The cells and proteases that mediate cigarette smoke-induced emphysema are controversial, with evidence favoring either neutrophils and neutrophil-derived serine proteases or macrophages and macrophage-derived metalloproteases as the important effectors. We recently reported that both macrophage metalloelastase (MMP-12) and neutrophils are required for acute cigarette smoke-induced connective tissue breakdown, the precursor of emphysema. Here we show how these disparate observations can be linked. Both wild-type (MMP-12 +/+) mice and mice lacking MMP-12 (MMP-12 -/-) demonstrated rapid increases in whole-lung nuclear factor-kappaB activation and gene expression of proinflammatory cytokines after cigarette smoke exposure, indicating that a lack of MMP-12 does not produce a global failure to upregulate inflammatory mediators. However, only MMP-12 +/+ mice demonstrated increased whole-lung tumor necrosis factor-alpha (TNF-alpha) protein or release of TNF-alpha from cultured alveolar macrophages exposed to smoke in vitro. Levels of whole-lung E-selectin, an endothelial activation marker, were increased in only MMP-12 +/+ mice. These findings suggest that, acutely, MMP-12 mediates smoke-induced inflammation by releasing TNF-alpha from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil-derived proteases. TNF-alpha release may be a general mechanism whereby metalloproteases drive cigarette smoke-induced inflammation.

Mice lacking tumor necrosis factor-alpha (TNF-alpha) receptors (TNFRKO mice) do not develop an inflammatory infiltrate or matrix breakdown after a single acute cigarette smoke exposure. To determine the role of TNF-alpha in the long-term development of emphysema, mice were exposed to smoke for 6 months. TNFRKO mice demonstrated an 11% increase in mean linear intercept; wild-type mice had a 38% increase. TNFRKO mice had 65% fewer neutrophils and no increase in macrophages in lavage fluid. Whole lung matrix metalloprotease (MMP)-2, MMP-9, MMP-12, MMP-13, and matrix type-1 (MT1)-MMP proteins were increased in wild-type mice, but smaller increases in MMP-12, MMP-13, and MT1-MMP were also seen in TNFRKO mice. Lavage matrix breakdown products were elevated in wild-type mice and only partially reduced by anti-neutrophil antibody, implying both neutrophil- and non-neutrophil-mediated matrix breakdown. We conclude that TNF-alpha-mediated processes, probably driving neutrophil influx, are responsible for approximately 70% of airspace enlargement and the majority of inflammatory cell influx/matrix breakdown in the mouse model. TNF-alpha causes increased MMP production, but some increased MMP activity is present even in TNFRKO mice. These findings imply a second TNF-alpha-independent process, possibly related to direct MMP attack on matrix, that produces the remaining 30% of airspace enlargement.

We have previously observed that mice exposed to cigarette smoke and treated with exogenous alpha(1)-antitrypsin (A1AT) were protected against the development of emphysema and against smoke-induced increases in serum TNF-alpha. To investigate possible mechanisms behind this latter observation, we cultured alveolar macrophages lavaged from C57 mice. Smoke-conditioned medium caused alveolar macrophages to increase secretion of macrophage metalloelastase (MMP-12) and TNF-alpha, and this effect was suppressed in a dose-response fashion by addition of A1AT. Macrophages from animals exposed to smoke in vivo and then lavaged also failed to increase MMP-12 and TNF-alpha secretion when the animals were pretreated with A1AT. Because proteinase activated receptor-1 (PAR-1) is known to control MMP-12 release, macrophages were treated with the G protein-coupled receptor inhibitor, pertussis toxin; this suppressed both TNF-alpha and MMP-12 release, while a PAR-1 agonist (TRAP) increased TNF-alpha and MMP-12 release. Smoke-conditioned medium caused increased release of the prothrombin activator, tissue factor, from macrophages. Hirudin, a thrombin inhibitor, and aprotinin, an inhibitor of plasmin, reduced smoke-mediated TNF-alpha and MMP-12 release, and A1AT inhibited both plasmin and thrombin activity in a cell-free functional assay. These findings extend our previous suggestion that TNF-alpha production by alveolar macrophages is related to MMP-12 secretion. They also suggest that A1AT can inhibit thrombin and plasmin in blood constituents that leak into the lung after smoke exposure, thereby preventing PAR-1 activation and MMP-12/TNF-alpha release, and decreasing smoke-mediated inflammatory cell influx.