Expression of elastolytic MMPs and elastolysis by differentiating MDMs. (A) Expression of elastolytic proteinases was assessed in monocytes (Mo) and MDMs after 1, 2, 3, 5, 7, and 9 d in culture by oligonucleotide array analysis. Elastase 1, 2, IIB, and IIIA are pancreatic elastase I, neutrophil elastase, pancreatic elastase II, and pancreatic endopeptidase E, respectively. Results are shown as a color-coded increase in mRNA content relative to that expressed by freshly isolated nonadherent monocytes from a single representative experiment of three performed. (B) Secretory phenotype of MDMs. Mature MDMs were incubated for 48 h in serum-free media. Aliquots of conditioned media or membrane extracts (for MT1-MMP) were resolved by SDS-PAGE and probed with specific antibodies. The ability of MT1-MMP to process gelatinase A was assessed by gelatin zymography (lower right-hand section of panel). MDMs were incubated for 48 h in serum-free media alone (lane 3) or cocultured with 0.5 μg/ml progelatinase A in the absence (lane 4) or presence (lane 5) of BB-94. Standards for the proforms and processed forms of gelatinase A as well as the gelatinase A proenzyme are shown in lanes 1 and 2, respectively. The proforms (open arrowheads) and processed (arrows) forms of enzymes are indicated. Results are shown from a single representative experiment of three performed. (C) MDMs were cocultured with [³H]elastin suspended in the upper well of a transwell insert to prevent direct cell–elastin contact in the absence (▪ and □) or presence (▴ and ▵) of 50 μg/ml exogenous plasminogen. Elastolysis was monitored over the course of a 5-d culture period. Elastolysis was inhibited by BB-94 under both plasminogen-free (□) or plasminogen-supplemented conditions (▵). Results are expressed as the mean ± SEM (n = 5). (D) Human MDMs were cocultured for 10 d with aortic tissue in the absence or presence of 50 μg/ml plasminogen and BB-94. Appearance of the aortic elastin is shown on cross sections by hematoxylin and eosin staining. Arrows indicate zones of elastin fragmentation and asterisks show intact elastic lamina (×100). The appearance of the full thickness sections is shown in the insets (×20).

Plasminogen-dependent elastolysis and processing of MMP zymogens by human MDMs. (A) Human MDMs were cocultured with [³H]elastin in the upper well of a transwell insert with 50 μg/ml plasminogen (plg) in the absence or presence of the indicated inhibitors. Results are shown as μg elastin degraded/10⁶ cells for 5 d (mean ± SEM; n = 3). Western Blots: MDMs and elastin were cocultured without contact in the presence of 50 μg/ml plasminogen and/or 100 μg/ml aprotinin for 5 d. The processing of progelatinase B (B), prometalloelastase (C), and promatrilysin (D) in the presence of plasminogen was assessed by Western blot analysis in samples recovered from conditioned media or the elastin surface. The proforms (open arrowheads) and mature (arrows) forms of enzymes are indicated. The processing of the proforms by 4-aminopherylmercuric acetate or trypsin are shown at the far right of each panel with the proforms (open arrowheads) and mature (arrows) forms depicted. Results are shown from a single representative experiment of three performed.

Cysteine proteinases complement MMP-dependent elastolysis by human MDMs. (A) MDMs were incubated with [³H]elastin to allow cell–substrate contact and elastolysis was monitored in the absence (▴) or presence (▪) of 50 μg/ml exogenous plasminogen (plg) over the course of a 5-d culture period. Results are expressed as the mean ± SEM (n = 4). (B) MDMs were cocultured in contact with elastin in the absence (gray bar) or presence (solid bar) of plasminogen for 5 d alone or with either 100 μM E-64 alone or E-64 in combination with either 5 μm BB-94, 5 μg/ml TIMP-2, or 100 μg/ml aprotinin. Results are shown as μg elastin degraded/10⁶ cells for 5 d (mean ± SEM; n = 3).

Plasminogen-independent proteolysis by human and mouse macrophages. (A) Human MDMs were cocultured with [³H]elastin (suspended in the upper well of a transwell insert) under plasminogen-free conditions in the absence or presence of inhibitors directed against metallo (5 μM BB-94 or 5 μg/ml TIMP-2), serine (100 μg/ml aprotinin), cysteine (100 μM E-64), or aspartate (5 μM pepstatin) proteinases as well as solvent controls (0.1% DMSO and 0.05% ethanol) as indicated. Results are shown as μg elastin degraded/10⁶ cells for 5 d (mean ± SEM; n = 6). (B and C) Left: Plasminogen-independent processing and elastolytic activity of MMP zymogens. Human macrophages and elastin were cocultured in the absence or presence of 5 μM BB-94 for 5 d. The processing of progelatinase B (B) and prometalloelastase (C) in the absence of plasminogen (plg) was assessed by Western blot analysis in samples recovered from conditioned media or the elastin surface. The proforms (open arrowheads) and mature (arrows) forms of enzymes are indicated. Results are shown from a single representative experiment of three performed. Right: Mouse macrophages obtained from gelatinase B^+/+ (Gel.B^+/+) or gelatinase B^−/− (Gel.B^−/−) mice (B) and metalloelastase^+/+ (MME^+/+) or MME^−/− mice (C) were incubated with [³H]elastin in the absence or presence of BB-94 for 5 d. Results are shown as μg elastin degraded/10⁶ cells for 5 d (mean ± SEM; n = 3).

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/10⁶ 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 [³H]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/10⁶ 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 [³H]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/10⁶ 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 [³H]elastin in serum-free media at 37°C. Results are expressed as μg elastin degraded for 5 d (mean ± SEM; n = 3).