Alpha-1-antitrypsin and a broad spectrum metalloprotease inhibitor, RS113456, have similar acute anti-inflammatory effects.

There is increasing evidence that antiproteases are able to affect the inflammatory response. To further examine this question, we administered human alpha-1-antitrypsin (alpha1AT) or a synthetic metalloprotease inhibitor (RS113456) to C57 mice followed by a single intratracheal dose of quartz, a dust that evokes a marked, lasting, polymorphonuclear leukocyte (PMN) infiltrate. At 2 hours after dust administration, both antiproteases completely suppressed silica-induced PMN influx into the lung and macrophage inflammatory protein-2 (MIP-2)/monocyte chemotactic protein-1 (MCP-1) (neutrophil/macrophage chemoattractant) gene expression, partially suppressed nuclear transcription factor kappaB (NF-kappaB) translocation, and increased inhibitor of NF-kappaB (IkappaB) levels. By 24 hours, PMN influx and connective tissue breakdown measured as lavage desmosine or hydroxyproline were still at, or close to, control levels after antiprotease treatment, and increases in NF-kappaB translocation and MIP-2/MCP-1 gene expression were variably suppressed. At both time points, neither agent prevented silica-induced increases in amount of whole lung MIP-2 or MCP-1 protein, but both did prevent increases in whole lung intercellular adhesion molecule-1 (ICAM-1) at 24 hours. Inactivating the alpha1AT by oxidation to the point that it no longer possessed antiproteolytic properties did not affect its ability to suppress inflammation. Both antiproteases also prevented the silica-induced acute inflammatory response in mice with knocked out genes for macrophage metalloelastase (MME -/-), mice that develop inflammation, but not connective tissue breakdown, and the pattern of alpha1AT breakdown fragments was identical in control and MME -/- animals. These findings suggest that, in this model of acute PMN mediated inflammation, a serine protease inhibitor and a metalloprotease inhibitor have similar anti-inflammatory properties, that inflammation is not mediated by proteolysis with generation of chemotactic matrix fragments, and that classic antiproteolysis (complexing of protease to antiprotease) probably does not play a role in suppression of inflammation. The antiproteolytic effects of these agents do not seem to be mediated by protection of endogenous alpha1AT.

TNF-alpha increases tracheal epithelial asbestos and fiberglass binding via a NF-kappaB-dependent mechanism.

Tumor necrosis factor (TNF)-alpha is released from alveolar macrophages after phagocytosis of mineral fibers. To determine whether TNF-alpha affects the binding of fibers to epithelial cells, we exposed rat tracheal explants to TNF-alpha or to culture medium alone, followed by a suspension of amosite asbestos or fiberglass (MMVF10). Loosely adherent fibers were removed from the surface with a standardized washing technique, and the number of bound fibers was determined by scanning electron microscopy. Increasing doses of TNF-alpha produced increases in fiber binding. This effect was abolished by an anti-TNF-alpha antibody, the proteasome inhibitor MG-132, and the nuclear factor (NF)-kappaB inhibitor pyrrolidine dithiocarbamate. Gel shift and Western blot analyses confirmed that TNF-alpha activated NF-kappaB and depleted IkappaB in this system and that these effects were prevented by MG-132 and pyrrolidine dithiocarbamate. These observations indicate that TNF-alpha increases epithelial fiber binding by a NF-kappaB-dependent mechanism. They also suggest that mineral particles may cause pathological lesions via an autocrine-like process in which the response evoked by particles, for example, macrophage TNF-alpha production, acts to enhance subsequent interactions of particles with tissue.

Acute cigarette smoke-induced connective tissue breakdown is mediated by neutrophils and prevented by alpha1-antitrypsin.

Recent studies have suggested that macrophage-derived metalloproteases are the critical mediators of cigarette smoke-induced emphysema, in contrast to earlier hypotheses that this process was mediated by neutrophil elastase. To determine whether smoke can acutely induce connective tissue breakdown in the lung and to examine the mediators of this process, we exposed C57-BL/6 mice to whole cigarette smoke and used high-performance liquid chromatography to examine lavage fluid levels of desmosine (DES), a marker of elastin breakdown, and hydroxyproline (HP), a marker of collagen breakdown. Smoke produced a dose-response increase in lavage neutrophils, DES, and HP, but not lavage macrophages (MACs). This effect was evident by 6 h after exposure to two cigarettes. Pretreatment with an antibody against polymorphonuclear leukocytes (PMNs) reduced lavage PMNs to undetectable levels after smoke exposure, did not affect MAC numbers, and prevented increases in lavage DES and HP. Intraperitoneal injection of a commercial human alpha1-antitrypsin (alpha1AT) 24 h before smoke exposure increased serum alpha1AT levels approximately 3-fold and completely abolished smoke-induced connective tissue breakdown as well as the increase in lavage PMNs, again without affecting MAC numbers. We conclude that in this model cigarette smoke can acutely induce connective tissue breakdown and that this effect is mediated by neutrophil-derived serine proteases, most likely neutrophil elastase. Exogenous alpha1AT is protective and appears to inhibit both matrix degradation and PMN influx, suggesting that alpha1AT has anti-inflammatory as well as antiproteolytic effects in this system.

