goat anti ccl21  (R&D Systems)

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: ( A , B ) A capture of a phase contrast/immunofluorescence live recording (Movie ) shows a DC transmigrating the VE-cadherin stained (magenta) lymphatic endothelial multicellular junction. ( B ) Quantification of DC transmigration sites in CCL21-mCherry expressing LEC cultures. Transmigration sites are shown as a percentage of all events from 8 biological replicates in three independent experiments. n = 361 transmigration events. ( C , D ) A capture of a live recording of a mouse ear pinna dermal explant (Movies – ) shows a DC (red), which is starting to transmigrate an α-CD31-FITC stained (gray) lymphatic endothelial multicellular junction. See more examples in Appendix Fig. . In ( D ), the transmigration sites are shown as a percentage of all events ( n = 33) from 3 independent experiments, representing, altogether, 6 mice. ( E – G ) Time-lapse series of a wild-type (WT) or CCR7 −/− DC migration on the CCL21-mCherry expressing LEC cultures prior to transmigration. Stained VE-cadherin junctions are shown in magenta, and nuclei in blue (Hoechst). Also, time-color-coded track of the full DC migration path is shown. See also the corresponding Movie and Fig. . Quantification in ( F ) shows the straightness of wild-type and CCR7 −/− DC tracks. The data represents 1403 wild-type and 543 CCR7 −/− tracks. Quantification in ( G ) shows the sites of wild-type or CCR7 −/− DCs arrest on CCL21-mCherry expressing LEC monolayers prior to transmigration. The data represents 633 wild-type and 396 CCR7 −/− DC arrest events. In ( F , G ) data is derived from 6 wild-type and 5 CCR7 −/− biological replicates and, altogether, three independent experiments. Data information: The yellow arrow in ( A ) indicates a transmigrating DC. In ( C ), yellow arrow indicates DC cell body, white arrowhead the DC leading edge, and magenta arrowhead the multicellular junction. In ( E ) the CCR7 −/− DC detachment is indicated with a yellow arrow. The p -value in ( F ) and ( G ) were calculated using the Chi-squared test. Scale bars are 20 µm in ( A ), ( C ) and ( E ). .

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Immunofluorescence, Staining, Transmigration Assay, Expressing, Migration, Derivative Assay

