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Published ahead of print on March 29, 2007, doi:10.1165/rcmb.2006-0345OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 144-151, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0345OC

{alpha}1-Antitrypsin Suppresses TNF-{alpha} and MMP-12 Production by Cigarette Smoke–Stimulated Macrophages

Andrew Churg, Xiaoshan Wang, Rong D. Wang, Scott C. Meixner, Edward L. G. Pryzdial and Joanne L. Wright

Department of Pathology, and Canadian Blood Services, Research and Development Department, Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada

Correspondence and requests for reprints should be addressed to Andrew Churg, M.D., Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, V6T 2B5 Canada. E-mail: achurg{at}interchange.ubc.ca


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
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.

Key Words: cigarette smoke • emphysema • {alpha}1-antitrypsin • MMP-12 • thrombin



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This article shows that {alpha}1-antitrypsin can suppress cigarette smoke–induced production of TNF-{alpha} and MMP-12 by alveolar macrophages, a novel mechanism that will lead to suppression of TNF-{alpha}–mediated inflammation and thus prevent emphysema.

 
The pathogenesis of cigarette smoke–induced emphysema is generally believed to involve a smoked-mediated influx of inflammatory cells into the lower respiratory tract, and release by those cells of proteases that overwhelm lower respiratory tract anti-proteolytic defenses and cause destruction of the alveolar wall matrix (1, 2).

The exact cells and proteases that are important in this process are a matter of dispute. Mice lacking macrophage metalloelastase (MMP-12) are completely protected against emphysema (3), and we have suggested that one role of MMP-12 in this setting is to release active TNF-{alpha} from latent TNF-{alpha}, leading to an influx of neutrophils that secrete neutrophil elastase and other proteases that are the major actors in matrix degradation (see Ref. 4, and DISCUSSION below). The importance of neutrophils in smoke-induced matrix destruction has been confirmed by the observation that mice with targeted deletion of the neutrophil elastase gene are ~ 60% protected against emphysema (5). Further, we have shown that exogenous administration of {alpha}1-antitrypsin (A1AT), a serine elastase inhibitor generally believed to be the major antiproteolytic agent in the lower respiratory tract, provided significant protection against emphysema in a mouse model (6). Consistent with this observation, Cavarra and coworkers (7) and Takubo and colleagues (8) have shown that mice genetically deficient in A1AT (pallid mice) develop emphysema more rapidly than wild-type mice.

While these findings support a role for neutrophil-derived serine proteases and their inhibitors in the pathogenesis of emphysema, a curious finding in our model of exogenous A1AT administration was that this serine elastase inhibitor also suppressed smoke-mediated increases in serum TNF-{alpha} and decreased inflammatory cell influx into the lung (6). These observations lead us to ask whether the mechanism of protection by A1AT might be related not only to direct inhibition of neutrophil elastase activity, but might also proceed via an anti-inflammatory effect that prevents TNF-{alpha} release and subsequent inflammatory cell infiltration. In this paper we test this hypothesis and show that A1AT does in fact suppress both MMP-12 and TNF-{alpha} release from alveolar macrophages.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Macrophage Culture and Smoke Exposure In Vitro
Untreated C57Bl/6 mice (Charles River, Montreal, PQ, Canada) were killed by CO2 inhalation, and a 20-gauge catheter was inserted into the trachea and the lungs lavaged six times with 1 ml of ice-cold saline. The saline lavage was centrifuged at 200 x g at 4°C for 2 min. The supernatants were decanted, the numbers of cells in the pellets counted, and the wells of 96-well plates filled with samples containing 4 x 105 alveolar macrophages. Each well was filled with macrophages from one mouse. The cells were allowed to adhere for 2 h. After 2 h the nonadherent cells were removed along with the supernatant, and one of the following was added: (1) 250 µl/well of RPMI 1640 (Cat no 12385–019; Gibco, Burlington, ON, Canada) culture medium (control); (2) 250 µl/well smoke-conditioned RPMI 1640 (stock solution 20 ml RPMI 1640 through which had been freshly bubbled the whole smoke of six 2R1 cigarettes; the solution was then filtered through a 0.2 µM filter to remove particulates). To assay the protective effect of A1AT, A1AT (Prolastin; Bayer, Toronto, ON, Canada) in RPMI 1640 was added to the macrophages 2 h before they were exposed to smoke-conditioned medium, and A1AT was also added to the smoke-conditioned medium. The timing of the experiments was adjusted so that all macrophages for all treatments had the same length of exposure to RPMI. For individual experiments, Western blots and casein zymography (below) used cells from two or three animals; TNF-{alpha} experiments used cells from three animals per experiment. All experiments were repeated at least once, and representative data are shown.

