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doi: 10.1152/ajplung.00390.2007294:L612-L631, 2008. First published 25 January 2008;Am J Physiol Lung Cell Mol Physiol

Andrew Churg, Manuel Cosio and Joanne L. Wrightfrom animal modelsMechanisms of cigarette smoke-induced COPD: insights

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Invited Review

Mechanisms of cigarette smoke-induced COPD: insights from animal models

Andrew Churg,1 Manuel Cosio,2 and Joanne L. Wright1

1Department of Pathology, University of British Columbia, Vancouver, British Columbia; and 2Respiratory Division, Royal

Victoria Hospital, and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada

Churg A, Cosio M, Wright JL. Mechanisms of cigarette smoke-induced COPD:insights from animal models. Am J Physiol Lung Cell Mol Physiol 294: L612L631, 2008.First published January 25, 2008; doi:10.1152/ajplung.00390.2007.Cigarette smoke-induced animal models of chronic obstructive pulmonary disease support theprotease-antiprotease hypothesis of emphysema, although which cells and proteasesare the crucial actors remains controversial. Inhibition of either serine or metallo-proteases produces significant protection against emphysema, but inhibition isinvariably accompanied by decreases in the inflammatory response to cigarettesmoke, suggesting that these inhibitors do more than just prevent matrix degrada-tion. Direct anti-inflammatory interventions are also effective against the develop-ment of emphysema, as are antioxidant strategies; the latter again decrease smoke-induced inflammation. There is increasing evidence for autoimmunity, perhapsdirected against matrix components, as a driving force in emphysema. There is

intriguing but controversial animal model evidence that failure to repair/failure oflung maintenance also plays a role in the pathogenesis of emphysema. Cigarettesmoke produces small airway remodeling in laboratory animals, possibly by directinduction of fibrogenic growth factors in the airway wall, and also producespulmonary hypertension, at least in part through direct upregulation of vasoactivemediators in the intrapulmonary arteries. Smoke exposure causes goblet cellmetaplasia and excess mucus production in the small airways and proximal trachea,but these changes are not good models of either chronic bronchitis or acuteexacerbations. Emphysema, small airway remodeling, pulmonary hypertension,and mucus production appear to be at least partially independent processes that mayrequire different therapeutic approaches.

animals; emphysema; small airway remodeling; pulmonary hypertension; chronicobstructive pulmonary disease

CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) is now thefifth leading cause of death worldwide (126). A meta-analysisusing data from 28 countries suggests that the prevalence ofCOPD based on spirometric measurements is 9 10% in adultsover age 40. It has been estimated that there are 1517 millionindividuals with COPD in the United States alone (62, 160).

By far, the most important risk factor for COPD in thedeveloped world is cigarette smoking; exposures to dusts,fumes, air pollution particles, and, in the developing world,biomass fuels, are also believed to cause COPD (62), but thereis much less information available about these etiologies.Genetic predisposition seems to play an important role in thedevelopment of COPD, and a variety of genetic polymor-phisms related to levels of antiproteases (1-antitrypsin, thebest established and the most clearly important), metallopro-teases, proinflammatory and profibrotic cytokines, and variousantioxidant enzymes and detoxifying enzymes have beenlinked to COPD (reviewed in Refs. 43, 81, 147, 178).

This review will concentrate on mechanistic insights fromlaboratory animal models of COPD caused by cigarettesmoke, both because of the overwhelming role of cigarette

smoke as a causative agent, and because of a lack of animalmodels based on dusts, air pollution particles, and biomassfuels. We will emphasize in vivo models and draw on datafrom tissue culture and explant studies where these appeardirectly relevant; for reviews that focus more on in vitroexperiments, molecular mechanisms, potential targets, andnon-smoke-induced COPD models, see Refs. 7, 8, 18, 40,41, 99, 156, 197. There are plans to cover, in a future reviewin this journal, the pros and cons of various different animalmodels of COPD, including cigarette smoke-induced, elas-tase-induced, and genetic manipulation-induced models, andso these are not addressed here.

Because human COPD really consists of four anatomiclesions (emphysema, small airway remodeling, vascular re-modeling with pulmonary hypertension, mucus overproductionand chronic bronchitis) and one functional lesion [acute exac-erbation (13, 26)], we have separated our discussion into thesebroad categories. In some senses, this separation is artificial,since many patients have airflow limitation because of bothemphysema and small airway remodeling, and they may havepulmonary hypertension and chronic bronchitis and developacute exacerbations as well. However, as will become appar-ent, the mechanisms behind these anatomic lesions may wellbe different, so separating them is useful in understandingpathogenesis and in devising therapeutic approaches.

Address for reprint requests and other correspondence: A. Churg, Dept. ofPathology, Univ. of British Columbia, 2211 Wesbrook Mall, Vancouver, BC,Canada V6T 2B5 (e-mail: [email protected]).

Am J Physiol Lung Cell Mol Physiol 294: L612L631, 2008.First published January 25, 2008; doi:10.1152/ajplung.00390.2007.

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Mechanisms Related to the Protease-Antiprotease Hypothesisof Emphysema

Chronic (generally 6 mo or longer) exposure of mice orguinea pigs to cigarette smoke produces lesions that morpho-logically and physiologically resemble a mild form of centri-lobular human emphysema (184) (Fig. 1), and emphysema is

the lesion that has received the most study, although theanatomic changes differ from those in humans in a subtle waybecause these animals do not normally have respiratory bron-chioles, the locus of initial destruction in human smoke-induced centrilobular emphysema.

The classic theory of emphysema is the protease antiproteasehypothesis. This hypothesis was formulated from the observa-tion that humans deficient in 1-antitrypsin (A1AT) developedearly emphysema, particularly if they smoked (85), and fromthe experiments of Gross and colleagues (59) showing thatinstillation of elastolytic enzymes produced emphysema inexperimental animals. These observations lead to the idea thatsmoke evokes an inflammatory cell reaction and that these cellsrelease proteases that overwhelm the antiproteolytic defenses

of the lower respiratory tract, leading to matrix destruction andemphysema. Despite a variety of new theories, the protease-antiprotease hypothesis (now expanded to include metallo andcysteine proteases as well as the original serine proteases)remains the generally accepted basis for the destruction ofmatrix that leads to emphysema, and is the basis of mostexperimental smoke exposure models, but exactly what cells/proteases are crucial to this process is a complex and contro-versial issue.

That smoke evokes an inflammatory response in both hu-mans (reviewed in Ref. 165) and animals is clear. Everyexperimental animal study that has looked at lavage/tissueneutrophils, macrophages, and lymphocytes in guinea pigs,rats, and mouse strains that develop emphysema has found an

increase, although the details of cell types, timing, and mag-nitude of the effect vary (Tables 1, 2, 3), and smoke inducesproinflammatory cytokine release from cultured macrophages,epithelial cells, and fibroblasts (90, 195, 198). Most but not all

(68, 97) authors report an increase in gene expression/proteinin whole lung/bronchoalveolar lavage (BAL) of chemoattrac-tant and proinflammatory mediators including TNF, IL-1,IL-8, MIP-2, MCP-1, MIP-1, MIP-1, MCP-3, KC, PGE2,IL-12, IL-18, RANTES, and IP-10 (28, 30, 32, 61, 78, 83, 92,98, 166).

Interference with/manipulation of serine proteases. The

original formulation of the protease-antiprotease hypothesispostulated the neutrophil, and in particular, neutrophil elastase,as the important effectors in emphysema (76). In recent years,the role of the neutrophil has become controversial; somereports have shown localization of neutrophils in areas of tissuedestruction in human emphysema (39) but others have failed tofind any correlations between neutrophil numbers in tissuesections and the severity of lung destruction (47, 49).