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: ( A ) VE-cadherin (red) and LYVE1 (green) staining of mouse ear pinna dermis. A flattened overview image and zoom-in of a single optical slice are shown. The images represent n = 3 mice. ( B ) The dot plot shows mean ± SD percentage of wild-type or CCR7 −/− DCs that detached, subsequent to the arrest at multicellular junction. The data point represents n = 6 wild-type and n = 5 CCR7 −/− biological replicates from three independent experiments, altogether, representing 401 wild-type and 140 CCR7 −/− DCs. The data is related to Fig. . ( C ) Schematic shows mCherry tagged full-length CCL21 and CCL21ΔC-mCherry that lacks the charged C-terminus (Hirose et al, ). ( D ) Quantification of wild-type DC transmigration sites in CCL21ΔC-mCherry expressing LEC cultures. The stacked bar graph shows transmigration sites as a percentage of all events from 15 biological replicates in three independent experiments and, altogether, n = 128 transmigration events. ( E – I ) The bar graphs show the mean percentage ± SD of DCs positive for ( E ) CD86, ( F ) CD11b, ( G ) CD11c, and ( H ) MHCII in wild-type versus CCR7 −/− DCs. In ( I ) the bar graph shows mean cell size ± SD of wild-type vs CCR7 −/− DCs normalized to the average of wild-type DCs, which was set at 1, in each experiment. In ( E , I ) The data points represent n = 4 biological replicates/genotype in two independent experiments. The number of analyzed DC singlets was at least 277000/sample. ( J ) A capture of a phase contrast/immunofluorescence microscopy showing wild-type (magenta) and CCR7 −/− DC (green), on a LEC monolayer. The images represent n = 3 biological replicates. ( K , L ) Western blot panel of non-muscle myosin heavy chain 2 A (NMH-IIA), actin, and HSC70 (for loading control) in wild-type versus CCR7 −/− DCs. Quantification of the band intensities is shown in the western blot data shown in ( L ). The bar graph in ( L ) shows, mean intensity ± SD. Data was normalized to the average of wild-type DCs, which was set at 1. The data in ( K , L ) represent n = 4 biological replicates/genotype and two independent experiments. ( M ) Quantification of human CCL21 mRNA levels in human specific siCCL21 transfected and mouse CCL21ΔC-mCherry expressing LECs. The dot plot shows the mean hCCL21 mRNA level ± SD normalized to the average of siControl samples, which was set at 1 (green line), in each experiment. Data points represent n = 4 biological replicates/ siRNA oligo in 2 independent experiments. P -values show the comparison to controls. ( N ) Images show mouse CCL21ΔC-mCherry expression in siControl or human CCL21-specific siCCL21 oligo transfection. Images represent n = 4 biological replicates in 2 independent experiments. ( O ) Quantification of transmigration sites of wild-type DCs in siControl or siCCL21 transfected and CCL21-mCherry or CCL21ΔC-mCherry expressing LEC cultures. Transmigration sites are shown as a percentage of all events from 2 independent experiments, consisting of siCTRL01 n = 209 transmigration events (3 biological replicates), siCTRL02 n = 187 (3 biological replicates), siCCL21-05 n = 137 (3 biological replicates), or siCCL21-08 n = 249 (4 biological replicates) transfected and CCL21-mCherry expressing LEC cultures and of siCTRL01 n = 96 (4 biological replicates), siCTRL02 n = 68 (4 biological replicates), siCCL21-05 n = 69 (4 biological replicates), or siCCL21-08 n = 223 (8 biological replicates) transfected and CCL21ΔC-mCherry expressing LEC cultures. ( P ) The bar graph shows a mean number of observed CCL21ΔC-mCherry exocytosis events/cell ± SD in control or BAPTA-AM treated LECs. The data points represent n = 37 DMSO control and n = 33 BAPTA-AM treated cells in 11 biological replicates across two independent experiments. Data information: In ( A ), yellow arrows indicate the site of zoom-in image and white arrowheads indicate the multicellular junctions. In ( J ), the white arrowheads indicate wild-type and yellow arrows CCR7 −/− DC dendrites. The p -values in ( B ), ( E – I ), and ( M ) were calculated using a parametric T-test with Welch’s correction, whereas in ( L ) and ( P ) p -values were calculated using the Mann–Whitney test. In ( O ), Chi-square test was used to test the significance of data. The scale bar is 20 µm in overview images ( A and J ), 5 µm in zoom-in images ( A ), and 50 µm in ( N ).