To ensure that any effects seen were not simply nonspecific protein-mediated effects, albumin, a molecule of molecular weight similar to A1AT, was used. To examine non–anti-protease effects of A1AT, A1AT was oxidized with hydrogen peroxide as described by us in (9). Similarly, A1AT was polymerized by boiling for 3 h at 60°C as described in (10), and the oxidized or polymerized molecules used as above.

The 96-well plates were then incubated for 18 h at 37°C in an air/6% CO2 incubator and the supernatants collected for TNF-{alpha} and MMP-12 assays. Trypan blue exclusion was used to assess cell viability and showed greater than 95% viability for all treatments at 18 h.

In Vivo Smoke Exposure
C57Bl/6 mice were exposed to air (control) or the smoke of four University of Kentucky 2R1 Research Cigarettes as previously described (6). Some animals were given 20 mg of A1AT by intraperitoneal injection 24 h before smoke exposure (11). At 2 h after starting smoke exposure, alveolar macrophages were lavaged and cultured in RPMI 1640 medium and supernatants collected over 18 h for TNF-{alpha} and MMP-12 assays. The animal studies were approved by the University of British Columbia Animal Care Committee.

TNF-{alpha} Assay
Supernatant TNF-{alpha} was assayed using the L929 cell assay as described by us previously (4).

Supernatant MMP-12
Macrophage supernatant MMP-12 was assessed primarily by Western blot using goat anti-mouse MMP-12 antibody (SC8839; Santa Cruz Biotechnologies, Santa Cruz, CA). Fifty microliters of supernatant was mixed with 10 µl of 6x loading buffer and 5 µl DTT, and then heated to 95°C for 3 min, cooled to 4°C for 1 min, and loaded onto polyacrylamide gels. The samples were separated in a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated in tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% Bio-Rad (Hercules, CA) dry milk powder for 30–60 min at room temperature. The membranes were washed three times and then incubated in 1:200 primary antibody against MMP-12 in TBS with 0.05% Tween-20 and 5% Bio-Rad dry milk powder overnight. The membranes were subsequently washed three times in TBS with 0.05% Tween-20 and incubated in a 1:2,000 dilution secondary antibody of donkey anti-goat IgG (Santa Cruz Biotechnology) for 45 min. The membranes were washed three times again and then incubated in Chemiluminescence Reagent (Cat No. RPN2106; Amersham Biosciences, Piscataway, NJ) for detection, and densitometry performed.

Since this is the only commercial antibody we could find that detects mouse MMP-12, and since it only detects the 54-kD pro-form, we also did casein zymography to look for the active form of the enzyme and to show that A1AT does not affect conversion of the pro-form to the active form of the enzyme. For casein zymography, the sample was mixed with sample buffer (50 mM Tris-HCl, 0.1 M NaCl, 10 mM CaCl2, 0.1% Brij 35) and incubated at 55°C overnight for enzyme activation. A 12% SDS-polyacrylamide gel with bovine-casein (Sigma-Aldrich, Oakville, ON, Canada) was prepared and prerun in 1x running buffer (125 mM Tris-HCl, 1.23M glycine, 0.5%SDS) with nonreducing sample buffer. The sample was then loaded with nonreducing sample buffer and run at 4°C. The gel was washed with 50 mM Tris-HCl(pH7.5) +2.5%Triton X-100 twice for 30 min and twice for 10 min with Tris-HCl buffer only, and then incubated overnight at 37°C with incubation buffer (50 mM Tris-HCl 6.05 g, 0.15 M NaCl 8.70 g, 10 mM CaCl2 1.47 g, 0.1% Triton X-100 1 ml, 0.02% NaN3 0.2 g). Finally the gel was stained with Comassie Blue. MMP-12 activity was assessed by densitometry of the 22-kD cleared band.

Modification of Proteinase-Activated Receptor-1 Activity
The G protein–coupled receptor inhibitor pertussis toxin (PTX; Sigma), which will inhibit proteinase-activated receptor-1 (PAR-1), was added to the macrophages 2 h before exposure to untreated or smoke-conditioned culture medium at a concentration of 1 mg/ml, and then was included in the medium during the 18-h collection period. To drive PAR-1 activity, the PAR-1 agonist, TRAP (SFLLRNPNDKYEPF; Sigma) was added to the macrophage culture medium for the 18-h collection period at a concentration of 100 nM.