Table 1 summarizes interventions related to serine proteases.Shapiro et al. (155) showed that mice lacking neutrophilelastase were 59% protected against emphysema, strong evi-dence for a role for neutrophil elastase (see below). In acutesmoke exposure studies, levels of lavage neutrophils correlated

with levels of lavage desmosine, a marker of elastin break-down, and lavage hydroxyproline, a measure of collagenbreakdown (30, 44, 186), and administration of anti-neutrophilantibodies before smoke exposure reduced both neutrophilsand matrix breakdown (44). In chronic studies, inhaled (128) orinjected (30) A1AT or the synthetic serine elastase inhibitorZD0892 (186) provided partial protection against emphysema(Table 1). Takubo et al. (163) and Cavarra et al. (24) showedthat pallid mice, which are naturally deficient in A1AT, devel-oped earlier emphysema than strains with normal A1AT levels.

While these reports support a role of the neutrophil/neutro-phil elastase in the genesis of emphysema, they produced thesurprising result that all interventions decreased the inflamma-tory response and A1AT suppressed smoke-induced elevations

of TNF as well (30). There is extensive evidence fromalveolar epithelial cell and alveolar macrophage cultures aswell as whole mouse and human lung tissue that smoke directlyevokes an inflammatory response by activating NF-B (112,

Fig. 1. Cigarette smoke-induced emphysema inguinea pigs. Photomicrographs of lung from a con-trol (A) and a 6-mo smoke-exposed (B) animalshowing the typical pattern of centrilobular-like em-physema seen in small laboratory animals chroni-cally exposed to smoke. Note that the process of airspace enlargement primarily affects alveolar ducts.

Invited Review

L613ANIMAL MODELS OF COPD

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195), and possibly the aryl hydrocarbon receptor (167) andToll-like receptor-4, at least early on (98). Recent data alsosuggest that smoke inactivates histone deacetylases, leading to

prolonged (and non-steroid-sensitive) inflammation (9, 74,112). TNF production is driven by NF-B and TNF isgenerally presumed to be a driver of inflammatory cell influx insmokers. Thus, a priori, one would expect continuing cigarettesmoke exposure to generate a continuing inflammatory re-sponse, and serine protease inhibitors should provide protec-tion against emphysema without decreasing inflammation; sup-pression of the inflammatory response thus implies additionalanti-inflammatory mechanisms are at work (see below).

Interference with/manipulation of metalloproteases. In thelast 15 years, there has been an increasing interest in metallopro-teases (MMP) as mediators of emphysema. This has stemmed inpart from the recognition that a number of metalloproteases,

including MMP-9 and MMP-12 (124), can degrade elastin; in partfrom reports of increased levels of MMPs including MMP-1, -2,-9, -14 (50, 72, 120, 150), and in some studies, MMP-12 (42, 60,

72, 111, 182), in BAL fluid, alveolar macrophage supernatant, orwhole lung tissue from smokers with emphysema compared withthose without; and in particular, from the report (63) that micewith a targeted deletion of MMP-12 (MMP-12/) failed todevelop emphysema after cigarette smoke exposure.

Cigarette smoke causes increased whole lung or alveolarmacrophage levels of MMP-2, -9, -12, -13, and -14 in mice(32) and MMP-1 in guinea pigs (151). Table 1 lists experi-mental studies using genetically targeted mice or MMP inhib-itors in smoke exposure models. Several broad conclusionsstand out. First, MMP inhibition or deletion can significantly oreven totally abrogate the development of emphysema, indicat-ing a clear role for MMPs in this process. In fact, with broad

Table 1. Effects of interference with proteases in chronic smoke exposure studies

Serine Protease Inhibition or Manipulation

Report (Ref. no.) Species/Strain Intervention Inflammatory Response% Protection Against

Emphysema Comments

Wright (186) Guinea pig ZD0892 (serine proteaseinhibitor)

Decreased 45% Decreased BAL desmosine,hydroxyproline; treatment for last 2

mo of smoke exposure did notprotect against emphysema

Churg (30) C57Bl/6 mouse A1AT injected (prolastin) Decreased 67% Decreased BAL desmosine,hydroxyproline; treatment roughlydoubled serum A1AT levels andsuppressed smoke-mediatedincreases in serum TNF

Shapiro (155) C57Bl/6 mouse Neutrophil elastaseknockout

Decreasedneutrophils,macrophages

59% MMP-12 and neutrophil elastasedegrade each others inhibitors

Pemberton (128) Mouse A1AT inhaled (recombinanthuman A1AT)

Decreased 72% Highest dose increased BAL PMN andgave less protection againstemphysema

Takubo (163) Pallid mouse A1AT deficient Only CD4 cellssignificantlyincreased in tissue

Early onsetemphysema

Pan-lobular emphysema

Cavarra (24) Pallid mouse A1AT deficient Not reported Early onset

emphysema

Pan-lobular emphysema

Metalloprotease and Cysteine Protease Inhibition or Manipulation


Emphysema Comments

Hautamaki (63)Shapiro (155)

129 mouseC57Bl/6 mouse

MMP-12 knockout Decreasedmacrophages butnot BALneutrophils

100% MMP-12 degrades A1AT andneutrophil elastase degradesTIMP-1

Mahadeva (99) Mouse MMP-9 knockout Not reported 0%Pemberton (127) Mouse Broad spectrum MMP

inhibitor GM6001Decreased 90% 90% average value for 2 highest

dosesMartin (105) Mouse Broad spectrum MMP

inhibitor RS113456Not reported 100% Started after 3 mo of smoke exposure;

no increase in mean air space sizebetween 3 and 6 mo

Martin (105) Mouse Broad spectrum MMPinhibitor RS132908

Not reported 75%

Selman (152) Guinea pig Broad spectrum MMPinhibitor CP471474

Decreased 30% Measured as alveolar area at 4 mo;decreased MMP-9 levels at 4 mo

Churg (34) Guinea pig MMP-9/-12 inhibitorAZ11557272

Decreased 68% Decreased BAL desmosine; strongcorrelation BAL desmosine withmean air space size; inhibitor alsoprevents small airway remodeling

Kang (78) Mouse (C57) IL-18R knockout Decreased 51% Decreased apoptosis; decreasedcytokines; decreased cathepsin B, S

All studies use 6 mo of smoke exposure, except as noted.

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L614 ANIMAL MODELS OF COPD



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spectrum MMP inhibitors, it is sometimes possible to achievegreater protection against emphysema than with serine elastaseinhibitors.

Second, the choice of which MMP to target is crucial. Micelacking MMP-12 are completely protected against emphysema(63, 155), whereas mice lacking MMP-9 show no protection atall (99). This latter observation is of particular interest, since it

has been suggested from studies of cultured alveolar humanmacrophages that MMP-9 is the major mediator of emphysemain humans (143, 144), and some have denied a role forMMP-12 in humans (72). Selman et al. (152) found littleprotection with a broad spectrum MMP inhibitor in guineapigs, but we (34) found 70% protection in guinea pigs givena combined MMP-9/-12 inhibitor, AZ11557272, indicatingthat MMPs are not just confined to murine emphysema models,and lending support to the idea that one or both of these MMPsmay be central players in humans. Animals exposed to smokeand AZ11557272 had 70% protection against decreases inairflow compared with animals exposed to smoke alone, thusshowing that prevention of anatomic changes in animal modelsconfers a corresponding physiological benefit.

