Figure 4.

Endothelial Ca Signaling Facilitates DC Transmigration In Vitro

(A) Transmission electron micrograph at a site of DC-LEC interaction at ear dermis lymphatic capillary. Black line indicates thinning of endothelial cell at site of interaction. Interstitium (I), lumen (L), LEC, and DC are marked.

(B) Immunoelectron micrograph of whole mount preparation labeled for CCL21 (silver amplified gold particles) at a site of DC-LEC interaction in ear dermis. The yellow arrows in (A) and (B) indicate the endothelial detachment from interstitium.

(C) Phase contrast imaging of an in vitro transmigration event from the apical to basolateral side of the LEC monolayer. White arrow points at leading edge, yellow arrow at cell body, and blue arrow at trailing edge of transmigrating DC. See Movie S4.

(D) Epifluorescence imaging of Oregon Green BAPTA-AM-treated LECs (green) and TAMRA-labeled DCs (red). Blue arrow indicates DC leading edge and yellow arrow LEC displaying a transient Ca peak upon DC contact. Below the image, the kymograph and the corresponding line graph of the Oregon green BAPTA-AM intensity in the LEC (highlighted with yellow arrow) are shown. See Movie S5.

(E) Quantification of LECs displaying Ca peaks upon addition of fresh media, DCs, DC-conditioned media, or Mycalolide B-treated DCs. Dot blot graph shows mean ± SD of pooled samples. Fresh media and DC samples represent eight independent experiments (n = 18 and 19, respectively), of which two experiments included Mycalolide B-treated DCs (n = 6), and three experiments included conditioned media samples (n = 4). Data are normalized to average of DCs sample, which is set as 1.

(F) Quantification of DC transmigration on control or 10 μM BAPTA-AM (B-AM)-treated CCL21-mCherry or CCL21ΔC-mCherry expressing monolayers. Dot blot graph shows mean ± SD of pooled samples from three independent experiments, n = 7 for BAPTA-AM-treated CCL21ΔC-mCherry expressing LECs and eight for all other conditions. Data are normalized to average of controls, which is set as 1. See Movie S6.

(G) Schematic of DC-induced Ca influx and CCL21 secretion. Scale bars, 500 nm (A and B); 50 μm (C); and 10 μm (D).

See also Figure S2.