Inhibition of Thrombin and Plasmin Activity
The thrombin inhibitor, hirudin, was added to the macrophages 2 h in advance, and also included in the medium during supernatant collection. Aprotinin (Sigma), a plasmin inhibitor, was added to the macrophages 2 h in advance and also included in the medium during supernatant collection.

Cell-Free Plasmin and Thrombin Functional Assay
These were performed as described in refs (12, 13). Briefly, 40 nM human plasmin or 16 nM human thrombin (both from Haematologic Technologies, Inc., Essex Junction, VT) in Hepes-buffered saline was combined with dilutions of A1AT in a 96-well plate. Two hundred microliters of 160 nM pyroGlu-Pro-Arg-p-nitroanalide (S2366, plasmin substrate) or H-D-Phe-Pip-Arg-p-nitroanalide (S2238, thrombin substrate, both from DiaPharma, West Chester, OH) diluted in Hepes-buffered saline containing 100 mM EDTA and 0.1% polyethylene glycol 8000 (Sigma-Aldrich) was added to each well. Color development was followed at 405 nm using a kinetic multiwell plate reader (SpectraMax190; Molecular Devices, Sunnyvale, CA).

Western Blot Procedures for Tissue Factor
Procedures similar to those used for MMP-12 were performed with primary rabbit anti-mouse tissue factor antibody (American Diagnostics, Stamford, CA) diluted 1:200 and goat anti-rabbit antibody diluted 1:2,000 as the secondary antibody.


   RESULTS
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Figure 1 shows the effects of A1AT on macrophage TNF-{alpha} and MMP-12 release. Exposure to smoke-conditioned medium increased TNF-{alpha} release by more than 2-fold and A1AT prevented TNF-{alpha} release in a dose–response fashion. Smoke-conditioned medium caused a parallel increase in 54-kD pro–MMP-12 release and this effect was also abolished by A1AT. Because the available antibody only detects pro-MMP-12, casein zymography was performed on macrophage supernatants to determine whether A1AT independently affected production of active (22 kD) enzyme. As shown in Figure 1C, the pattern of active enzyme concentration was similar to the concentration of the pro-form shown in Figure 1, suggesting that, while A1AT prevents secretion of pro–MMP-12, it does not additionally affect conversion of the pro-form to the active form.


Figure 1
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  		Figure 1. Effects of in vitro exposure to A1AT. Alveolar macrophage supernatant TNF-{alpha} (A) and MMP-12 (Western blot for the 54-kD pro-form) (B) after in vitro exposure to smoke-conditioned medium and A1AT. A1AT decreases smoke-mediated increases in TNF-{alpha} and MMP-12 release in a dose–response and parallel fashion. (C) Casein zymography of macrophage supernatant for active MMP-12 after exposure to smoke-conditioned medium and A1AT. A1AT decreases active MMP-12 production and the pattern of reduction is similar to that for the pro-form of MMP-12 shown in Figure 1B, indicating that A1AT does not affect conversion of the pro-form to the active form. A1–100 = A1AT 100 µg/ml; A1–1mg = A1AT 1 mg/ml. Values are mean ± SD, *P < 0.05 compared with control.

 
To show that the effects of A1AT were not just caused by protein, we ran a similar experiment with albumin; as shown in Figure 2, albumin did not prevent either MMP-12 or TNF-{alpha} release. To determine whether the effects of A1AT required an active protease inhibitory site, experiments similar to those in Figure 1 were run using either oxidized or polymerized A1AT, but neither of these modified proteins was protective (data not shown).


Figure 2
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  		Figure 2. Effects of albumin. (A) Alveolar macrophage supernatant TNF-{alpha} and (B) MMP-12 after in vitro exposure to smoke-conditioned medium and A1AT or albumin (Alb). A1AT protects against smoke-mediated increases in TNF-{alpha} and MMP-12 release, whereas albumin is not protective, indicating that the changes seen are not simply nonspecific protein effects. Values are mean ± SD, *P < 0.05 compared with control.

 
Figure 3 shows the effects of A1AT when mice were exposed to smoke and the A1AT administered in vivo, and the lavaged alveolar macrophages simply cultured without exposure to smoke-conditioned medium. In vivo smoke exposure produced an increased release of TNF-{alpha} and MMP-12, and A1AT administered in vivo prevented these increases.


