Chemokine production and chemokine receptor expression of DCs stimulated with recombinant Hsp70L1. (A) Dose- and time-dependent secretion of chemokines by human DCs stimulated with recombinant Hsp70L1. For the dose-dependent assay DCs were stimulated with a 2-fold titration series of Hsp70L1, while for the time-dependent experiment, DCs were stimulated with 10 μg/mL Hsp70L1 and supernatants harvested at different times for ELISA assay. Data are mean chemokine concentration (pg/mL) ± SEM of 3 independent experiments. In comparative experiments (B), DCs were cultured with the stimuli shown in the key. Supernatants were collected and assayed for IP-10, MIP-1α, MIP-1β, and RANTES by ELISA. The data are presented as mean ± SEM of 6 independent experiments. For RANTES, MIP-1α, and MIP-1β, P < .05 between Hsp70L1 and human Hsp70 stimulation; for IP-10, P < .01 between Hsp70L1 and human Hsp70 stimulation. (C) Expression of the chemokine receptors CCR7 and CXCR4. DCs were stimulated with LPS, His-CK, Hsp70L1, or boiled Hsp70L1, Hsp70, or boiled Hsp70 for 40 hours, then cells' total RNA was extracted and used for analysis of CCR7 and CXCR4 expression by semiquantitative RT-PCR, or cells were stained with FITC-conjugated mAbs to CCR7 and CXCR4 for FACS analysis.