Effect of assay conditions on measurement of elastolytic activity of alveolar macrophages in culture and characterization with proteinase inhibitors.
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.
Ofulue
A F
AF
Department of Medicine, Vancouver General Hospital, British Columbia, Canada.
Sansores
R M
RM
Abboud
R T
RT
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*
D015853:Cysteine Proteinase Inhibitors; D004549:Elastin; D008666:Metalloendopeptidases