Figure 3
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  		Figure 3. Effects of in vivo treatment with A1AT. Alveolar macrophage supernatant TNF-{alpha} (A) and MMP-12 (B) after treatment with A1AT and exposure to cigarette smoke, both in vivo. A1AT treatment completely abolishes smoke-mediated increases in TNF-{alpha} and MMP-12 production. Values are mean ± SD, *P < 0.05 compared with control.

 
Because secretion of MMP-12 from alveolar macrophages is known to be mediated through proteinase activated receptor-1 (PAR-1) (see Ref. 14, and DISCUSSION), macrophages were treated with the G protein–coupled receptor inhibitor, pertussis toxin (PTX, Figures 4A and 4B) or the PAR-1 agonist, TRAP (Figures 4C and 4D). PTX partially inhibited release of both TNF-{alpha} and MMP-12. TRAP caused increased release of both TNF-{alpha} and MMP-12.


Figure 4
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  		Figure 4. (A, B) Effects of the G protein–coupled receptor inhibitor, pertussis toxin (PTX) on macrophage TNF-{alpha} (A) and MMP-12 production (B). PTX partially inhibits release of both mediators. (C, D) Effects of the PAR-1 agonist, TRAP, on macrophage TNF-{alpha} (C) and MMP-12 production (D). TRAP increases release of both mediators Values are mean ± SD, *P < 0.05 compared with control.

 
Thrombin and plasmin are known to activate PAR-1 and lead to MMP-12 release (14), and cigarette smoke has been shown to cause increased alveolar permeability with leakage of blood proteins into the lung (15, 16). To investigate the possibility that thrombin or plasmin activity generated as a consequence of blood protein leakage after smoke exposure may be involved in secretion of TNF-{alpha} and MMP-12, hirudin, a specific inhibitor of thrombin, or aprotonin, a serine protease inhibitor with high affinity for plasmin (Kd = 0.2 nM), was added to the macrophages. Both hirudin (Figures 5A and 5B) and aproptinin (Figures 5C and 5D) provided protection against smoke-mediated TNF-{alpha} and MMP-12 release.


Figure 5
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  		Figure 5. (A, B) Effects of the thrombin inhibitor, hirudin, on macrophage TNF-{alpha} (A) and MMP-12 release (B). Hirudin blocks TNF-{alpha} release and blocks MMP-12 release. (C, D) Effects of the plasmin inhibitor, aprotinin, on macrophage TNF-{alpha} (C) and MMP-12 release (D). Aprotinin (Apro 1 or 10 µg/ml) partially blocks release of both mediators in a dose response fashion. Values are mean ± SD, *P < 0.05 compared with control.

 
To confirm that A1AT could inhibit thrombin and plasmin, a chromogenic activity assay using purified thrombin and plasmin was conducted (Figure 6). A1AT inhibited the activity of these proteases in a dose–response fashion, supporting the idea that A1AT-mediated inhibition of thrombin and plasmin could be playing a role in preventing smoke-induced macrophage release of TNF-{alpha} and MMP-12.


Figure 6
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  		Figure 6. Effects of A1AT on plasmin (A) and thrombin (B) activity in a cell-free assay. A1AT inhibits both proteases in a dose–response fashion. Data from three separate preparations. Values are mean ± SD, *P < 0.05 compared with control (no A1AT).

 
To determine whether increased thrombin activity might result from increased macrophage production of the coagulation initiator, tissue factor, tissue factor was assayed in the macrophage supernatant by Western blot. Smoke-conditioned medium caused an increase in release of tissue factor from alveolar macrophages (Figure 7).


Figure 7
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  		Figure 7. Effects of smoke-conditioned medium on release of the thrombin activator, tissue factor (Western blot). Smoke-conditioned medium increases release of tissue factor from alveolar macrophages. Values are mean ± SD, *P < 0.05 compared with control.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
A1AT is generally viewed as an antiprotease with a primary function in the lower respiratory tract of inhibiting inflammatory cell-derived serine proteases such as neutrophil elastase. While A1AT certainly can function in this "classical" fashion, there is increasing data that A1AT can also function in a wide variety of other ways (reviewed in 10). Of particular interest is evidence that A1AT can act in some contexts as an anti-inflammatory agent. For example, Janciauskiene and coworkers (10) and Nita and colleagues (17) have shown that A1AT inhibits LPS-mediated increases in TNF-{alpha} and IL-1beta release from human blood monocytes. Similarly, administration of A1AT to patients with A1AT deficiency not only decreased sputum elastase activity, but also decreased sputum levels of leukotriene B4, an inflammatory cell chemoattractant (18). As noted, we observed that administration of A1AT to mice markedly decreased smoke-mediated increases in lavage inflammatory cells (6) and we previously found that A1AT also inhibited silica-induced inflammation (9). Petrache and coworkers (19, 20) recently reported that A1AT has an anti-apoptotic function that appears to be driven by direct inhibition of caspace-3; apart from preventing airspace enlargement secondary to endothelial cell loss, this function could have an anti-inflammatory effect in the context of smoke-induced emphysema, where it has been proposed that apoptotic cells are not cleared properly and instead evoke an inflammatory response (21).

