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.

Serine elastase inhibitors have been proposed as a treatment for cigarette smoke-induced emphysema, but little is known about whether such agents actually are effective. We recently reported that a synthetic serine elastase inhibitor, ZD0892, provided some protection against emphysema in a guinea pig model. For these experiments, we used transgenic mice that expressed extremely low levels of human alpha-1-antitrypsin (A1AT) but were tolerant of exogenous human A1AT. Mice were exposed to daily cigarette smoke for up to 6 months; some animals received 20 mg of human A1AT (Prolastin) every 48 hours. Treatment with A1AT produced an approximate twofold increase in serum A1AT levels and elastase inhibitory capacity and abolished smoke-induced elevations in lavage neutrophils and matrix breakdown products (desmosine and hydroxyproline) measured from 2 to 30 days of smoke exposure. A1AT oxidized to remove antiproteolytic activity did not increase serum elastase inhibitory capacity but did prevent neutrophil influx. Treatment with A1AT for 6 months provided 63% protection against increased airspace size (emphysema) and abolished smoke-mediated increases in plasma tumor necrosis factor-alpha. We conclude that A1AT therapy ameliorates smoke-induced inflammation and matrix breakdown, possibly via an antiinflammatory mechanism related to tumor necrosis factor-alpha suppression, and provides partial protection against emphysema.

Small airway remodeling (SAR) is an important cause of airflow obstruction in cigarette smokers, but whether SAR represents a response to smoke-evoked inflammation or is directly mediated by smoke-induced growth factor production is disputed. To examine this process, we exposed rat tracheal explants, a model free of exogenous inflammatory cells, to cigarette smoke in vitro. Cigarette smoke caused release of active transforming growth factor (TGF)-beta1, and this was prevented by the oxidant scavenger tetramethythiourea. Nuclear immunostaining for phospho-Smad2, a TGF-beta downstream signaling molecule, was present in epithelial and interstitial cells within 1 h after exposure. Smoke caused upregulation of gene expression of connective tissue growth factor (CTGF), a mediator of TGF-beta fibrogenic effects, within 2 h, and upregulation of procollagen gene expression at 24 h; both changes could be prevented by the TGF-beta antagonist fetuin (alpha2-HS-glycoprotein). In a cell-free system, recombinant human TGF-beta latency-associated peptide was oxidized by cigarette smoke, and smoke released active TGF-beta1 from recombinant latent TGF-beta1 via an oxidant mechanism. These experiments suggest that SAR in cigarette smokers may be caused by direct, smoke-mediated, oxidant-driven induction of growth factor signaling in the airway wall, and that SAR does not necessarily require exogenous inflammatory cells.

No abstract

Small airway remodeling ("small airways disease") is a common finding in cigarette smokers and is an important cause of airflow obstruction. Airway remodeling is usually attributed to the effects of cigarette smoke-induced inflammation in the airway wall, but little is actually known about its pathogenesis. We exposed rat tracheal explants to cigarette smoke and then maintained them in air organ culture. At 24 hours after smoke exposure, there was a dose-dependent increase in gene expression of procollagen and a significant increase in tissue hydroxyproline, a measure of collagen content. Greater increases in procollagen gene expression were found with repeated smoke exposures. Increased procollagen gene expression could be prevented with SN50, a selective inhibitor of nuclear factor-kappaB activation, and superoxide dismutase, catalase, and tetramethylthiourea, scavengers of active oxygen species. AG1478, an inhibitor of epidermal growth factor receptor signaling, also prevented increased procollagen gene expression, but PD98059 and SB203580, inhibitors of mitogen-activated protein kinases, did not. These findings indicate that cigarette smoke can directly induce airway remodeling, specifically airway wall fibrosis, probably through active oxygen species-dependent transactivation of the epidermal growth factor receptor and subsequent nuclear factor-kappaB activation. Smoke-evoked inflammatory cells are not required for this process.

Hogg: Bronchiolitis in COPD 49332. Ingram JL, Rice AB, Geisenhoffer K, Madtes DK, Bonner JC. and IL-1beta promote lung fibroblast growth through coordinated upregulation of PDGF-AA and PDGF-Ralpha. FASEB J 2004;18:1132-1134. Cigarette Smoke Causes Small Airway Remodeling by Direct Growth Factor Induction and ReleaseAndrew Churg, Rong D. Wang, and Joanne L. WrightDepartment of Pathology, University of British Columbia, Vancouver, British Columbia, CanadaSmall airway remodeling (SAR) is one of the anatomic causes of airflow obstruction in patients with chronic obstructive pulmonary disease. The pathogenesis of SAR is poorly understood, but there are two general theories: (1 ) SAR is caused by smokeinduced inflammatory cells that damage the airway and evoke a repair process and (2 ) SAR is caused by direct induction of growth factors by cigarette smoke. To investigate this process, we briefly exposed rat tracheal explants, a model system free of exogenous inflammatory cells, to whole cigarette smoke, and then maintained the explants in organ culture for varying periods. With explants cultured in air, smoke induced up-regulation of both transforming growth factor (TGF)-␤ 1 and procollagen gene expression at 24 h after initial exposure. Increased procollagen gene expression was prevented in a dose-response fashion by the TGF-␤ antagonist fetuin, indicating that TGF-␤ was driving fibrosis. Collected supernatant from explants exposed to smoke and cultured in fluid medium showed increased release of TGF-␤ 1, and this was abolished by the oxidant scavenger tetramethylthiourea. Gene expression of connective tissue growth factor (CTGF) was sharply increased 2 h after smoke exposure. Using pure recombinant proteins, smoke-conditioned medium oxidized TGF-␤ latency-associated peptide and also caused release of active TGF-␤ 1 from latent TGF-␤ 1 . We conclude that cigarette smoke directly induces release of preformed TGF-␤ 1 by oxidizing the latency-associated peptide in rat tracheal explants, and that release must be extremely rapid, since gene expression of CTGF, the downstream fibrogenic driver of TGF-␤ effects on collagen synthesis, is elevated very shortly after smoke exposure. Although tracheal explants are only an approximate model of small airways, these findings suggest that smoke-induced SAR may reflect direct induction of growth factor release and signaling and does not require smoke-evoked inflammatory cells. Conflict of Interest Statement :None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. We have reported previously that MARCKS protein is a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo, and presented evidence supporting a mechanism whereby MARCKS regulates secretion by translocating from plasma membrane to cytoplasm, where it binds to membranes of intracellular mucin granules (1, 2). The actual mechanism whereby MARCKS is targeted to and attaches onto membranes of mucin granules ha...