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Staining, Transmigration Assay, Expressing, Immunofluorescence, Microscopy, Western Blot, Control, Transfection, Comparison, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: Figure 2 is shown with alternative colors (non-red and -green) in Appendix Fig. . ( A ) LEC monolayer expressing CCL21ΔC-mCherry (red) and stained for VE-cadherin (magenta) and nuclei (DAPI, blue). The data in ( A ) represents at least n = 3 independent experiments. ( B , C ) A capture of immunofluorescence live recording (Movie ) shows primary LEC monolayer expressing CCL21ΔC-mCherry (red) and stained for VE-cadherin (green). Exocytosis events were analyzed within a 7 µm wide region. ( C ) A histogram showing exocytosis events (mean number of secretions/cell + SD) at the LEC junctions, as a function of distance (in µm) from the nearest multicellular junction. The data in ( B and C ) represents 241 secretion events from n = 22 LECs in 5 biological replicates across two independent experiments. ( D – F ) LECs expressing chemokine CCL21ΔC-mCherry (red) and the indicated EGFP-tagged RAB-GTPase (green). The nuclei are stained with DAPI (blue). The images are representative of n = 3 biological replicates in three independent experiments. Quantification in ( E ) shows percentage of CCL21ΔC-mCherry+ vesicle colocalization with the indicated EGFP-RAB GTPases in the whole LEC area. The dot plot shows the mean percentage ± SD. Each data point represents a single analyzed cell (EGFP-RAB3D ( n = 20); EGFP-RAB27A ( n = 18); EGFP-RAB37 ( n = 25) and EGFP-RAB6A ( n = 24)), from a total of 3 biological replicates in three independent experiments. The histogram ( F ) shows the distribution (mean percentage) of the colocalized vesicles as a function of distance from a multicellular junction. The number of samples was the same as in ( E ). ( G ) A TNF-α-treated LEC, expressing chemokine CCL21ΔC-mCherry (red) and, stained for endogenous CCL2 (green), and nuclei (DAPI, blue). The images represent n = 2 independent experiments. ( H ) A LEC expressing chemokine CCL21ΔC-mCherry (red) and stained for endogenous RAB6 (green). The images represent n = 2 independent experiments. ( I ) A TNFα-treated LEC monolayer stained for endogenous CCL2 (red), RAB6 (green), and nuclei (DAPI, blue). The images represent n = 2 independent experiments. ( J ) Mouse-ear pinna dermis stained for CCL21 (red), RAB6 (green), LYVE1 (magenta), and nuclei (DAPI, blue). For clarity, the overview image shows only staining of LYVE1+ and nuclei. The images are representative of 3 mice. ( K ) Representative images show a mouse ear pinna dermis lymphatic pre-collector (continuous junctions, quantified in ( M , N ) and ( L ) capillary (discontinuous junctions, quantified in ( O , P ) stained for CCL21 (red), RAB6 (green), VE-cadherin (magenta). The overview images show VE-cadherin-only or CCL21 and RAB6. The zoom-in images show images of peripheral and perinuclear areas of the LEC. Figures 2K and L are shown with more examples in Appendix Fig. . ( M ) Quantification of CCL21 vesicle colocalization (mean percentage ± SD) with RAB6, in LECs showing continuous junctions in mouse ear pinna dermis in vivo. Each data point represents a single analyzed cell. ( N ) The histogram shows the distribution (mean percentage + SD) of CCL21 and RAB6 colocalized vesicles and non-colocalized CCL21 vesicles, as a function of distance from the multicellular junction (for LECs displaying continuous junctions). In ( K ) and ( M , N ), n = 29 cells representing 6 mice. ( O ) Quantification of CCL21 vesicle colocalization (mean percentage ± SD) with RAB6, in LECs showing discontinuous junctions in mouse ear pinna dermis in vivo. Each data point represents a single analyzed cell. ( P ) The histogram shows the distribution (mean percentage + SD) of CCL21 and RAB6 colocalized vesicles and non-colocalized CCL21 vesicles, as a function of distance from the VE-cadherin stained cell border (for LECs displaying discontinuous junctions). In ( L ) and ( O , P ), n = 15 cells representing 3 mice. Data information: In ( A ), ( D ), ( G – I ), and ( J – L ), the yellow arrow indicates the peripheral, and the white arrowheads the perinuclear area shown in the zoom-in image. The cell borders in ( D ), ( G – I ), and ( K , L ) are marked with white dotted lines. The cyan arrow shows an example of colocalization and the magenta arrowheads examples of non-colocalizing vesicles. In ( B ), the yellow arrow indicates LEC multicellular junction (shown in the zoom-in image and in Movie ). The vesicles that were exocytosed are marked with white arrowheads. The p -value in ( E ) was calculated using one one-way ANOVA test. Scale bars in ( A , B ), ( D ), and ( G – J ) are 20 µm in the overview images and 5 µm in the zoom-in images. In ( K , L ) the scale bars in overview images are 10 µm and 2 µm in the zoom-in images. .