However, as is true of serine proteases, the exact role ofMMPs in the pathogenesis of emphysema is unclear, particu-larly since neutrophils/serine proteases and MMPS appear tointeract, and MMP inhibition reduces smoke-induced neutro-phil and macrophage influx (29, 34, 128, 155). In an acutesmoke model using MMP-12/ mice, we (29) showed thatMMP-12 was required for smoke-induced neutrophil influxand neutrophils were required for matrix breakdown. Thislatter idea was also supported by the finding that the broadspectrum MMP inhibitor RS113456 acutely inhibited neutro-phil influx and matrix breakdown (44). But in a 6-mo smokemodel, Shapiro et al. (155) found that MMP-12/ micedeveloped a BAL neutrophilia comparable to that of wild-

type animals, implying that proinflammatory mechanismschange over time; we have seen a similar late neutrophilrecruitment effect in animals treated with A1AT and in TNFreceptor (p55 p75/) knockout mice (30, 32). It is of interestin this regard that Stevenson et al. (161), using gene microar-rays, recently reported that gene expression patterns in ratschange from an early proinflammatory to a later pattern ofenhanced acquired immunity, although these data must beviewed with great caution, since rats are extremely susceptibleto (and the Stevenson paper indeed found) the phenomenonknown as particle overload (reviewed in Ref. 119), and particleoverload leads to prolonged generation of inflammatory, fibro-genic, and probably mutagenic mediators by alveolar macro-phages.

Additional evidence for interactions of neutrophils andmacrophages comes from Shapiro et al. (155) who showedthat neutrophil elastase activates pro-MMP-12 and destroystissue inhibitor of metalloprotease (TIMP)-1; conversely,MMP-12 degrades A1AT. Thus neutrophil elastase andMMP-12 cooperate to increase each others proteolyticpotential.

There appear to be a variety of mechanisms by which serineand metalloprotease inhibitors exert anti-inflammatory effects.We (186) found that the serine elastase inhibitor ZD0892inhibited acute smoke-mediated increases in gene expressionof the neutrophil/macrophage chemoattractants MIP-2 andMCP-1 in mice. We also observed that alveolar macrophages

from MMP-12/ mice did not increase TNF secretion aftersmoke exposure, apparently because MMP-12 functions as aform of TNF converting enzyme that liberates active TNF,(Figs. 2 and 3) and lack of TNF appeared to be one reason fora lack of neutrophil infiltration in the lungs of MMP-12/animals (31). As well, A1AT suppresses smoke-mediated in-creases in TNF release by inhibiting the serine proteases

thrombin and plasmin that leak into the air spaces after smokeexposure (31, 35); thrombin and plasmin in turn activateproteinase-activated receptor-1 (PAR-1), thereby causingMMP-12 and TNF release (32, 133) (Fig. 2). This whole setof observations would remove MMP-12 from a matrix destruc-tive role to a signaling role in the context of cigarette smokeexposure, and signaling roles are now recognized as majorfunctions of MMPs (125).

An alternate explanation for decreases in inflammatorycell influx is that inhibition of matrix destruction by eitherneutrophil or macrophage-derived proteases decreases thegeneration of chemotactic matrix fragments (1, 69, 129,153, 154). Houghton et al. (68) observed that the lavagefluid of wild-type mice contained elastin fragments thatwere chemoattractants for monocytes and that this chemoat-tractant activity was absent from the lavage of MMP-12/mice. They suggested that matrix breakdown in emphysemais driven by both neutrophil elastase and MMP-12, with thelatter the major player because of the relatively large num-bers of monocytes that migrate into the lung after smokeexposure. In practice, MMP-12 may well play both a sig-naling and a direct matrix destructive role.

More recently, Maeno et al. (97) have proposed that thiswhole process of smoke-driven matrix destruction is initiatedby CD8 lymphocyte-mediated production of IFN/IP-10,with resulting neutrophil and macrophage infiltration and in-

Fig. 2. Postulated mechanisms of cigarette smoke-induced MMP-12 release.Smoke directly activates Toll-like receptor-4 (TLR4) and probably other (asyet unidentified) cell surface receptors that drive MMP-12 release. In addition,smoke causes leakage of plasma proteins into the alveolar spaces. Theseinclude prothrombin and plasminogen, which are converted to thrombin andplasmin by alveolar macrophage-derived tissue factor (TF) and plasminogenactivator (PA); thrombin and plasmin in turn activate proteinase-activatedreceptor-1 (PAR-1), which causes MMP-12 release. IP-10 derived fromsmoke-evoked T cells also causes increased macrophage production ofMMP-12 (see Fig. 3). Modeled from Refs. 35, 97, 98, 133.

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creases in MMP-12 production (Fig. 3). Of note, these data aresimilar to findings reported in human lungs with emphysemaby Grumelli et al. (60).

Cantor et al. (20, 21) showed that inhaled hyaluronan (HA)can bind to elastin fibers in vivo and in vitro and preventselastolysis by elastases. HA reduced air space enlargementcaused by porcine pancreatic elastase (20), provided 100%

protection against emphysema in smoke-exposed DBA/2Jmice, and reduced smoke-mediated increases in lavage desmo-sine. These findings support matrix attack as a fundamentalfeature of smoke-induced emphysema.

Cysteine proteases. Kang et al. (78) showed that smokeinduces production of the cysteine proteases cathepsins B andS in mice via a mechanism involving IL-18, and, probably,interferon-. These proteases can degrade matrix componentsand thus might play a role in emphysema. The same reportfound greater levels of cathepsins B and S in alveolar macro-phages from cigarette smokers compared with controls. IL-18R/ mice were significantly protected against emphy-sema and had decreases in smoke-induced lavage neutrophilsand macrophages as well as a variety of chemokines andcathepsins, along with decreased levels of MMP-12. Thesedata thus present the same mix of possible effectors seen withstudies inhibiting/deleting serine and metalloproteases.

Anti-inflammatory interventions. If the protease-antiproteasehypothesis (i.e., that the smoke-driven inflammatory response

leads to proteolytic matrix destruction) is correct, one couldinhibit smoke-mediated inflammatory responses instead of pro-teases, and a number of studies have directly or indirectlytargeted the production and/or signaling of proinflammatorymolecules (Table 2).

TNF is consistently elevated in human smokers (10, 165).In mice, knockout of TNF receptors 1 and 2 greatly reduced

inflammatory infiltrates, emphysema, levels of some MMPs,and gene expression of proinflammatory cytokines (28, 32, 46)after smoke exposure; TNF receptor 2 appeared to be themajor mediator of the inflammatory response (46). Conversely,inducible overexpression of TNF produced an increase inneutrophils, parenchymal B cell nodules, MMP-12, cathepsinK, and emphysema in the absence of smoke (176). There wasan extremely strong correlation (R 0.89, P 0.0001)between serum TNF levels and air space size after 6 mo ofsmoke exposure in guinea pigs (34). While these reports implyan important role for TNF, two studies in humans usingTNF antagonists failed to show a benefit (10, 134, 172),suggesting that translation of anti-inflammatory therapies from

animal models to humans is not straightforward.Using a different anti-inflammatory approach, Thatcher et al.(166) prevented neutrophil influx by administration of SCH-N,a CXCR2 inhibitor, to mice, with an 50% reduction inparenchymal cells after a 3-day smoke exposure. SCH-N didnot decrease smoke-mediated increases in TNF, IL-6, orPGE2, and levels of the neutrophil chemoattractant CXCR2ligands KC and MIP-2 were considerably elevated. Maes et al.(98) showed that, in mice, Toll-like receptor-4 plays a role inthe early but not the chronic inflammatory response to cigarettesmoke.

Phosphodiesterase-4 degrades the anti-inflammatory nucle-otide cyclic 35-adenosine monophosphate. In C57Bl/6 miceexposed to smoke for 7 mo, Roflumilast, a phosphodiesterase-4

inhibitor, provided 100% protection against smoke-inducedincreases in air space size, reversed smoke-induced loss of lungdesmosine, reduced neutrophil and especially macrophage in-flux, and more than doubled levels of the anti-inflammatorycytokine, IL-10 (106). One of the effects of Roflumilast is todecrease macrophage production of TNF, and in a humantrial, 4 wk of Roflumilast decreased blood/sputum TNF, IL-8,and neutrophil elastase, and improved FEV1 (58).

