D6 regulates lymphatic interactions with dCs. (A) D6 expression can be knocked down by D6-specific siRNA, but not by irrelevant siRNAs (irr siRNA) or fluorescent transfection control siRNA (siGLO), as determined by QPCR. (B) siRNA knock-down of D6 downregulates the endogenous Alexa-CCL2 uptake ability of HDLECs. Red, baseline autofluorescence of HDLECs; blue, endogenous uptake ability of control HDLECs; green/pink, repeats of D6 siRNA–treated HDLECs exposed to Alexa-CCL2. Functional D6 knockdown is represented as a “left-shift” in the flow cytometry profiles. (Ci) siRNA knockdown of D6 in HDLECs increases the number of iDC adhering (8 × 10⁴ added) to tumor necrosis factor (TNF)-stimulated HDLEC monolayers as assessed by flow cytometry. (Cii) siRNA knockdown of D6 has no effect on the number of mDC adhering (2 × 10⁴ added) to TNF-stimulated HDLEC monolayers. (D) D6 is strongly overexpressed in HDLECs following transduction with an adenovirus expressing D6 (AdD6) vector (at 100 and 1000 viral particles/cell), as determined by QPCR. (E) AdD6 induces Alexa-CCL2 uptake. Red, baseline autofluorescence of HDLECs; green, control HDLECs treated with Alexa-CCL2; blue, adenovirus-infected HDLECs treated with Alexa-CCL2. (F) mDC and iDC compete for binding sites at HDLEC:HDLEC cell junctions. This figure visualizes unlabeled DC interacting with the underlying LEC monolayer. (G) HDLECs were either treated with siRNA to knockdown D6 or transduced with AdD6 to increase expression. Differentially dye-labeled iDC and mDC were mixed at a ratio of 4:1 (numbers as in C) and allowed to adhere to HDLEC monolayers; the relative numbers of iDC and mDC binding were assessed by FACS and expressed as an iDC/mDC ratio. All HDLECs used in the experiments represented in this figure were between passages 4 and 7.