Functional expression of CCR3 on CD30⁺cutaneous lymphoma cells.

(A) CCR3 surface expression measured by flow cytometry on freshly isolated tumor cells (patient no. 8) after incubation with corresponding ligand eotaxin or medium control. Cells were incubated in pH 3.0 acid buffer to remove receptor-bound chemokine on noninternalized receptor. Internalization was not seen after incubation with low-affinity ligands RANTES/CCL5 or MCP-3/CCL7 (data not shown). Data shown are representative of 3 experiments. (B) The F-actin content was measured in the CD30⁺ cutaneous lymphoma cell line Mac-1 upon stimulation with 100 ng/mL eotaxin for the indicated times. Fluorescein phalloidin was used to stain the cells, and the filamentous actin content was then measured by means of flow cytometry. Data show means of 3 different experiments. (C) To assess the chemotactic responses of the CD30⁺cutaneous lymphoma cell line Mac-1 to different concentrations of eotaxin, migration across a 5-μm pore size polycarbonate membrane during 3 hours was measured by counting migrated cells by means of a flow cytometer. Migrated cells at each time point were measured in triplicate. Data are expressed as the mean numbers of migrated cells per well. Error bars indicate SEM.