Journal: The Journal of Experimental Medicine
Article Title: Matrilysin-dependent Elastolysis by Human Macrophages
Figure Lengend Snippet: Matrilysin “deficiency” abrogates plasminogen-dependent elastolysis by human MDMs and mouse peritoneal macrophages. (A) Mouse peritoneal macrophages or human monocytes differentiated for 9 d atop the surface of either bacteriologic plastic dishes (Plastic) or a cell-derived ECM (Matrix; phase micrographs are shown on the right of A) were cultured with elastin suspended in transwell inserts as described in Fig. 1 in the absence or presence of 50 μg/ml plasminogen (plg). Where indicated, macrophages were incubated with or without 5 μM BB-94. In the presence of plasminogen and BB-94, the release of radiolabel from elastin is due primarily to plasmin activity and can be reduced to background levels with aprotinin (unpublished data). Results are shown as μg elastin degraded/10 6 cells for 5 d (mean ± SEM; n = 3). (B) Secretion of elastolytic MMPs by human MDMs and murine macrophages as a function of in vitro culture conditions. Human MDMs differentiated atop surfaces of bacteriologic plastic (Plastic) or the cell-derived matrix (Matrix) were cultured under serum-free conditions for 48 h. Conditioned media was collected and analyzed by immunoblot for gelatinase B, human metalloelastase (HME), and matrilysin. Mouse macrophages were cultured in bacteriologic plastic dishes for 48 h under serum-free conditions and conditioned media was analyzed for MMPs by immunoblot as described in Fig. 1 . (C) Human MDMs were cocultured with [ 3 H]elastin and incubated alone (lane 1), with 50 μg/ml plasminogen (plg; lane 2), or plasminogen in the presence of aprotinin (lane 3), BB-94 (lane 4), anti–gelatinase B antibody (lane 5), or normal mouse IgG (both 50 μg/ml; lane 6). MMP-9 processing was assessed by gelatin zymography (top) and the effects on degradative activity were quantified (bottom). Results are expressed as μg elastin degraded/10 6 cells for 5 d (mean ± SEM; n = 3). (D) Human MDMs differentiated atop a cell-derived matrix (Matrix) or mouse peritoneal macrophages cultured under standard conditions (Plastic) were incubated with [ 3 H]elastin and 50 μg/ml plasminogen in the absence or presence of 5 μM BB-94. Where indicated, 20 nM recombinant promatrilysin was added to the culture system in the absence or presence of BB-94. 20 nM recombinant promatrilysin and 25 μg/ml plasmin were also incubated with elastin under cell-free conditions (Cell-Free). Inset shows the relative levels of matrilysin detected in media conditioned by human MDMs (Mφ CM) and the amount of recombinant matrilysin added (rMMP-7). Results are expressed as μg elastin degraded/10 6 cells for 5 d (mean ± SEM; n = 3). (E) Equimolar quantities (1.0 pmol) of active site–titrated gelatinase B, human metalloelastase, or matrilysin alone, or all three enzymes together were incubated with [ 3 H]elastin in serum-free media at 37°C. Results are expressed as μg elastin degraded for 5 d (mean ± SEM; n = 3).
Article Snippet: Where indicated, macrophage/[3 H]elastin cocultures were incubated alone, with 50 μg/ml glu-type human plasminogen, 100 μg/ml mouse-neutralizing antibody against human uPA (American Diagnostica, Inc.), mouse anti–gelatinase B IgG (ab-2; reference ), 50 μg/ml normal mouse IgG (both from Oncogene Research Products and Calbiochem-Novabiochem), recombinant human pro–MMP-7 (Chemicon International, Inc.), recombinant human pro–gelatinase B and A (R & D Systems), 5 μM of the synthetic MMP inhibitor, BB94 (0.1% DMSO final; provided by British Biotechnology; reference 22), 5 μg/ml endotoxin-free recombinant human tissue inhibitor of metalloproteinases-2 (TIMP-2; Fuji Chemical Industries, LTD.; reference 22), 100 μM the pan-specific cysteine proteinase inhibitors, E-64 (0.05% ethanol final; Sigma-Aldrich; reference 16), 100 μg/ml aprotinin (Roche; reference 22), 5 μM the aspartate protease inhibitor, pepstatin (0.05% ethanol final; Roche; reference 23), or solvents (0.1% DMSO and 0.05% ethanol) alone.
Techniques: Derivative Assay, Cell Culture, Incubation, Activity Assay, In Vitro, Zymography, Recombinant