IFN appears to drive many proinflammatory cytokines viaCCR5 (92). IFN/ or CCR5/ mice exposed to smokefor 6 mo were completely protected against emphysema andshowed decreased inflammatory cells, apoptosis, levels ofMIP-1, MIP-1, and RANTES (16, 92). Protection may have

been mediated through decreased MMP-12, since transgenicmice overexpressing IFN increased MMP-12 production (92).Administration of the 3-hydroxy-3-methyl-glutaryl-coen-

zyme-A reductase inhibitor, simvastatin (87), to rats com-pletely abolished smoke-induced emphysema and preventedperibronchial and perivascular accumulation of lymphocytes.This report is difficult to interpret: the authors reported aremarkable degree of air space enlargement (80% increase inmean air space size) after a relatively short exposure period (16wk). Furthermore, statins have been reported to increase mac-rophage production of MMP-12 (4), which should make em-physema worse. However, there are reports of beneficial ef-fects of statins in patients with COPD (102, 159) and reports

Fig. 3. Postulated mechanisms of matrix attack and emphysema. In this model,smoke causes release of a variety of chemokines from structural cells andalveolar macrophages, leading to an influx of neutrophils (PMN), macro-phages, lymphocytes, and dendritic cells. MMP-12 causes conversion ofpro-TNF to active TNF, thereby amplifying the inflammatory process.Neutrophil elastase and macrophage-derived MMP-12, as well as other pro-

teases, can directly attack the alveolar wall, leading to both matrix destructionand liberation of elastin fragments, which are chemoattractants for macro-phages, further amplifying the inflammatory response. The scheme shown hereencompasses the findings of Lee et al. (94) that COPD patients have anti-elastin antibodies, so that protease-derived elastin fragments evoke an auto-immune response, leading to further matrix attack and production of IFN/IP-10, which itself increases MMP-12 release by macrophages and also increasesmatrix destruction. Other matrix fragments or smoke-modified non-matrixproteins might function in a similar fashion. In addition, neutrophil elastasedegrades TIMPs and MMP-12 degrades 1-antitrypsin (A1AT), thus removinganti-proteolytic defenses, and oxidants in the smoke may inhibit A1AT andother antiproteases such as SLPI as well. This scheme thus incorporates aninnate immune response to smoke, a protease feedback amplification systemdriven by matrix destruction, and an acquired immune response, also driven bymatrix destruction. The relative importance of each process probably changesover time. Modeled from Refs. 31, 38, 88, 92, 97, 155.

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that statins enhance clearance of apoptotic cells (114), so thistype of compound may be worth further investigation.

In aggregate, both interventions directed against proteases(serine, cysteine, or metalloproteases) and interventions di-rected against the development of an inflammatory influxprovide significant protection against emphysema in animalmodels and thus support the protease-antiprotease hypothesis,although the mechanism(s) involved are complex and notentirely clear.

Effects of Smoke on Different Mouse Strains: Evidence for aGenetic Propensity to Emphysema

As noted above, susceptibility to COPD likely results frommultiple genetic and environmental effects. Cavarra et al. (24)showed that mouse strains with differing antioxidant/antipro-tease capacity reacted differently to cigarette smoke (see Mech-anisms Related to Oxidative Stress in Emphysema). Guerassi-mov et al. (61) exposed five different strains of mice to smokein an attempt to define genetically susceptible and resistantvarieties. The strains were chosen based on differences in theMHC haplotype, a major determinant of the inflammatoryresponse in mice. After 6 mo of smoking, NZWLac/J mice had

no increase in mean air space size (Lm), whereas AJ, SJL,C57BL/6, and AKR mice had 17.9%, 23.8%, 13.2%, and 38%increases, respectively. Because, as noted by Henson andVandivier (64), it is remarkably easy to induce alveolar en-largement with a very wide variety of manipulations in themouse, Guerassimov et al. (61) defined emphysema as anincrease in Lm along with an increase in lung compliance(Fig. 4). By these criteria, only the AKR strain had significantemphysema, whereas A/J, SJL, and C57BL6 strains appeared

to be mildly susceptible to smoke and NZW were resistant.Hoshino and Cosio (unpublished data) then investigated the

genetic response to cigarette smoking in resistant (NZW) andsusceptible (AKR) strains utilizing expression microarrays.There were striking constitutive differences between the twostrains, mainly in the expression of genes that encode forproteins with immune function. The NZW mice had higherconstitutive expression of genes that inhibit differentiation andproliferation of T and B cells and protect against apoptosis andT cell activation (Ifi203, CD72, C4, Klra-1, 8, and 13), alongwith higher levels of several antioxidant genes that were notprominently expressed in AKR mice. In contrast, the constitu-tive inflammatory genes expressed in AKR mice were proin-

Table 2. Effects of manipulation of the immune/inflammatory response in chronic smoke exposure studies


Emphysema Comments

Churg (32) Mouse (C57Bl/6) TNF R1 R2/ Decreased BALmacrophages, neutrophils

71% Increased MMP-2, -9, -12, -13, -14with smoke exposure; knockoutmice: decreased MMP-12, -13,-14; decreased gene expression

of TNF, MCP-1, MIP-2Dhulst (46) Mouse (C57) TNF R1/ Not affected NoneDhulst (46) Mouse (C57) TNF R2/ Decreased BAL neutrophils,

macrophages,lymphocytes

100% Decreased CD4 and CD8 cells

Martorana (106) Mouse (C57) PDE4 inhibitorRoflumilast

Decreased 100% 7-mo smoke exposure; increasedIL-10

Lee (87) Rat (Sprague- Dawley) Simvastatin Decreased peribronchiallymphoid aggregates

100% 4-mo smoke exposure; alsodecreased pulmonary arterypressure; possible technicalproblems with study

Cantor (21) Mouse (DBA/2) Hyaluron aerosol Not reported 100% Putative protection of elastin fibers;possible developmental problems

Bracke (13) Mouse (C57Bl/6) CCR6/ Decreased 67% No protection against small airwayremodeling; decreased MMP-12,BAL PMN, T lymphocytes,macrophages

Ma (92) Mouse (C57Bl/6) IFN/ Not reported 100% Decreased apoptosis; MMP-12probably involved

Ma (92) Mouse (C57Bl/6) CCR5/ Decreased (primarilydecreased macrophages)

100% Decreased cytokines, apoptosis

Bracke (16) Mouse (C58Bl/6) CCR5/ Decreased 25% Decreased PMN, macrophages,dendritic cells, lymphocytes; noprotection against small airwayremodeling

Maeno (97) Mouse (C57Bl/6) CD8/ Decreased 100% Some persisting elevation in BALPMN

Maeno (97) Mouse (C57Bl/6) CD4/ Not decreased NoneDHulst (45) SCID mouse No acquired immune

responseIncreased neutrophils,

macrophages, dendriticcells

None No T or B lymphocytes; nolymphoid follicles

Maes (98) Mouse (C3H/HeJ) TLR4/ Increased BAL PMNmacrophages at 26 wk

None Decreased BAL lymphocytes anddendritic cells at 26 wk

van der Deen (172) Mouse (FVB) Multi-drug resistanceprotein/ Decreased inflammation andinflammatory mediators No emphysema produced in wildtype or knockout

All studies use 6 mo of smoke exposure, except as noted.

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flammatory, and several were related to adaptive immunity.

