Figure 6.

Alpha-1 antitrypsin (AAT) induces the release of a bioactive factor. U937 cells were primed with lipopolysaccharide (LPS) and stimulated with Prolastin^® (AAT-P 1 mg/ml) for 30 min before collection of the cell-free cell culture supernatant (SN). A low molecular mass fraction (LF) and a high molecular mass fraction (HF) of the cell culture supernatant were separated by ultrafiltration with a cut-off of 10 kDa. For the production of control LF, either no AAT-P was added to LPS-primed U937 cells (LF C1) or AAT-P was added to the cell-free cell culture supernatant (LF C2) shortly before ultrafiltration. (A) The SN, the LF, and the HF were separated in a 15% SDS-polyacrylamide gel along with a molecular mass marker followed by staining with Brilliant Blue. Bands with a molecular mass of about 52 kDa correspond to AAT. (B,C) LPS-primed U937 cells were stimulated with 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP) in the presence or absence of AAT-P. The experiments were performed in medium, LF C1, LF C2, or in different concentrations of LF diluted in medium. Interleukin (IL)-1β released to the supernatant was measured after 30 min. (C) Nicotinic receptor antagonists mecamylamine (Mec), α-bungarotoxin (α-Bun), strychnine (Stry), RgIA4, or ArIB [V11L, V16D] were added together with BzATP in the presence of LF. *p ≤ 0.05 versus cells treated with LPS, BzATP, and AAT-P (B) or LF (C). Data are presented as individual data points, bar represents median, whiskers encompass the 25th to 75th percentile, n-numbers of independent experiments are indicated in the figure. Experimental groups were compared by Kruskal–Wallis followed by Mann–Whitney rank sum test (B,C).