Our current results add another yet another new observation to the list of A1AT effects, since we show that A1AT can inhibit both thrombin and plasmin (see below), and by doing so prevent release of MMP-12 and TNF-{alpha}. Here A1AT functions as a serine protease inhibitor and the process requires an intact A1AT molecule, since oxidized and polymerized A1AT are ineffective. However, the end result in this particular context is that A1AT acts as an anti-inflammatory agent and modulates the development of smoke-induced emphysema in an unexpected fashion.

It is necessary to ask whether the concentrations of A1AT used here in vitro are realistic ones in terms of normal alveolar lining fluid concentrations or concentrations that could be achieved by A1AT infusion. The most effective in vitro concentration of 1 mg/ml is less than the normal plasma concentration of A1AT, and thus is probably achievable locally (i.e., microscopically) in areas of cigarette smoke–induced leakage of plasma (15, 16) into the lung. Fairly similar concentrations have been shown to inhibit LPS-induced TNF-{alpha} and IL-1beta release (10). The strongest evidence for the in vivo relevance of our observations is the finding that alveolar macrophages lavaged from mice given A1AT in vivo fail to release excess MMP-12 and TNF-{alpha} when the dose of A1AT used is one that, as reported previously (6), acutely suppresses smoke-induced increases in inflammation and serum TNF-{alpha} levels, and that over the long term ameliorates the development of emphysema in cigarette smoke–exposed mice.

As noted earlier in this article, mice lacking MMP12 (MMP12–/–) do not develop emphysema after cigarette smoke exposure (3), and in a previous study (4), we showed that alveolar macrophages from such mice failed to increase supernatant TNF-{alpha} release after cigarette smoke exposure, as opposed to macrophages from wild-type mice, and similarly, that, in vivo, there was no up-regulation of whole lung TNF-{alpha} in the MMP-12–/– mice after cigarette smoke exposure. When exposed to cigarette smoke, wild-type mice up-regulated whole lung e-selectin, an adhesion molecule that is a downstream target of TNF-{alpha} and is a mediator of inflammatory cell tethering to endothelial cells and subsequent migration into the lung (22), whereas MMP-12–/– mice did not up-regulate e-selectin, and these mice failed to increase lavage inflammatory cells. Based on these observations, we proposed that MMP-12 can function as a TNF-{alpha}–converting enzyme, liberating active TNF-{alpha} from the pro-form, a process that has been demonstrated with MMP-12 and synthetic fusion proteins (23) and that can be seen with other MMPs as well (24). Under this hypothesis, liberation of TNF-{alpha} then evokes an influx of inflammatory cells, and in particular neutrophils that release neutrophil elastase, leading to matrix breakdown and eventual emphysema. The importance of TNF-{alpha} as a mediator of emphysema, at least in the mouse model, was confirmed by using mice lacking TNF-{alpha} receptors; these animals were found to be ~ 70% protected against smoke-mediated airspace enlargement (11).

Our present results further support the linkage between MMP-12 and TNF-{alpha}. There is a clear and parallel correlation between suppression of MMP-12 release and suppression of TNF-{alpha} release; thus A1AT, inhibition of G protein–coupled receptors (here PAR-1) with pertussis toxin, inhibition of thrombin with hirudin, and inhibition of plasmin by aprotinin, each decrease macrophage MMP-12 and TNF-{alpha} production. Conversely, when MMP-12 secretion was increased by stimulating PAR-1, TNF-{alpha} secretion was also increased. These results are not just confined to cell cultures exposed to smoke-conditioned medium, since we observed that in vivo smoke exposure and in vivo treatment with A1AT suppressed smoke-induced increases in release of MMP-12 and TNF-{alpha} from lavaged alveolar macrophages.