With smoke exposure there was a striking difference in generesponse: the NZW strain demonstrated decreased expressionof 77 of the 82 genes that were significantly changed inresponse to smoke, and 44% of the genes attenuated by smokeexposure influence immune and/or inflammatory processesincluding immunoglobulins, complement, chemokines, cyto-kines, and other proinflammatory factors that enhance thefunctions of neutrophils, macrophages, and T and B cells.NZW mice also upregulated antioxidant genes. In contrast,AKR mice showed increased expression of 52 of the 57 genesthat were changed by smoke exposure, and 25% of the genesincreased after smoking had functions related to the immuneresponse; 13% were proapoptotic (Fig. 5).

In another study aimed at investigating the potentiation of

inflammation by cigarette smoke, Reynolds et al. (137) inves-tigated the expression of Egr-1 (early growth response gene 1)in NZW and AKR mice. Egr-1 gene induces IL-1 and TNF,cytokines that contribute to the recruitment of inflammatorycells after smoke exposure. Egr-1 expression was marginallydetected by immunochemistry in the lungs of nonsmokingmice, but increased markedly in the susceptible AKR strainafter smoke exposure. However, Egr-1 was only minimallyinduced in the lungs of the resistant NZW.

These studies suggest that resistant animals do not increaseor actively decrease the proinflammatory response to smokewhile increasing the antioxidant response, thus preventingmatrix breakdown and a potential acquired immune response to

matrix fragments. In contrast, inflammation in the susceptible

strain is progressive, probably due to ongoing stimulation ofinnate and adaptive immunity by the expression of genesinvolved in antigen presentation and T and B cell activation.These findings support the possibility of an autoimmune pro-cess triggered by smoke exposure as a factor important in themaintenance of the inflammation and the development ofemphysema.

Evidence for Acquired Immunity in Animal Modelsof Smoke-Induced Emphysema

An abnormal inflammatory response is an important com-ponent in the definition of COPD (131). Besides neutrophilsand alveolar macrophages, both CD4 (T helper) and CD8

(cytotoxic) T cells are increased in the airways and lungparenchyma of patients with COPD with a predominance ofCD8 T cells, and Finkelstein et al. (49) showed that in humanlungs, there is a correlation between the number of T lympho-cytes/mm3 of lungs and the extent of emphysema. This infil-tration with T cells, seen in smokers who develop COPD, butnot in normal smokers, represents an activation of the adaptiveimmunity that presumably follows from the initial and thensustained innate immune response characterized by increasednumbers of macrophages and neutrophils.

The T cells found in patients with COPD are fully activated(60, 146), expressing a large array of Th1 chemokines andcytokines. This inflammatory process most likely also involves

Fig. 4. Pressure volume curves from control and cigarette smoke-exposed during 6 mo NZW, C57Bl/6, Pallid, and AKR/J mice. The animals were anesthetizedand paralyzed, and lung mechanics were measured at PEEP levels of 19 cmH2O by delivering a broad-band volume perturbation to the lungs for a period of16 s, and the fast Fourier transforms of the data windows were used to calculate the input impedance of the lung. The parameter Htis is equal to lung elastance(1/compliance) at a frequency of 1 rad/s 0.16 Hz, and we used Htis vs. PEEP to calculate an equivalent pressure-volume curve for the lungs between1 and 9 cmH2O. Only the Pallid and AKR/J show a significant compliance increase. [From Guerassimov et al. (61).]

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the migration of dendritic cells, since it has been recentlyreported that T cells in humans with emphysema are beingpresented with antigens derived from the breakdown of elastin(88). These findings support a role for autoimmunity in thepathogenesis of COPD.

Guerassimov et al. (61) investigated the inflammatory re-sponse to long-term cigarette smoking in the mice with differ-ent susceptibilities to emphysema described in EFFECTS OFSMOKE ON DIFFERENT MOUSE STRAINS: EVIDENCE FOR A GENETICPROPENSITY TO EMPHYSEMA. To approximate human studies, theyused morphometric methods to quantitate the percentage ofimmunostained inflammatory cells in the alveolar walls. After6 mo of smoking, only the susceptible AKR strain had a floridcellular inflammatory infiltrate comprising CD4, CD8, and

T cells, along with macrophages and neutrophils. Neitherthe resistant nor the mildly susceptible strains exhibited aninflammatory infiltrate containing T cells other than some cells in the NZW strain (Fig. 6). Similar results were found byTakubo et al. (163) in the Pallid mouse, a strain that alsodevelops marked emphysema after 6 mo of smoking exposure.Analysis of inflammatory chemokines and cytokines in thelungs (Fig. 7) in the three groups with different susceptibilitiesconfirmed that a true Th1 inflammatory response, secondary toT cell activation, had developed in the susceptible AKR micebut was not present in the resistant or mildly susceptiblestrains. These findings are of interest since they mimic the inflam-matory changes that smokers develop, and, as in the human

smokers, only the susceptible animals develop the adaptive im-mune reaction seen in humans.

The importance of CD8 T cell inflammation in the devel-opment of cigarette-induced emphysema in mice has beenrecently demonstrated by Maeno et al. (97). In contrast towild-type mice that showed macrophage, neutrophil, and lym-phocyte inflammation and emphysema after 6 mo of exposure,

CD8/ mice had a blunted inflammatory response and didnot develop emphysema when exposed to long-term cigarettesmoke. They showed that the CD8 T cell product IFN-inducible protein-10 induces production of MMP-12 that de-grades elastin, causing lung destruction and generating elastinfragments that attract more macrophages and probably act asan antigenic stimulus to maintain the adaptive immune reactionbringing more T cells into the lung.

It should be noted that some of the literature in this area iscontradictory. SCID mice, which completely lack B and Tcells, develop emphysema of a degree comparable to that of thewild type (Balb/C), along with an inflammatory influx ofneutrophils, macrophages, and dendritic cells, and increasedlevels of MMP-12 (45). However, some workers have foundthat SCID mice develop hemorrhage after smoke exposure,making emphysema difficult to measure (Shapiro SD, personalcommunication). As well, reported laboratory to laboratoryvariations in response to a given mouse strain seem to translateinto different molecular responses. For example, two of theauthors of this review (Churg and Wright) have consistentlyfound an 3540% increase in air space size and a smoke-induced elevation in gene and protein expression of TNF andother proinflammatory mediators in C57Bl/6 mice (28, 30), butthe third author of this paper (Cosio) finds much smallerincreases in air space size and has not observed such inflam-matory responses in C57Bl/6 mice. The mice used by Maenoet al. (97), which developed a CD8 T cell response (see

above), were also C57Bl/6 based. It is unclear if these findingsindicate the presence of small genetic differences in the samemouse strain obtained from different sources, or mean thatdifferent smoke exposure conditions and smoke exposure sys-tems can force animals of the same notional strain into the typeof proinflammatory and adaptive immune response just de-scribed.

Mechanisms Related to Oxidative Stress in Emphysema

Cigarette smoke is an extremely concentrated source ofreactive oxygen species (ROS) and reactive nitrogen species(130). The inflammatory response to smoke potentially aug-ments oxidative stress, since neutrophils and macrophages

release ROS, and those from smokers release even greateramounts (17, 66, 91, 95, 96).

There is considerable biochemical evidence of oxidativestress in cigarette smokers and greater levels in those withCOPD (reviewed in Refs. 14, 95, 96). These changes includeincreased exhaled H2O2 and 8-isoprostane, decreased plasmaantioxidants, and increased plasma and tissue levels of oxi-dized proteins, various lipid peroxidation products such as4-hydroxynonenal, as well as protein tyrosine residues/3-nitro-tyrosine, indicators of attack by reactive nitrogen species.Antioxidant enzyme levels are also altered. Oxidative attackmay inactivate antiproteases such as SLPI (25), and oxidativedamage is believed to decrease production of/inactivate some

Fig. 5. Overall differences in gene expression profiles in mice resistant, NZW,and susceptible, AKR, to the development of emphysema after 6 mo ofcigarette smoke exposure. Gene expression profiles were investigated in 3animals per control and exposed groups using Affymetrix Microarray Analy-sis. Compared with their controls, NZW mice largely downregulated geneexpression, whereas AKR mice showed the opposite pattern.

