FIG. 5.

RT-PCR analysis of RNA extracts obtained from NALT after MALP-2 treatment. (A) Kinetic transcriptional analysis of the genes coding for IP-10, MCP-3, and CCR2. Results are expressed as the fold increase with respect to the untreated controls (mice which received only PBS). (B) Electrophoretic analysis of the β-actin, CCR5, CCR6, CCR7, and CCR9 products obtained by RT-PCR. For these specific products the profile of the melting curves obtained by real-time RT-PCR did not allow exact quantification. The gel shows the results obtained with mRNA isolated from the macrophage-like cell line J774A.1 (ATCC TIB 67) after activation with LPS (10 μg/ml) or MALP-2 (0.5 μg/ml) during 16 h in comparison to results in nontreated control cells. Twenty microliters of each PCR mixture was loaded per lane. The relative amount of mRNA for β-actin was 304, 189, and 142 ng/μl for nonactivated, LPS-activated, and MALP-2-activated cells, respectively. (C) The surface expression of CCR5 and CCR6 was evaluated on J774A.1 cells after 18 h of treatment with MALP-2 by flow cytometry, using anti-CCR5-PE and anti-CCR6-Alexa Fluor 647 antibodies (BD Pharmingen). The percentages of CCR5- and CCR6-positive cells are indicated in each quadrant for MALP-2-activated and nonactivated control cells (in brackets).