DARC immunoreactivity in MDCK-DARC monolayers. (a–c) Confocal laser-scanning microscopy of the subcellular distribution of DARC immunoreactivity in cross-sections of unstimulated (a) and CCL2-stimulated (b,c) MDCK-DARC monolayers grown on Transwell inserts; in b,c, 35 nM CCL2 was added below the monolayers, followed by incubation for 30 min (b) or 2 h (c). DARC immunoreactivity was detected by indirect immunofluorescence with Alexa Fluor 488 (quantification of DARC distribution, Supplementary Table 1). Scale bars, 10 μm. (d) Immunoelectron microscopy of DARC immunoreactivity (electron-dense dots of colloidal gold) on apical microvillus extensions of MDCK-DARC cells 2 h after the application of CCL2 to the basolateral side. Arrows indicate apical microvilli. Scale bar, 500 nm. (e) Immunoelectron microscopy of the colocalization of DARC (large black dots; 15-nm colloidal gold) with CCL2 (small black dots; 5-nm colloidal gold) on an apical microvillus extension (arrow). Scale bar, 200 nm. Images in d,e were acquired with a transmission electron microscope. Results are representative of at least three independent experiments.

Chemokine degradation by MDCK cells expressing DARC or D6. Degradation of ¹²⁵I-labeled CCL2 (detected as the TCA-soluble fraction) by monolayers of MDCK-mock cells, MDCK-DARC cells and MDCK cells transfected with D6 (D6), assessed 3 h after placement below (a) or above (b) the monolayers. Data are representative of three independent experiments (mean and s.d. of three samples).

CCL2-induced monocyte migration across MDCK cells and HUVECs. Migration of human monocytes (isolated from healthy donors) across monolayers of DARC- and mock-transfected MDCK cells (a) and HUVECs (b) seeded onto Transwell inserts; after 4 h of incubation with 35 nM CCL2, transmigrated monocytes were counted in three different compartments: adherent to the bottom side of the filter (Filter), in suspension in the lower chamber (Suspension) and adherent to the bottom of the chamber (Bottom). (a) CCL2 PMFC, replacement of fluid in the upper chamber three times during incubation. Data are representative of three (a) or two (b) independent experiments (mean and s.d. of three samples).

Acute CHS reaction to oxazolone in mDARCtg and wild-type mice. (a) Ear thickness of mDARCtg mice (n = 5) and wild-type mice (n = 8) sensitized with 2% oxazolone on the shaved abdomen and challenged with 0.2% oxazolone on the ear, measured before and after two challenges (1 and 2) and presented as the difference (Δ mm) between the unchallenged ear and the oxazolone-challenged ear. (b) Ear weight after the second oxazolone challenge in a, presented as the difference (Δ mg) between the unchallenged ear and the oxazolone-challenged ear. (c) Histology of CHS lesions in mDARCtg and wild-type mice. Scale bars, 100 μm. Data are from one of two independent experiments (error bars (a,b), s.e.m.).

Colocalization of DARC and CXCL8 in venular endothelial cells in human skin. Ultrastructural colocalization of DARC immunoreactivity (large black dots; silver-enhanced 15-nm colloidal gold) with CXCL8 immunoreactivity (small black dots; silver-enhanced 5-nm colloidal gold) in plasmalemmal vesicles (a) and on the luminal membrane (inset, a), in large intracellular electron-lucent vesicles (b) and on the abluminal membrane (c) in venular endothelial cells 120 min after the injection of CXCL8 (12 pmol) into intact pieces of normal human skin immediately after removal during elective surgery. Large arrows, small arrow and arrowhead indicate colocalization intracellularly, luminally and abluminally, respectively. Scale bars, 200 nm. Results are representative of two experiments with skin samples from two people.

Chemokine transport by DARC across MDCK-DARC monolayers. Analysis of ¹²⁵I-labeled CCL2 (¹²⁵I-CCL2) placed above or below monolayers of MDCK-mock cells (Mock) and MDCK-DARC cells (DARC) and, after incubation, detected in three different compartments: intracellular (IC), cell surface bound (CS) and in the fluid (Fluid). (a) Basolateral-to-apical transport of intact ¹²⁵I-labeled CCL2 TCA-precipitable. (b) Autoradiography of MDCK-DARC cell–associated ¹²⁵I-labeled CCL2 after its basolateral-to-apical transport. Ctrl, native ¹²⁵I-labeled CCL2 (control). kDa, molecular size in kilodaltons. (c,d) Binding and internalization (c) and transport (d) of intact ¹²⁵I-labeled CCL2 from the apical side to the basolateral side. (d) Soluble chemokine in the bottom Transwell chamber. Data are representative of three (a,c,d) or two (b) independent experiments (mean and s.d. of three samples).

Chemokine transport by DARC across HUVEC-DARC monolayers. (a) Basolateral-to-apical transport of ¹²⁵I-labeled CCL2 across monolayers of HUVECs transfected with empty vector (Mock) or DARC (DARC) and seeded onto Transwell inserts, assessed after 3 h of incubation. (b,c) Binding and internalization (b) and transport (c) of chemokine placed above monolayers of HUVECs transfected with empty vector or DARC. (c) Soluble chemokine in the bottom Transwell chamber. Data are representative of two independent experiments (mean and s.d. of three samples).

Recruitment of neutrophils into chemokine injection sites in mDARCtg and wild-type mice. (a) Peritoneal neutrophils 4 h after intraperitoneal injection of saline (NaCl; control) or mouse CXCL1 (350 pmol per site) into mDARCtg mice and wild-type (WT) mice (n = 4). (b) Dermal neutrophil infiltration 4 h after intradermal injection of saline or mouse CXCL1 (350 pmol per site) into mDARCtg and wild-type mice (n = 4), assessed as myeloperoxidase (MPO) activity associated with dermal injection sites. Activity in untreated skin biopsies, 0.03 ± 0.01 units per biopsy. Data are representative of three (a) or two (b) independent experiments (mean and s.e.m.).