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 c onflicting reports on measured elastolytic activity of alveolar macrop hages (AM) cultured in direct contact with the H-3-elastin substrate c oated onto 16-mm wells in serum-containing media. The data indicate th at measured elastolytic activity in this assay system was dependent on the amount of H-3-elastin/culture well. H-3-elastin >350 mu g/well, i n contrast to the less than or equal to 200 mu g/well commonly used in this assay system, resulted in optimal measurement of elastolytic act ivity that was linear with respect to culture time (up to 72 h examine d) and was directly proportional to number of AM/well (up to 1.0 x 10( 6) examined). The sensitivity of measured elastolytic activity to tiss ue inhibitor of metalloproteinases (TIMP) and to Z-phe-phe (a specific cysteine proteinase inhibitor) was not affected by amount of H-3-elas tin/well, but appears to be dependent on the time period of AM culture . TIMP (at 5 mu M, maximal dose examined) inhibited the measured elast olytic activity by 25% in 24-h cultures compared to 69% in 72-h cultur es; Z-phe-phe (at 10 mu M, dose at which maximal effect was obtained) inhibited the elastolytic activity by 45% in the 24-h cultures compare d to 34% in the 72-h cultures. These findings indicate that difference s in substrate levels and in culture time have a significant effect on the results obtained in measurement of AM-mediated elastolytic activi ty in culture, which may account for the conflicting reports in the li terature. Thus standard optimal assay condition are required for valid interpretation of results of AM-mediated elastolytic activity measure ments.