Effects of cigarette smoke on nitric oxide synthase expression in the rat lung.

To examine the effects of cigarette smoke on nitric oxide synthase (NOS) gene expression and protein production, groups of rats were exposed to smoke once only or daily and were sacrificed after 1, 2, 7, or 28 days of exposure. NOS-1, NOS-2, and NOS-3 mRNAs in whole lung were quantified using reverse transcription polymerase chain reaction (RT-PCR), and NOS protein levels were determined by Western blots. Neither NOS-1 gene expression nor protein levels changed with smoke exposure. Levels of NOS-2 expression were more than doubled in smokers at Day 1 and decreased to control values during 1 month with daily smoke exposure, while protein levels did not change. NOS-3 expression was increased approximately 35% after 2 days of smoke exposure and remained increased to 28 days, whereas protein levels were increased by approximately 60% at Day 7 and remained elevated. In situ hybridization showed that NOS-2 was diffusely expressed in the lung parenchyma, airways, and vessels, and that NOS-3 was strongly expressed in vascular endothelium. Protein distribution, as determined by immunohistochemical staining, was identical to mRNA tissue distribution, and these distributions were not changed by smoke. We conclude that smoke exposure induces a rapid but transient increase in transcription of NOS-2, and a sustained increase in transcription and translation of NOS-3; up-regulation of NOS occurs within the anatomic compartment where these genes are normally expressed. These findings indicate that cigarette smoke can directly and rapidly affect NOS expression, and thus potentially affect the function of the pulmonary vasculature.

Pulmonary epithelial expression of human alpha1-antitrypsin in transgenic mice results in delivery of alpha1-antitrypsin protein to the interstitium.

Alpha1-antitrypsin (alpha1AT) therapy is used as a treatment for alpha1AT deficiency. It has also been proposed as a therapy for cigarette smoke-induced emphysema, although the efficacy of such therapy is as yet unproven. Moreover, the optimal route of delivery of alpha1AT to the lung interstitium, the crucial locus of action, is unknown. We created transgenic mice with expression of the human alpha1AT gene directed by a human surfactant protein C (SpC) promoter fragment or a rat Clara cell 10-kDa protein (CC10) promoter fragment in order to examine the ability of pulmonary epithelial cell expression of alpha1AT to deliver protein to the interstitium, and to produce a model that would allow studies on the efficacy of alpha1AT in preventing lung damage after cigarette smoke exposure. Four transgenic lines were studied. In situ hybridization and light microscopic immunohistochemistry showed that two CC10 driven lines expressed human alpha1AT in type 11 alveolar cells and airway epithelial cells; alpha1AT expression was seen in the alveolar parenchyma in two SpC driven lines, and in small airway epithelium in one of the SpC lines. Electron microscopic immunochemistry showed the presence of the human alpha1AT protein in the interstitium in all lines. Mean levels of human protein varied from 0.37 to 2.9 microg/g lung protein and serum levels from 0.72 to 1.3 microg/ml, compared to normal human serum alpha1AT levels of 2-5 mg/ml. We conclude that transgene-mediated expression of alpha1AT in pulmonary epithelial cells results in diffuse expression of the transgene in the alveolar parenchyma and reproducibly leads to transfer of protein to the interstitium. The present model is, however, limited by low levels of protein production; limited protein production may be a problem in other forms of gene therapy in which relatively large amounts of extracellular protein are needed in the lung for a therapeutic effect.

Role of neutrophils and alpha1-antitrypsin in coal- and silica-induced connective tissue breakdown.

Mineral dusts produce emphysema, and administration of dust to rats results in the rapid appearance of desmosine and hydroxyproline in lavage fluid, confirming that dusts directly induce connective tissue breakdown. To examine the role of neutrophils and alpha1-antitrypsin (alpha1-AT) in this process, we instilled silica or coal into normal rats or rats that had been pretreated with antiserum against neutrophils. One day after dust exposure, lavage fluid neutrophils and desmosine and hydroxyproline levels were all elevated; treatment with antiserum against neutrophils reduced neutrophils by 75%, desmosine by 40-50%, and hydroxyproline by 25%. By 7 days, lavage fluid neutrophils and desmosine level had decreased, whereas macrophages and hydroxyproline level had increased. By ELISA analysis, lavage fluid alpha1-AT levels were increased four- to eightfold at both times. On Western blot, some of the alpha1-AT appeared as degraded fragments, and by HPLC analysis, 5-10% of the methionine residues were oxidized. At both times, lavage fluid exhibited considerably elevated serine elastase inhibitory capacity and also showed elevations in metalloelastase activity. We conclude that, in this model, connective tissue breakdown is initially driven largely by neutrophil-derived proteases and that markedly elevated levels of functional alpha1-AT do not prevent breakdown, thus providing in vivo support for the concept of quantum proteolysis proposed by Liou and Campbell (T. G. Liou and E. J. Campbell. Biochemistry 34: 16171-16177, 1995). Macrophage-derived proteases may be of increasing importance over time, especially in coal-treated animals.

