Time course of mast cell–mediated NAP-2 formation as detected by immunoreactivity and biologic activity. A fixed concentration (1 × 10⁴/mL) of either α-IgE–stimulated (□) or unstimulated (▪) MCs was incubated with 3 μM CTAP-III for increasing periods of time. Thereafter, 2 μL cell-free supernatant of each sample and standard preparations of CTAP-III and NAP-2 (50 ng per lane) were separated by SDS-PAGE, subjected to Western blotting, and immunochemically stained with rabbit antiserum Rα-βTG to visualize CTAP-III and potential degradation products (A). Migration of standard CTAP-III (CT) and NAP-2 (NA) is indicated. Data from 1 representative experiment of 3 are shown. For quantification of NAP-2 biologic activity, CTAP-III and derivates were isolated from recovered cell-free supernatants by immunoaffinity chromatography, and the amount of NAP-2–like activity formed was assessed by its neutrophil-stimulating capacity as determined by the elastase release assay against a standard of purified NAP-2 (B). Shown are means ± SD of data obtained in 3 independent experiments. In the approach using α-IgE–stimulated MCs, the initial velocity (V_i) of NAP-2 formation was calculated as the increase in NAP-2 concentration per minute on the basis of the first time point at which significantly elevated NAP-2-levels could be detected.