh et al. Blood 2008;111:
©2008 by American Society of Hematology