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Science 339 (6117): 328-332

Copyright © 2013 by the American Association for the Advancement of Science

Interstitial Dendritic Cell Guidance by Haptotactic Chemokine Gradients

Michele Weber1, Robert Hauschild1, Jan Schwarz1, Christine Moussion1, Ingrid de Vries1, Daniel F. Legler2, Sanjiv A. Luther3, Tobias Bollenbach1, and Michael Sixt1,*

1 IST Austria (Institute of Science and Technology Austria), Am Campus 1, A-3400 Klosterneuburg, Austria.
2 Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Unterseestrasse 47, CH-8280 Kreuzlingen, Switzerland.
3 Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland.


Figure 1
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Fig. 1. DCs move directionally toward CCL21-expressing LVs in the dermal interstitium. (A to D) Tracks of DCs migrating in ear explants. (A) Migratory paths (lines in gray) tracked from a representative 60-min movie, overlaid onto the LV mask (dark gray). (B) Selected tracks (gray) from five movies (n = 200, three independent experiments) reorientated to nearest LV (LV margin at distance = 0 indicated by the black horizontal line). (C and D) Directionality and velocity toward LV as a function of distance to the nearest LV. Mean ± SEM is shown. n = 200, three independent experiments. a.u., arbitrary units. (E) Wild-type DC (highlighted by the white arrow) migrating between two adjacent vessels (indicated by yellow dotted lines). Scale bar indicates 50 μm. (F) (Left) Z-stack projection showing wild-type (red) and Ccr7–/– DCs (green) after 120-min incubation with ear sheets stained for LYVE-1 (blue). Scale bar, 100 μm. (Right) DC migration paths tracked from a representative 60-min movie, overlaid onto the LV mask (dark gray).

 

Figure 2
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Fig. 2. Visualization and quantification of an interstitial CCL21 gradient. (A) Z-stack projection of permeabilized ear dermis stained for CCL21 (red) and laminin (green). Scale bar, 100 μm. (B) CCL21 (green) LYVE-1+ (red) co-staining of permeabilized ear dermis after treatment with 25 μg/ml brefeldin A. Scale bar, 25 μm. (C) Z-stack projection of nonpermeabilized ear dermis stained for CCL21. Gray scale shows maximum intensity projection (left). Right image shows same staining as color-coded average projection. LV boundaries are indicated by the blue dotted line based on LYVE-1 staining (as shown in fig. S2). Scale bars, 100 μm. A representative image from n = 9 out of four independent experiments is shown. (D) Quantification of interstitial CCL21 and CCL19 staining as function of distance from the nearest LV margin. Mean signal intensities relative to average maximum CCL21 signal ± SEM are shown (red, CCL21; green, CCL19 in Ccl19+/+ ear; n = 9; four independent experiments; black, CCL19 in Ccl19–/– ear; n = 5; two experiments). (E) Vector maps of local chemokine gradients. Arrow length and direction indicate concentration rise in CCL21 after averaging over circular surface area with indicated diameter (virtual cell size). Gray scale indicates averaged intensities. Scale bar, 15 μm. (F) Directionality of CCL21 gradients (Dc) for indicated virtual cell size as cosine of angle between direction toward closest point of LV and direction of increasing chemokine concentration. Means ± SEM for n = 6 from three independent experiments are shown. (G) Average local CCL21 concentration delta ({Delta}) as a function of distance to nearest LV, calculated for indicated virtual cell size. Means ± SEM for n = 7 from four independent experiments are shown.

 

Figure 3
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Fig. 3. The CCL21 gradient is functional and immobilized to heparan sulfates. (A) (Left) Z-stack projection of CCL21 immunostaining (false color-coded) of nonpermeabilized ear dermis after incubation with 0.6 μg/ml CCL21. LYVE-1–positive LVs are indicated by the blue dotted line. Scale bar, 100 μm. (Right) cCCL21 after incubation with exogenous CCL21. Gray line indicates endogenous CCL21 as shown in Fig. 2. n = 3, two independent experiments. (B) Z-stack projections of DCs (red) and LYVE-1 immunostaining (green) after a 120-min co-incubation of DCs with ear sheets. Tissue was either vehicle-treated or incubated with CCL21 before addition of DCs. n = 3, two independent experiments. (C) DC tracks (light gray lines) from representative 60-min movie with CCL21 pre-incubation, overlaid onto LV mask (dark gray). (D) Number of DCs associated with LVs after 120-min crawl-in. Indicated chemokine was pre-incubated at 0.6 μg/ml before addition of DCs. Control versus CCL21, **P = 0.006; control versus CCL21trunc, P = 0.382; control versus CCL21mut, P = 0.8163, control versus CXCL13, P = 0.458; control versus CXCL12, P = 0.0642. n = 3, at least two independent experiments. (E) Z-stack projection of nonpermeabilized ear dermis stained for CCL21 (false color-coded, left) and quantification of cCCL21 (right) in nonpermeabilized ear dermis after treatment with 50 mIU heparitinase. Gray line in graph indicates endogenous CCL21 as in Fig. 2. (F) Numbers of DCs associated with LVs in ear dermis after 120 min of crawl-in. n = 5, three independent experiments (**P = 0.004). All scale bars, 100 μm. Error bars indicate SEM.

 


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