SECRETION OF NEUTROPHIL ATTRACTANT ACTIVATION PROTEIN BY LIPOPOLYSACCHARIDE-STIMULATED LUNG MACROPHAGES DETERMINED BY BOTH ENZYME-LINKED-IMMUNOSORBENT-ASSAY AND N-TERMINAL SEQUENCE-ANALYSIS

Alveolar macrophages contribute to acute pulmonary inflammation by secretion of neutrophil chemoattractants. We determined if one of these attractants is neutrophil attractant/activating protein (NAP-1), which is secreted by blood monocytes stimulated by lipopolysaccharide (LPS). Alveolar macrophages were stimulated in tissue culture with 10 μg/ml LPS. Culture fluids collected at 24 h were assayed for both neutrophil chemotactic activity and the concentration of NAP-1 as determined by a sandwich ELISA. The concentration of NAP-1 in culture fluid of LPS-stimulated macrophages was 860 ± 40 ng/ml (SEM for six normal subjects). NAP-1 in fluid of unstimulated macrophages was 40 ± 15 ng/ml. We confirmed the presence of NAP-1 in culture fluid of LPS-stimulated lung macrophages by immunoaffinity and HPLC-CM column purification. The HPLC-CM elution profile of macrophage NAP-1 was identical to that of monocyte NAP-1, and the N-terminal sequence of the protein in one of the isolated peaks corresponded to that of monocyte-derived NAP-1β. Two lines of evidence show that NAP-1 does not account for all neutrophil chemotactic activity in culture fluid of 24-h, LPS-stimulated macrophages. At a dilution of culture fluid that elicited the same chemotactic response as a known concentration of pure NAP-1, the concentration of culture fluid NAP-1 was only one-tenth that of pure NAP-1. Furthermore, after removal by immunoabsorption of more than 95% of culture fluid NAP-1, the amount of neutrophil chemotactic activity remaining exceeded that which could be accounted for by residual NAP-1.