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histone deacetylases (9, 74), leading to a prolonged inflamma-tory response.

Evidence of oxidant attack is also seen in animal models.Aoshiba et al. (2) showed that even a 1-h exposure of mice tocigarette smoke resulted in immunochemically detectable4-hydroxynonenal and 8-hydroxy-2-deoxyguanosine, a markerof oxidative DNA damage, in the airway and alveolar epithelialcells. We (27) found that a 10-min smoke exposure producedlipid peroxidation in rat tracheal explants. Rangasamy et al.

(132), using microarrays, showed upregulation of a variety ofantioxidant enzymes after smoke exposure of ICR mice.McCusker and Hoidal (109) found increases in superoxidedismutase and catalase expression in alveolar macrophagesfrom smoke-exposed hamsters, whereas Cavarra et al. (25)showed depletion of protein thiols, ascorbic acid, and glutathi-one, all antioxidant substances, in smoke-exposed C57Bl/6mice.

Interference with the antioxidant response by deletion ofNrf2 (Nrf2/) (71, 132), a redox-sensitive transcriptionfactor that upregulates a variety of detoxification and antioxi-dant genes, caused increased smoke-induced inflammation andearlier and more severe emphysema than in wild-type (ICR orICR/Balb/c) mice, and Nrf2/ mice had impaired upregula-

tion of antioxidant enzymes (157). As well, Nrf2/ micewere deficient in A1AT and secretory leukoprotease inhibitor(SLPI). These findings link a lack of antioxidant protection tothe protease-antiprotease hypothesis (Fig. 3).

Conversely, several investigators have acutely increasedantioxidant protection (Table 3) with consequent decreases ininflammatory cell influx, protein oxidation, inflammatory me-diators, and airway squamous metaplasia after short-termsmoke exposure (5, 116, 158). Rubio et al. (142) found that theantioxidant N-acetyl cysteine ameliorated elastase-induced em-physema in rats.

Cavarra et al. (24) reported that ICR mice, which increasedantioxidant levels in BAL after smoke exposure, were pro-

tected against emphysema, whereas C57Bl/6 and DBA/2,which did not increase antioxidant levels, developed emphy-sema; of note, levels of antioxidant protection appeared to bemore important than BAL levels of elastase inhibitory capacityin determining the severity of emphysema. Foronjy et al. (52)showed that chronically smoke-exposed transgenic CuZnSODanimals had much smaller increases in neutrophil and macro-phage infiltration than did wild-type animals, did not showincreases in production of a variety of MMPs including MMP-

12, were protected against generation of lipid peroxidationproducts, and were 100% protected against the development ofemphysema. There was substantial protection against elastase-induced emphysema as well.

These studies thus support a role for oxidant attack in thegenesis of smoke-induced emphysema, but it is noteworthythat the immediate mechanism of attack appears to be throughincreases in inflammatory cells, and, probably, decreases inantiprotease protection, thus linking oxidant attack to theprotease-antiprotease hypothesis.

Mechanisms Related to Repair of Alveolar Structurein Emphysema

Retinoids accumulate in the lung during embryogenesis andare essential to alveolar septation; mice lacking retinoid recep-tors have lower levels of elastin and enlarged alveolar spaces(108). Massaro and Massaro (107) reported that treatment withall-trans retinoic acid reversed the emphysematous changesseen in rats after instillation of elastase, and this was confirmedin rats by Belloni et al. (12). However, reports in otherspecies and with cigarette smoke have been discouraging.March et al. (103) did not find any protective effects ofretinoids against smoke-induced emphysema in either A/J orB6C3F1 mice, and Meshi et al. (110) did not observe anybenefit in smoke-exposed guinea pigs. A human trial ofretinoid therapy did not show any clear improvement in

Fig. 6. Inflammation in the alveolar wall inmouse strains with different susceptibilitiesto the development of emphysema after 6 moof cigarette smoke exposure. In these exper-iments, only the susceptible AKR miceshowed a full inflammatory profile. PMN, neu-trophil; AM, alveolar macrophage; CD4,CD4 T cell; CD8, CD8 T cell. [FromGuerassimov et al. (61).]

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emphysema (141). But this issue still needs further investi-gation because the dose of retinoic acid may be crucial.Stinchcombe and Maden (162) have recently observed thatretinoic acid will restore alveolar architecture that is dis-rupted by steroid treatment of newborn mice if a highenough dose is used.

Mechanisms Related to Failure to Repair/Failure of Lung

Maintenance in Emphysema

While there appears to be overwhelming evidence thatcigarette smoke-induced damage to the alveolar wall matrix asa result of inflammatory cell-derived protease, and, probably,oxidant, attack (but, again, with oxidant-driven increases in

inflammatory cells) is the driving force behind emphysema,one of the curious features of emphysema is that the alveolarwall largely fails to regenerate new matrix. This is in sharpcontradistinction to the small airways and the intrapulmo-nary arteries, where the response to smoke is a markedincrease in matrix and/or structural cells (see Mechanisms ofSmall Airway Remodeling and Mechanisms of VascularRemodeling and Pulmonar y Hype rtension), despite the factthat these anatomic compartments are separated by only afew micrometers.

A variety of theories, conveniently grouped as failure torepair/failure of lung maintenance have been advanced toaccount for this phenomenon (reviewed in Refs. 134, 170,171). Increases in apoptotic cells, sometimes accompanied bydecreases in proliferating cells, have been found in severelyemphysematous compared with nonemphysematous humanlungs (73, 100, 170, 196). Administration of agents that causeendothelial or epithelial cell apoptosis to animals results inrapid air space enlargement, although, unlike smoke-inducedemphysema, this process is also rapidly reversible and is notaccompanied by an inflammatory response (3, 79). In tissueculture models, smoke exposure interferes with cell prolifera-tion, chemotaxis, and production/remodeling of matrix com-ponents by fibroblasts (23, 134) and depresses the productionand activity of lysyl oxidase (53, 86), an enzyme crucial to theformation of stable insoluble elastin and collagen. Fibroblastsfrom emphysematous human lungs proliferate more slowly

than those from nonemphysematous lungs (67, 117) and showincreased expression of a variety of senescence-associatedmarkers (118, 169).

Only a few animal models have looked at this issue usingsmoke exposure. Mice lacking senescence-associated marker30, an anti-aging calcium binding protein, show increased

Fig. 7. Cytokine and chemokine gene expression in mouse strains withdifferent susceptibilities to the development of emphysema. In these experi-ments, NZW mice tend to downregulate gene expression, C57Bl/6 mice tendnot to change expression patterns, and AKR mice tend to upregulate expres-sion. [From Guerassimov et al. (61).]