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Expressing, Staining, Immunofluorescence, In Vivo

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: ( A – E ) Shows colocalization of full-length CCL21-mCherry (red) with the indicated EGFP-tagged RAB-GTPase (green). The nuclei were stained with DAPI (blue). ( C , D ) Quantification of CCL21-mCherry+ vesicle colocalization with the indicated EGFP-RAB GTPases in the whole LEC area. The dot plots show the mean percentage ± SD. Each data point represents a single analyzed cell with n = 6 cells for each of EGFP-RAB3D, EGFP-RAB27A, EGFP-RAB37, EGFP-RAB6, EGFP-RAB10, and EGFP-RAB13 representing two independent experiments. ( E ) The histogram shows the distribution (mean percentage) of CCL21ΔC-mCherry and the indicated EGFP-RAB-GTPase colocalized vesicles as a function of distance from a multicellular junction. The number of cells and independent experiments is the same as in ( C ). Data information: In ( A , B ), the perinuclear and peripheral areas (shown in the zoom-in images below the overviews) are indicated with white arrowheads and yellow arrows, respectively. Colocalization is indicated with cyan arrows and non-colocalizing CCL21-mCherry vesicles are indicated with magenta arrowheads. The cell borders are indicated with white dotted lines. Scale bars are 20 µm in the overview images; 3 µm in zoom-in images.

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Staining

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: Figure 3 is shown with alternative colors (non-red and -green) in Appendix Fig. . ( A – C ) Transmission electron micrograph of anti-RFP or anti-EGFP immunogold labeled ( A ) CCL21-mCherry, ( B ) CCL21ΔC-mCherry, ( C ) EGFP-RAB6A, or EGFP-RAB27A expressing LECs. The images represent n = 2 independent experiments. Electron micrographs with control labeling are shown in Appendix Fig. . ( D ) Shows a LEC expressing CCL21ΔC-mCherry (red) and stained for endogenous RAB7 (green). The images represent n = 2 independent experiments. ( E ) LEC expressing CCL21ΔC-mCherry (red) and stained for endogenous LAMP3 (CD63; green) and RAB7 (gray). The nuclei are stained with DAPI (blue). The zoom-in images, show either CCL21ΔC-mCherry and LAMP3 channels (images 2 and 4) or RAB7 together with CCL21ΔC-mCherry, and LAMP3 channels (images 3 and 5). The images represent n = 3 independent experiments. Quantification shown in ( G ). ( F ) Shows LEC expressing CCL21ΔC-mCherry (red) and stained for endogenous LAMP1 (cyan), RAB7 (green), and nuclei (DAPI; blue). The zoom-in images, show either CCL21ΔC-mCherry and LAMP1 (images 2 and 4) or also with RAB7 (images 3 and 5). The images represent n = 3 independent experiments. Quantification is shown in ( H ). ( G , H ) Quantification of CCL21ΔC-mCherry vesicle colocalization with endogenous RAB7A or LAMP3 in ( G ) and RAB7A or LAMP1 in ( H ). The dot plots show the mean percentage ± SD. Each data point represents a single analyzed cell with n = 15. The data represents 5 biological replicates in three independent experiments. ( I ) Shows expression of EGFP-RAB3D (green) with mCherry-RAB27A (red) in TNF-α treated LECs. The images represent n = 3 independent experiments. Data information: In ( A – C ), cyan arrows indicate dense core granules, yellow arrows low-electron density vesicles, and magenta arrowheads multivesicular bodies. In ( D – F ) and ( I ), the cell borders are marked with a white dotted line. Yellow arrows and white arrowheads indicate the site of the peripheral and perinuclear areas, respectively, shown in the zoom-in images. Cyan arrows indicate examples of CCL21ΔC-mCherry+ colocalizing vesicles and magenta arrowheads non-colocalizing CCL21ΔC-mCherry+ vesicles. Scale bars are 200 nm in transmission electron micrographs in ( A – C ); 20 µm in overview image and 5 µm in zoom-in images in ( D – F ) and ( I ). .

