title: Effect of assay conditions on measurement of elastolytic activity of alveolar macrophages in culture and characterization with proteinase inhibitors.
abstract: We have investigated whether varied assay conditions account for the conflicting reports on measured elastolytic activity of alveolar macrophages (AM) cultured in direct contact with the 3H-elastin substrate coated onto 16-mm wells in serum-containing media. The data indicate that measured elastolytic activity in this assay system was dependent on the amount of 3H-elastin/culture well. 3H-elastin > 350 micrograms/well, in contrast to the < or = 200 micrograms/well commonly used in this assay system, resulted in optimal measurement of elastolytic activity that was linear with respect to culture time (up to 72 h examined) and was directly proportional to number of AM/well (up to 1.0 x 10(6) examined). The sensitivity of measured elastolytic activity to tissue inhibitor of metalloproteinases (TIMP) and to Z-phe-phe (a specific cysteine proteinase inhibitor) was not affected by amount of 3H-elastin/well, but appears to be dependent on the time period of AM culture. TIMP (at 5 microM, maximal dose examined) inhibited the measured elastolytic activity by 25% in 24-h cultures compared to 69% in 72-h cultures; Z-phe-phe (at 10 microM, dose at which maximal effect was obtained) inhibited the elastolytic activity by 45% in the 24-h cultures compared to 34% in the 72-h cultures. These findings indicate that differences in substrate levels and in culture time have a significant effect on the results obtained in measurement of AM-mediated elastolytic activity in culture, which may account for the conflicting reports in the literature. Thus standard optimal assay condition are required for valid interpretation of results of AM-mediated elastolytic activity measurements.
authors.affiliation: Department of Medicine, Vancouver General Hospital, British Columbia, Canada.
authors.forename: A F
authors.initials: AF
authors.lastname: Ofulue
authors.forename: R M
authors.initials: RM
authors.lastname: Sansores
authors.forename: R T
authors.initials: RT
authors.lastname: Abboud
mesh_terms: D001681:Biological Assay; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004549:Elastin / Q000378:metabolism*; D006801:Humans; D016676:Macrophages, Alveolar / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D004549:Elastin; D008666:Metalloendopeptidases