DC-SIGN ligation in myeloid and lymphoid cell lines results in ERK activation. (A) Cell surface expression of DC-SIGN in THP-1 cells, as determined by flow cytometry. The percentage of marker-positive cells and the mean fluorescence intensity are indicated in each case. (B) DC-SIGN on THP-1 cells was engaged by the anti–DC-SIGN MR-1 antibody (20 μg/mL), and the cells were incubated at 37°C for 5 minutes. As a control, cells were either incubated with an anti-CD70 monoclonal antibody or with PMA (10 ng/mL). After cell lysis, phosphorylated ERK (pERK, top panel) and total ERK (ERK, middle panel) were detected using specific polyclonal antisera. The bottom panel illustrates the level of pERK relative to the level of total ERK under each condition, as determined by densitometric analysis. NS indicates not stimulated. (C) Cell surface expression of DC-SIGN in mock-transfected (Jurkat) and DC-SIGN–transfected Jurkat cells (Jurkat-DC-SIGN) as determined by flow cytometry, using an isotype-matched anti–c-Myc antibody (9E10) as control. The mean fluorescence intensity is indicated in each case. (D) Homotypic aggregation of mock-transfected (Jurkat) and DC-SIGN–transfected (Jurkat-DCSIGN) Jurkat cells, as observed by reverse-phase microscopy at 2 different amplifications (5 ×, 10 ×). (E) DC-SIGN on Jurkat-DC-SIGN transfectants was ligated by the anti–DC-SIGN MR-1 antibody alone or in the presence of an anti-CD3 monoclonal antibody as control, and the cells were incubated at 37°C for 1 or 5 minutes. Mock-transfected Jurkat cells were subjected to the same treatments for control purposes. After cell lysis, phosphorylated ERK (pERK), phosphorylated ZAP-70 (pZAP-70), and total content of ERK and ZAP-70 were detected using specific polyclonal antisera. Each experiment was done 3 times with similar results. Representative results are shown.