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Transmission Assay, Labeling, Expressing, Control, Staining

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: Figure 6 is shown with alternative colors (non-red and -green) in Appendix Fig. . ( A , B ) The dot plot in ( A ), shows a mean number of observed exocytosis events/cell + SD in siControl and siRAB6 samples. Whereas, in ( B ), the histogram shows the distribution of the mean number of exocytosis events/cell + SD at LEC junctions, as a function of distance from a multicellular junction. In ( A , B ), the data points represent n = 39 cells in 13 biological replicates from three independent experiments. ( C – F ) Images of a LEC monolayer expressing CCL21ΔC-mCherry (red), treated with siControl or siRAB6 oligos and stained for VE-cadherin (green) and nuclei (DAPI, blue). The dot plot in ( D , E ) shows the mean CCL21ΔC-mCherry intensity ± SD measured at the multicellular junctions per ( D ) biological replicate and ( E ) per multicellular junction, whereas the dot plot in ( F ) shows the mean CCL21ΔC-mCherry intensity ± SD measured in the whole LEC. Data points represent n = 5 biological replicates or n = 129 (siControl) and n = 129 (siRAB6) multicellular junctions in ( D , E ) and 160 (siControl) and 164 (siRAB6) LECs in ( F ), altogether, in 4 independent experiments. ( G – J ) Images of TNF-α treated LEC monolayer transfected with siControl, siRAB6, or siELKS oligos, and stained for endogenous CCL2 (red), VE-cadherin (green), and nuclei (DAPI, blue). The dot plot shows the mean CCL2 intensity ± SD, measured at the multicellular junctions, ( H ) per experiment and ( I ) per multicellular junction. Whereas, in ( J ) the dot plot shows the mean CCL2 intensity ± SD, measured in the whole imaged area. Data points represent n = 4 independent experiments or n = 337 (siControl pool), n = 268 (siRAB6 pool), and n = 384 (siELKS pool) multicellular junctions from 5 (siControl pool and siRAB6 pool) or 6 (siELKS pool) biological replicates in ( H , I ), and 37 (siControl pool and siRAB6 pool), and 42 (siELKS pool) images from 6 (siControl pool and siRAB6 pool) or 7 (siELKS pool) biological replicates in ( J ). ( K ) Quantification of the mean DC transmigration efficiency ± SD on LEC monolayer transfected with siControl or siRAB6 and transduced with CCL21-mCherry or CCL21 ΔC-mCherry. The results were normalized to the average of control samples (set at 1) in each experiment. The data points represent biological replicates: CCL21-mCherry + siControl n = 11 (4359 DCs), CCL21-mCherry + siRAB6 n = 9 (4091 DCs), CCL21ΔC-mCherry + siControl n = 10 (3319 DCs), CCL21ΔC-mCherry + siRAB6 n = 10 (3766 DCs), across three independent experiments. The data is related to Movies and . Data information: In ( C ) and ( G ) yellow arrows indicate the multicellular junction (area shown in the zoom-in images below), and white arrowheads indicate the accumulation. White dotted lines represent the cell borders. In ( A ), ( D ), ( F ), ( H ), ( J ), and ( K ), the p -values were calculated using a parametric T-test with Welch’s correction and in ( E ) and ( I ) the p -values were calculated using Mann–Whitney’s test. The scale bar in ( C ) and ( G ) is 20 µm in overview images and 5 µm in zoom-in images. .

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Expressing, Staining, Transfection, Transmigration Assay, Transduction, Control

