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 micrograms/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 to 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-1 beta. 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 elcited 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. (ABSTRACT TRUNCATED AT 250 WORDS)
Sylvester
I
I
Department of Internal Medicine, Yale University School of Medicine, West Haven, Connecticut.
Rankin
J A
JA
Yoshimura
T
T
Tanaka
S
S
Leonard
E J
EJ
D000328:Adult; D002478:Cells, Cultured; D002630:Chemotactic Factors / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D016209:Interleukin-8; D007378:Interleukins / Q000032:analysis / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000166:cytology*; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D009000:Monocytes / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D002630:Chemotactic Factors; D016209:Interleukin-8; D007378:Interleukins; D008070:Lipopolysaccharides; D010455:Peptides