These findings are of potential importance, because they lend further support to the idea that interfering with the secretion or function of MMP-12 will down-regulate TNF-{alpha} and therefore smoke-induced inflammation. We have found that this is indeed the case in guinea pigs exposed to smoke and treated with an MMP-9/MMP-12 inhibitor; in those animals the inhibitor prevented both increases in lavage inflammatory cells and also increases in serum TNF-{alpha}, and there was a strong correlation of serum TNF-{alpha} levels with airspace size (25). It is interesting in this regard that Warner and colleagues (26), using a model of cockroach antigen–induced allergic response, also found that MMP-12–/– mice had a significantly reduced inflammatory cell response and decreased levels of a number of inflammatory mediators including TNF-{alpha}.

The proteinase activated receptors are activated in an unusual fashion by proteinase-mediated cleavage of their extracellular N-terminus, creating a new terminal sequence that functions as a tethered ligand to activate the receptor (27). Thrombin and plasmin are well-established serine protease activators of PAR-1, and activation can also be achieved with synthetic peptides such as TRAP that mimic the tethered ligand. Raza and coworkers (14) previously reported that PAR-1 activation drives MMP-12 release from macrophages; they found that plasmin/plasminogen and thrombin both caused increased secretion of MMP-12 by a post-translational mechanism that involved protein kinase C but not tyrosine kinase. PAR-1 is a G protein–coupled receptor, and Raza and colleagues showed that, similar to our present results, MMP-12 secretion could be inhibited by pertussis toxin, an inhibitor of G protein–coupled receptors, or by hirudin, a selective thrombin inhibitor, and could be stimulated by synthetic PAR-1 agonists.

The Raza data prompted us to look at plasmin and thrombin as possible targets of A1AT. Although A1AT is not believed to have a significant inhibitory effect on plasmin or thrombin activity in the context of other physiologic inhibitors in blood, in isolated systems A1AT has been reported to inhibit both of these serine proteases (28, 29) (association rate constants: thrombin 4.8 x 101, plasmin 1.9 x 102, neutrophil elastase 6.5 x 107 M-1 s-1 [29]), and we confirmed this in a cell-free system (Figure 6). Our observations that both hirudin, a very selective inhibitor of thrombin, and aprotinin, a relatively selective inhibitor of plasmin (30), decrease MMP-12 and TNF-{alpha} production, support a role for thrombin and plasmin in MMP-12 and TNF-{alpha} release.

In our cell culture model, 5% fetal calf serum is added to the culture medium, and this provides a source of plasminogen and prothrombin. Since our inhibitory results with hirudin strongly imply a thrombin effect, we propose that serum-derived prothrombin is likely being activated during the experiment. To generate thrombin, other coagulation enzymes must be activated. Factors Xa and VIIa are present in serum and are known to activate prothrombin. Macrophages produce tissue factor (31, 32), and Factors Xa and VIIa can themselves be activated on the surface of macrophages by tissue factor (33, 34). As indicated in Figure 7, tissue factor is produced at a basal level by unstimulated macrophages, and this provides one explanation for the PAR-1–mediated effects seen in control (non–smoke-exposed) macrophages. In addition, alveolar macrophages produce plasminogen activator (35), which can explain the generation of active plasmin from plasminogen and the inhibitory effects of aprotinin in our system, as well as providing another mechanism for basal PAR-1 activation.

The relevance of adding serum to our culture model relates to the long-standing observation that, in vivo, cigarette smoke rapidly causes increased lung capillary permeability, with leakage of a variety of blood proteins into the alveolar spaces (15, 16). Cigarette smoke has been shown to increase tissue factor release from macrophages in atherosclerotic plaques (31, 32), and alveolar macrophages from patients with COPD have been reported to produce considerably more plasminogen activator than those from smokers without COPD (35). Here we observed that cigarette smoke also increased tissue factor release from our cultured alveolar macrophages. Thus we propose that, in vivo, plasminogen and prothrombin leak into the airspaces after smoke exposure and are converted to their active forms by mechanisms involving macrophage-derived plasminogen activator and tissue factor.

In summary, our data reinforce the idea that MMP-12 and TNF-{alpha} secretion are closely linked in alveolar macrophages, and suggest that one mechanism of MMP-12 production is driven through activation of PAR-1 by serum constituents that leak into the smoke-exposed lung. A1AT prevents these effects by inhibiting thrombin and plasmin, and thus functions here as an anti-inflammatory agent.


   Footnotes
 
This study was supported by grant MOP 42539 from the Canadian Institutes of Health Research.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0345OC on March 29, 2007

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.

Received in original form September 11, 2006

Accepted in final form March 20, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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