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: Figure 7 is shown with alternative colors (non-red and -green) in Appendix Fig. . ( A ) Quantification of the CCL21ΔC-mCherry exocytosis events at the LEC junctions in EGFP or EGFP-RAB8A DN expressing LECs. The dot plot shows the mean number of exocytosis events/cell ± SD. Data points represent n = 3 independent experiments, comprising of, altogether, 24 LECs in 12 biological replicates. ( B ) Quantification of the effect of EGFP-RAB8A DN expression on CCL21ΔC-mCherry intensity in the media. The dot plot shows CCL21ΔC-mCherry mean intensity ± SD, and normalized to the average of controls, which was set at 1 in each experiment. The data points represent n = 11 biological replicates in three independent experiments. ( C – F ) Shows a LEC monolayer expressing CCL21ΔC-mCherry (red) and EGFP, EGFP-RAB8A DN, or EGFP-RAB8A WT (green). LECs were stained for VE-cadherin (magenta) and nuclei (DAPI, blue). Quantification of the mean CCL21ΔC-mCherry intensity ± SD at the multicellular junctions of EGFP-mCherry double-positive LECs ( D ) per biological replicate and ( E ) per multicellular junction. Whereas the dot plot in ( F ) shows the mean CCL21ΔC-mCherry intensity ± SD measured in the whole LEC. The results were normalized to the average of controls, which was set at 1, in each experiment. The data points represent n = 6 biological replicates in three independent experiments or n = 149 (EGFP), n = 153 (EGFP-RAB8A DN), and n = 147 (EGFP-RAB8 WT) multicellular junctions in ( D , E ) and 212 (EGFP), 188 (EGFP-RAB8A DN), and 170 (EGFP-RAB8 WT) LECs in ( F ). ( G ) Immunofluorescence images of a LEC monolayer expressing CCL21ΔC-mCherry (red) and either EGFP or EGFP-RAB8A DN (green). The cells were stained for endogenous RAB6 (gray), and nuclei (DAPI, blue). The data represents n = 4 biological replicates from 2 independent experiments. ( H – K ) Shows TNF-α treated LEC monolayer expressing EGFP (control), EGFP-RAB8A DN, or EGFP-RAB8A WT (green). LECs were stained for CCL2 (red), VE-cadherin (magenta), and DAPI (blue). Quantification of the mean CCL2 intensity ± SD at the multicellular junctions ( I ) per biological replicate and ( J ) per multicellular junction. Whereas the dot plot in ( K ) shows mean CCL2 intensity ± SD measured in the whole LEC. The data was normalized to the average of controls (set at 1) in each experiment. The data points represent n = 6 biological replicates from 3 independent experiments or n = 204 (EGFP), n = 202 (EGFP-RAB8A DN), and n = 192 (EGFP-RAB8 WT) multicellular junctions in ( I , J ), and 242 (EGFP), 240 (EGFP-RAB8A DN), and 136 (EGFP-RAB8 WT) LECs in ( K ). (L , M ) A capture of live recording of LEC monolayer expressing CCL21-mCherry and either of EGFP or EGFP-RAB8A DN (green) and stained for VE-cadherin (magenta). The nuclei of dendritic cells (DC) are stained with Hoechst (blue). ( M ) Quantification of the mean DC transmigration efficiency ± SD on LEC monolayer co-expressing either EGFP (control) or EGFP-RAB8A DN together with either CCL21-mCherry or CCL21 ΔC-mCherry. The results were normalized to the average of control (set at 1) in each experiment. The data points represent n = 10 CCL21-mCherry + EGFP (total of 3285 DCs), n = 9 CCL21-mCherry + EGFP-RAB8A DN (2552 DCs), n = 12 CCL21 ΔC-mCherry + EGFP (7123 DCs), and n = 12 CCL21 ΔC-mCherry + EGFP-RAB8A DN (5535 DCs) biological replicates, across 4 independent experiments. The data in ( L , M ) is related to the Movie . Data information: In mCherry channel-only images in ( C ), the cell borders of all the EGFP-mCherry, double-positive LECs, are indicated with green dashed lines. Similarly, in ( H ) the green dashed lines indicate the cell borders of all EGFP-CCL2 double-positive LECs. Cell borders in ( G ) are shown with white dashed line. In ( C ), ( G ), and ( H ), yellow arrows indicate the multicellular junction shown in the zoom-in images and the white arrowheads the accumulation. In ( A , B ), ( D ), ( F ), ( I ), ( K ), ( M , CCL21 ΔC-mCherry samples), the p -values were calculated using a parametric T-test with Welch’s correction, and in ( E ), ( J ), and ( M , CCL21-mCherry samples) using Mann–Whitney’s test. Scale bars in ( C , G and H ) are 20 µm in overview images and 5 µm in zoom-in images and in ( L ) 50 µm. .