Table 3. Effects of antioxidant manipulation in acute and chronic smoke exposure studies

Report (Ref. no.) Species/Strain Intervention Inflammatory Response Effect on Emphysema Comments

Nishikawa (116) Guinea pig rhSOD Decreased Acute study only Decreased IL-8 gene expression,NF-B activation

Smith (158) Rat Catalytic antioxidant

AEOL 10150

Decreased Acute study only Decreased MIP-2, ICAM-1,

squamous metaplasia inairways

Banerjee (5) Guinea pig Black tea (antioxidant)in drinking water

Not reported Acute study only Decreased oxidation of lungproteins

Cavarra (24) ICR, C57, DBA/2 mice ICR mice have increasedantioxidant protection,cf. to C57, DBA/2

Not reported 5% increase in meanair space size inICR, not significant;C57 and DBA/2 getemphysema

Elastin content decreased in C57and DBA/2 but not in ICR

Rangasamy (32) M ouse (ICR) Nrf2 knockout Increased Earlier and moresevere

Decreased A1AT; increasedapoptosis

Iizuka (71) Mouse (Balb/c) Nrf2 knockout Increased Earlier and moresevere

Decreased SLPI

Foronjy (52) Mouse Tg ZnCuSOD Decreased 100% protection Protects against smoke-mediatedincrease in lipid peroxidation

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emphysema when exposed to smoke (148) and also showincreased numbers of apoptotic lung structural cells (113). Inelastase instillation emphysema, concurrent smoke exposureinhibits new elastin synthesis, whereas with elastase alone,there is brisk production of new protein (121). We haveobserved, using laser capture microdissection, that the paren-chyma initially responds to smoke by upregulation of genes

that produce matrix components, but that over time most ofthese genes are downregulated, suggesting that failure to repairis not a simple yes or no proposition, but a process that changeswith increasing exposure (Churg, unpublished data). Kanget al. (78) showed that smoke activates IL-18 and causesapoptosis by direct or IL-18R-mediated activation of a num-ber of caspases. IFN/ mice exposed to smoke for 6 mohad decreased levels of inflammatory cells, apoptosis, andemphysema (92). In Nrf2/ mice (132), there was an in-crease in apoptotic parenchymal cells with smoke exposure.Smoke disrupts lung VEGF signaling in rats, and VEGFappears to be an important trophic mediator of lung endo-thelial cell survival (79).

Tuder et al. (171) have proposed that oxidative damage andapoptosis are the primary events in emphysema and thatinflammation is secondary, but this idea is contradicted byevidence that smoke directly drives inflammation, even inmonolayer cultures where smoke causes cytokine release with-out apoptosis (see Mechanisms Related to the Protease-Anti-protease Hypothesis of Emphysema). Guinea pigs and mostgenetically intact mice that develop emphysema do not developincreased apoptosis, even with prolonged smoke exposure(Ref. 51 and Churg, unpublished data), although Bartalesi et al.(11) reported focal areas of increased apoptosis in DBA/2 butnot C57Bl/6 mice.

The question of whether apoptosis is a primary or secondaryevent in emphysema (and even whether it occurs in excess) is

unresolved and is not straightforward, because apoptosis ofepithelial cells (anoikis) may also occur as a result of destruc-tion of the underlying matrix (55). It should also be noted thatthere is not a total absence of repair in emphysema, sincehuman centrilobular emphysema is associated with increasedamounts of collagen (22). Nonetheless, the data suggest thatthere is ineffective repair in the parenchyma after long-termsmoke exposure.

Mechanisms of Small Airway Remodeling

Small airway remodeling (increases in bronchiolar wallfibrous tissue, muscle, inflammatory cells, and luminal mucus)(SAR) is now recognized as an important cause of airflow

obstruction in cigarette smokers (65, 122). There are only a fewreports that have examined smoke-induced SAR in animals.The changes found are subtle and easily missed on casualexamination, and some investigators deny that SAR occurs(104). However, careful morphometric studies from severallaboratories have confirmed that there is indeed SAR, largelymanifest as increases in airway wall collagen, in both guineapigs (34) and mice (15, 33, 16) after chronic smoke exposure(Fig. 8). Wright and colleagues (192) reported an increase inthick collagen fibers (Fig. 8), an effect that probably increasesairway stiffness, in the small airways of guinea pigs exposedto cigarette smoke for 6 mo. They found negative correla-tions of the amount of thick collagen fibers with peak

expiratory flow and FEV0.1/FVC, and positive correlationswith airway resistance, thus indicating that smoke-inducedairway remodeling in animals is associated with abnormalphysiology.

In humans, the usual assumption has been that, since em-physema is driven by inflammatory cells and their proteases,SAR should follow the same pathways (77). In fact, little is

known about the pathogenesis of SAR in humans. One piece ofevidence against a primary role of inflammation in SAR arereports (15, 16) that mice lacking CCR6 and CCR5 (receptorsfor various chemoattractant chemokines) had decreased in-flammation and decreased emphysema after smoke exposure,but no decrease in SAR compared with wild-type animals.SAR was also prevented in smoke-exposed guinea pigs by anMMP-9/-12 inhibitor, again evidence that mechanisms otherthan inflammation are important (34).

Using laser capture microdissection of small airways (bron-chioles) from the lungs of smoke-exposed C57Bl/6 mice,Churg et al. (33) found that smoke persistently upregulatedgene expression of type I procollagen and profibrotic cyto-kines, particularly those related to TGF- signaling. With asingle acute exposure, elevations in gene expression were seenwithin 2 h of starting smoke exposure and mostly decreasedover 24 h, as opposed to numbers of lavage inflammatory cellsthat increased slowly over 24 h (44), implying that, at least inthe short term, upregulation of the fibrotic response is inde-pendent of inflammation.

In rat tracheal explants, an airway model system that is freeof smoke-evoked inflammatory cells, a very brief (15-min)exposure to smoke resulted in increases in the same set ofgenes mentioned above, along with collagen (hydroxyproline)by 24 h, and these increases could be prevented with aninhibitor of TGF- receptor 1 (Churg, unpublished observa-tions) or a TGF- competitor, fetuin (179). The smoke-ex-

posed explants released increased amounts of TGF-1 via anoxidant-driven mechanism (Fig. 9).In short, the animal models suggest that small airway re-

modeling is driven by direct smoke induction of fibrogenicgrowth factors, and the role of inflammatory cells is uncertain;rather, the initial driving force appears to be oxidant-mediatedactivation of TGF-. However, since neutrophils and macro-phages release oxidants in response to smoke (17, 66, 91, 95,96), such cells might potentiate TGF- release and hencepotentiate small airway remodeling (Fig. 9).

Both B and T lymphocytes are increased in the small airwaywalls in human COPD (37, 65, 145, 173). Whether they play adirect role in the pathogenesis of SAR or reflect colonization ofthe small airways by infectious agents in patients with ad-

vanced COPD is unclear (65). In cigarette smoke-exposedanimals, peribronchial lymphoid aggregates increase in num-ber (Fig. 10), but in CCR6/ and CCR5/ mice, a lack ofperibronchial lymphoid aggregates with chronic smoke expo-sure did not protect against SAR (15, 16).

Mechanisms of Vascular Remodeling and PulmonaryHypertension

Pulmonary hypertension (PHT) is a relatively common formof cigarette smoke-induced lung disease and develops in 6%of subjects with COPD, but is present in 40% of patients withan FEV1 of less than 1 l (6, 19, 93, 94, 180). PHT is animportant complication since it is a significant predictor of

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mortality, and is a major cause of morbidity, in patients withCOPD (36, 168, 189).

The mechanism(s) of PHT in smokers is not known. Althoughit is often stated that PHT arises as a result of loss of vascular bedsecondary to emphysematous lung destruction and/or hypoxicvasoconstriction, recent data indicate that this is not true (re-viewed in Ref. 75). Guinea pigs exposed to cigarette smokedevelop an 25% increase in mean pulmonary artery pressure(191). Using plastic vascular casts in such animals, we showedthat PHT is not associated with significant alveolar capillary

destruction (193, 194). Likewise, although there is a correlationbetween PHT and PO2, PO2 has not been found to be an indepen-dent predictor of pulmonary arterial pressure (149).

In guinea pigs, smoke-increased arterial muscularization(Fig. 11) correlates with pulmonary arterial pressure, andinterestingly, also correlates with increased mRNA and proteinlevels of the vasoactive mediators endothelin and VEGF inthese vessels (198). Smoke-induced vascular remodeling alsoappears to be related to matrix reorganization, since in guineapigs, increases in pulmonary arterial pressure and vascular

Fig. 8. Small airway remodeling in guineapigs. Photomicrographs of Picrosirius Red-stained small airways (membranous bronchi-oles) from control (A and B) and 6-mo smoke-exposed (C and D) guinea pigs. Note thedistinct increase in airway wall collagen.

