CGRP induction as LPS response in human monocytes. (A) RT-PCR-amplified CGRP gene transcripts (i) from monocytes after 24 hours of incubation. Us indicates unstimulated monocytes without (-) or with 100 ng/mL NGF (+NGF); LPS, LPS-treated monocytes without (-) or with 10 μg/mL neutralizing goat anti-NGF antibody (+abNGF) or goat IgG as control (+IgG). Southern blotting analysis (ii) and actin expression (iii) of the electrophoresed RT-PCR-amplified samples shown in panel i. These ethidium bromide-stained gels are representative of 4 independent experiments. (B) CGRP expression was quantified in CD14⁺ cells by flow cytometry analysis. A specific CGRP immunoreactivity was detected in gated CD14⁺ cells after 24 hours of culture in different conditions. LPS-stimulated monocytes (LPS) showed a higher immunopositivity for CGRP than unstimulated monocytes (Us) but treatment with anti-NGF antibodies (LPS+abNGF) decreased CGRP expression, which was not affected when goat IgG was used (LPS+IgG). The darker area in the graphs represents the unspecific signal obtained in CD14⁺-permeabilized cells incubated with rabbit IgG and anti-rabbit FITC antibodies. In these graphs, representative of 6 independent experiments, the values of ΔMIF for CGRP positivity are included for each culture condition.