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Expressing, Staining, Immunofluorescence, Control, Transmigration Assay

Journal: Scientific Reports

Article Title: Cholinergic anti-inflammatory pathway ameliorates murine experimental Th2-type colitis by suppressing the migration of plasmacytoid dendritic cells

doi: 10.1038/s41598-021-04154-2

Figure Lengend Snippet: The inhibitory effect of nicotine on BMpDC migration was evaluated with a chemotaxis assay. ( A ) BMpDCs matured by treatment with CpG oligodeoxynucleotides chronologically migrated in response to an established concentration gradient of CCL21 (a representative experiment is shown). ( B ) The number of migrating BMpDCs was dose-dependently inhibited by the addition of nicotine (1–100 μM) to the chemotaxis assay medium ( n = 3–7). ( C , D ) The velocity and directionality of BMpDC migrating in response to CCL21 were calculated. The velocity and directionality of BMpDC migration were significantly inhibited by the addition of nicotine (10 µM) to the chemotaxis assay medium, and pretreatment with MLA blocked the inhibitory effect of nicotine ( n = 20 for each group, ** P < 0.01 vs nicotine). Data are represented as the mean value ± SEM. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test ( C , D ).

Article Snippet: CCL21, a goat IgG anti-mouse CCL21 antibody, AZ-10417808, GM-CSF and FLT3 ligand were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Chemotaxis Assay, Concentration Assay, Comparison

Journal: Scientific Reports

Article Title: Cholinergic anti-inflammatory pathway ameliorates murine experimental Th2-type colitis by suppressing the migration of plasmacytoid dendritic cells

doi: 10.1038/s41598-021-04154-2

Figure Lengend Snippet: The expression of active Rac 1 (Rac1-GTP) and total Rac 1 in BMpDCs was measured by western blotting. The ratio of active Rac 1 to total Rac 1 was significantly increased by stimulation with CCL21, and the increase in the ratio induced by CCL21 was inhibited by pretreatment with nicotine (10 µM) (a representative band pattern is shown; n = 4 for each group, * P < 0.05 vs CCL21). The full-length blots are shown in supplementary Fig. . Data are represented as the mean value ± SEM. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: CCL21, a goat IgG anti-mouse CCL21 antibody, AZ-10417808, GM-CSF and FLT3 ligand were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Comparison

Journal: Scientific Reports

Article Title: Cholinergic anti-inflammatory pathway ameliorates murine experimental Th2-type colitis by suppressing the migration of plasmacytoid dendritic cells

doi: 10.1038/s41598-021-04154-2

Figure Lengend Snippet: The proportions of pDCs in the colonic mucosa and MLN of OXZ mice were analyzed. ( A , B ) The proportions of pDCs in the MLN and LPMCs of the middle colon in normal mice, OXZ mice, and OXZ mice treated with nicotine were compared. The frequency of pDCs in the MLN of OXZ mice was significantly decreased compared with that in the MLN of normal mice. The frequency of pDCs in LPMCs from OXZ mice was increased compared with that in the LPMCs from normal mice. The frequencies of pDCs in the MLN and LPMCs of OXZ mice treated with nicotine were equivalent to the corresponding frequencies of OXZ mice ( A : a representative experiment is shown, B : n = 4–6, ** P < 0.01 vs OXZ, n.s.: not significant). ( C ) The localization of CCL21 in OXZ mice was investigated by immunohistochemistry. CCL21 was mainly expressed around the T cell zone in ILFs (a representative result is shown). The scale bars represent 50 μm. Data are represented as the mean value ± SEM. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test ( B ).