Cigarette smoke increases amosite asbestos fiber binding to the surface of tracheal epithelial cells.

Binding of asbestos fibers to the cell surface appears to be important in the initiation of intracellular signaling events as well as in initiation of particle uptake by the cell. We have previously shown that cigarette smoke increases the uptake of asbestos fibers by tracheal epithelial cells in explant culture. Whether smoke acts by increasing surface binding of fibers is not known. In this study, we exposed rat tracheal explants to air or cigarette smoke and then to a suspension of amosite asbestos. Explants were harvested after 1 or 24 h of dust exposure and washed by repeated dips in culture medium to remove loosely bound fibers, and the number of fibers adhering to the apical cell surfaces was determined by scanning electron microscopy. Smoke-exposed explants retained significantly more surface fibers than air-exposed explants. After four washes, binding levels were similar at 1 and 24 h. The smoke effect was still present when incubations were carried out at 4 degrees C, but binding was decreased approximately 25%. Preincubation of the asbestos fibers with iron chloride to increase surface iron increased fiber binding in both air- and smoke-exposed explants, whereas preincubation of the fibers with the iron chelator deferoxamine decreased binding after air exposure and completely eliminated the smoke effect. Inclusion of mannitol or catalase in the medium or preincubation of the explants with GSH produced decreases in binding of 10-25% in air-exposed explants and generally greater decreases in smoke-exposed explants. We conclude that 1) amosite binding is a very rapid process that does not require active cellular metabolism, 2) cigarette smoke increases adhesion of fibers to the epithelial surfaces, and 3) iron on the asbestos fiber appears to play an important role in binding, probably through an active oxygen species-mediated process.

Mechanisms of mineral dust-induced emphysema.

Mineral dust exposure can result in emphysema and chronic airflow obstruction. We postulated that dust-induced emphysema has a pathogenesis similar to that in cigarette smoke-induced emphysema, namely, excess release of proteolytic enzymes from dust-evoked inflammatory cells, and inactivation of alpha-1-antitrypsin (A1AT) by dust-catalyzed formation of oxidants. To test this theory we examined the antiproteolytic activity of A1AT exposed to quartz in vitro and found that it was decreased in a dose-response fashion. Catalase prevented this effect, which suggested that it was mediated by quartz-generated hydrogen peroxide. We also showed that a variety of dusts could oxidize methionine to methionine sulfoxide in vitro, using either pure amino acid or whole protein. The relative order of activity was coal > quartz > titanium dioxide. Lastly, we used a new high-performance liquid chromatography technique to demonstrate that quartz, coal, and titanium dioxide produced connective tissue breakdown in rat lungs, as determined by the appearance of desmosine and hydroxyproline in lavage fluid after dust instillation. On a particle-for-particle basis, the order of dust potency was similar to that for methionine oxidation. Connective tissue breakdown was associated with elevations of both polymorphonuclear leukocytes and macrophages in lavage fluid, and it is unclear whether one or both of these types of inflammatory cell mediates this process. These observations support our theory that dust-induced emphysema and smoke-induced emphysema occur through similar mechanisms.

Quartz inactivates alpha 1-antiproteinase: possible role in mineral dust-induced emphysema.

Occupational exposure to some types of mineral particles has been shown to be associated with the development of emphysema, but the mechanism of this process is unknown. Because many mineral particles are known to catalyze the formation of active oxygen species in aqueous solution, we hypothesized that mineral particles could oxidatively inactive antiproteinases, leading to an imbalance between protease and antiprotease activities, events similar to those believed to occur with cigarette smoke. To test this hypothesis, human alpha 1-antiproteinase (alpha 1-AP) was incubated with suspensions of freshly ground or aged quartz, and antiproteolytic activity was determined by using porcine pancreatic elastase. Increasing concentrations of quartz were associated with increasing losses of antiproteolytic activity; this effect could be prevented by catalase. Freshly ground quartz was more active than aged quartz. Western blot analysis for alpha 1-AP showed abnormal banding, suggesting that porcine pancreatic elastase-alpha 1-AP complex formation was impaired by silica exposure. Chemical assay of aqueous quartz suspensions demonstrated production of hydrogen peroxide; incubation of alpha 1-AP with hydrogen peroxide caused a dose-dependent loss of antiproteolytic activity, and this also could be prevented by catalase. We conclude that, at least in vitro, quartz can inactivate alpha 1-AP through a hydrogen peroxide-mediated mechanism and that oxidative loss of antiproteinase activity could play a role in mineral dust-induced emphysema.