Article Snippet: CCL21, a goat IgG anti-mouse CCL21 antibody, AZ-10417808, GM-CSF and FLT3 ligand were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunohistochemistry, Comparison

Journal: Scientific Reports

Article Title: Cholinergic anti-inflammatory pathway ameliorates murine experimental Th2-type colitis by suppressing the migration of plasmacytoid dendritic cells

doi: 10.1038/s41598-021-04154-2

Figure Lengend Snippet: Graphical abstract. The cholinergic anti-inflammatory pathway (nicotine and vagus nerve stimulation) ameliorated Th2-type OXZ colitis through α7nAChRs on pDCs, which was attributed to the suppression of pDC migration toward CCL21 in ILFs of the colonic mucosa of OXZ mice by α7nAChR-mediated JAK2-STAT3 activation, subsequent nonapoptotic caspase-3 activation and eventual Rac 1 inactivation.

Article Snippet: CCL21, a goat IgG anti-mouse CCL21 antibody, AZ-10417808, GM-CSF and FLT3 ligand were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Activation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis.

doi: 10.1016/j.biopha.2021.111881

Figure Lengend Snippet: Fig. 4. Effects of 1 μM As-IV or Oxy on RAC1 activation. RAC1 activation was detected by evaluating the expression of the GTP-RAC1 protein by using a western blot analysis kit. A representative image and the quantification of the band intensity for GTP-RAC1 relative to that of total RAC1 are shown in A-C. (A) Representative western blot analysis of GTP-RAC1 in BMpDCs induced with CCL21 and quantification of the band intensity are shown (5 independent experiments). Data are expressed as the mean ± SE (*p < 0.05, n = 5). Representative western blot analysis of GTP-RAC1 in CCL21-induced migrated BMpDCs following treatment with As- IV (B) or Oxy (C) and the quantification of band intensity are shown (7 independent experiments). Data are expressed as the mean ± SE (*p < 0.05, n = 7). Microarray analysis of BMpDCs induced with a CCL21 gradient following treatment with As-IV or Oxy was performed by using the Clariom S Array Mouse. The differential expression of genes in BMpDCs induced with a CCL21 gradient following treatment with vehicle (control), As-IV or Oxy is displayed in the heatmap (D). The red and blue colors indicated the up-regulation normalized intensity values (log2) and down-regulation normalized intensity values (log2) of each RNA in each sample.

Article Snippet: 30 μm sections cut by using a cryostat (Leica, Nussloch, Germany) were soaked in 0.3% Triton X (Sigma, Missouri, USA) for 2 h and 2% Block Ace (DS Pharma Biomedical, Osaka, Y. Zhang et al. Biomedicine & Pharmacotherapy 141 (2021) 111881 Japan) for 1 h. Then, the colon sections were stained with the primary antibodies rat IgG anti-mouse B220 (1:200, BioLegend, San Diego, CA, USA), hamster IgG anti-mouse CD11c (1:100, BioLegend) and goat IgG anti-mouse CCL21 (1:200, R&D Systems).

Techniques: Activation Assay, Expressing, Western Blot, Microarray, Quantitative Proteomics, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis.

doi: 10.1016/j.biopha.2021.111881

Figure Lengend Snippet: Fig. 6. Effects of As-IV or Oxy on the distribution of CCL21 in the DSS-induced colitis model. The distribution of CCL21 in the colonic ILFs of DSS-induced colitis mice treated with saline (A), As-IV (B) or Oxy (C) was identified by immunohistochemical staining of the colon. Immunohistochemical staining was performed on samples from 3 mice/group, and representative images are presented.

Article Snippet: 30 μm sections cut by using a cryostat (Leica, Nussloch, Germany) were soaked in 0.3% Triton X (Sigma, Missouri, USA) for 2 h and 2% Block Ace (DS Pharma Biomedical, Osaka, Y. Zhang et al. Biomedicine & Pharmacotherapy 141 (2021) 111881 Japan) for 1 h. Then, the colon sections were stained with the primary antibodies rat IgG anti-mouse B220 (1:200, BioLegend, San Diego, CA, USA), hamster IgG anti-mouse CD11c (1:100, BioLegend) and goat IgG anti-mouse CCL21 (1:200, R&D Systems).

Techniques: Saline, Immunohistochemical staining, Staining