B and D are taken with polarized light; thebright white birefringence indicates that this isthick fiber collagen, and increases in thick fibercollagen cause airway wall stiffness, leading toincreased resistance to airflow (see Ref. 192).

Fig. 9. Postulated mechanisms of small airway remodel-ing. In this model, oxidants in cigarette smoke, and perhapsoxidants released by smoke-evoked inflammatory cells,cause activation of latent TGF- on the airway epithelialcell surface and also TGF- bound to matrix, leading toTGF- signaling through Smads and connective tissuegrowth factor (CTGF), and increased collagen production.In addition, oxidant attack may activate MMP-9, which inturn can activate latent TGF-. Modeled from Refs. 33,34, 179.

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muscularization could be reduced by administration of theserine elastase inhibitor ZD0892 (188), and in smoke-exposedmice, there was an early TNF-dependent upregulation ofMMP-2, -9, -12, and -13 in the small intrapulmonary arteries,

with only MMP-12 persisting over a 6-mo period (190).However, an MMP-9/-12 inhibitor did not prevent PHT inguinea pigs (34).

In rats (164), simvastatin reduced pulmonary arterial pres-sure, perhaps through suppressing inflammation and MMP-9induction, or perhaps, as in other models of pulmonary hyper-tension (56), through mechanisms involving increased apopto-

sis or the Rho-kinase pathway. Figure 12 shows some of thefactors that appear to drive PHT in COPD.

Mechanisms of Goblet Cell Metaplasia, MucusHypersecretion, and Exacerbations

In humans, goblet cell metaplasia is a frequent finding in thelarge and small airways of cigarette smokers (115, 183), as areincreases in the number and size of mucus-producing glands inthe large airways. Indeed, the amount of intraluminal mucus inthe small airways has been shown to represent a major differ-ence between subjects with significant [GOLD Stage 3 and 4(48)] COPD compared with subjects with a lesser degree ofairflow obstruction [GOLD Stage 1 and 2 (48)] (65), and theexcess mucus production that defines chronic bronchitis is alsothought to be important in the pathogenesis of acute exacerba-tions of COPD (reviewed in Ref. 13).

As opposed to humans, bronchial glands are concentratedin the proximal trachea in rats and mice, whereas in guineapigs, they are more diffusely distributed but relatively sparsebeyond the proximal trachea (57, 181). This anatomic distri-

bution means that it is difficult to produce a model of smoke-induced chronic bronchitis in these animals, and for thisreason, the literature tends to emphasize instead goblet cellmetaplasia. However, goblet cell metaplasia, mucin produc-tion, mucin secretion, and bronchial gland hypertrophy areprobably independently regulated processes (reviewed in Ref.140). There are also a variety of types of mucin (140), andthese may not correspond exactly between humans and ani-mals. All of these factors make pathological smoke-inducedchanges related to mucin production somewhat difficult tomodel in animals.

In the guinea pig model, chronic cigarette smoke exposureinduces secretory cell metaplasia of the small non-cartilaginousairway epithelium (185) (Fig. 13), a finding reasonably analo-

gous to that seen in humans, but does not produce the luminalmucous plugs seen in patients with COPD (185). Smokingcessation reduces the degree of metaplasia (187). By contrast,in mice, tobacco smoke has a relatively minor effect, with onlya few secretory cells appearing in the small airways (11).

Some studies report significant degrees of goblet cell meta-plasia in smoke-exposed rats (84, 138, 139, 177), and this can

Fig. 10. Peribronchiolar lymphoid aggregate in a mouse exposed to smoke for6 mo. Note the pigmented smokers macrophages (arrows), a finding similar tothat seen in human smokers.

Fig. 11. Vascular remodeling. Smooth muscle actinstains of small intrapulmonary arterial branchesfrom control (A) and animal exposed to smoke for 6mo (B). Note the marked increase in smooth muscle.Such increases correlate with increased pulmonaryarterial pressure.

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be prevented, or postexposure regression enhanced, with theantioxidant/mucolytic agent N-acetyl cysteine, as well as avariety of nonsteroidal anti-inflammatory agents, and steroids(138, 139). In rats, the gastro-protective agent rebamipide(OPC-12759) decreased TNF production and reducedMUC5AC production, reduced inflammatory cells in BAL, andinhibited smoke-induced goblet cell metaplasia in the trachea(89). Tracheal goblet cell metaplasia in guinea pigs was in-duced by a 2-wk smoke exposure and could be attenuated by

use of a platelet-activating factor antagonist (82). By contrast,a PDE4 inhibitor had no significant effect on the minimalgoblet cell metaplasia induced by cigarette smoke in the mousemodel (106). These findings suggest that goblet cell metapla-

sia/mucin production is driven by a variety of mechanisms.Some of these have been elucidated in tissue culture systems(reviewed in Refs. 140, 175).

Investigation of exacerbations of COPD is an area of greatinterest, as exacerbations are a major (poor) prognostic featurein human smokers and are a major health care burden (174).This phenomenon is complicated by a lack of a universally

accepted definition (13, 101), and the triggers of exacerbationare not definitively known; bacterial and viral agents aresuggested instigators. A recent human model of virus infectionusing rhinovirus recapitulated the symptoms and signs of anexacerbation (123). Regardless of etiology, there is evidence ofupper and lower respiratory tract inflammation during exacer-bations (70).

Since chronic bronchitis is a clinical definition, involvingcough and sputum production, it is difficult to establish ananimal model as sputum production is difficult to monitor inanimals and probably sparse; the lack of diffuse increases inmucus production likewise makes it difficult to make models ofacute exacerbations. Potential models of exacerbation includeadministration of lipopolysaccharide or administration of bac-terial or viral agents with or without cigarette smoke. Forrecent reviews on models of acute exacerbations, the reader isreferred to Refs. 54, 80, 1017.

Conclusions

Taken as a whole, the data on proteases and anti-inflamma-tory agents still support the idea that inflammatory cell-derivedproteases are the major mediators of emphysema in animalmodels. However, which cells/proteases are most important inthis process remains uncertain, and there are clearly complexinteractions among them (Fig. 3). Antioxidant protection isimportant as well, and oxidative stress is linked to increases ininflammation and decreases in antiproteolytic protection,

thereby connecting oxidative damage to the protease-antipro-tease hypothesis (Fig. 3). As opposed to these various initiatingfactors, failure of the parenchyma to properly repair alsoappears to play a role in the genesis of emphysema, but the data

Fig. 12. Postulated mechanisms involved in the development of pulmonaryhypertension in COPD. Smoke (probably oxidant)-driven increases in vaso-proliferative and vasoconstrictive agents (endothelin and VEGF) and TNF-driven increases in MMP activation result in vascular remodeling and endo-thelial dysfunction. The normally vasorelaxant endothelial nitric oxide syn-

thase (eNOS) pathway is disrupted by smoke-generated oxidants and alsoby TNF, thus increasing the vasoconstrictive effects and endothelial dysfunc-tion. Vascular remodeling and endothelial dysfunction act in a vicious circleproducing pulmonary hypertension. Modeled from Refs. 189, 190, 191.

Fig. 13. Goblet cell metaplasia. Periodic acid Schiff/diastase-stained sections of small airways from controlguinea pig (A) and guinea pig exposed to smoke for 6mo (B). Note the increased numbers of mucin-contain-ing gobletcells in the epithelium of thesmoke-exposedanimal.

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are more controversial and most failure-to-repair models havenot used cigarette smoke.

This wealth of data in fact produces the interesting conun-drum that there are too many potential actors, and sorting outwhich processes are fundamental to the development of em-physema and which are epiphenomena is a crucial and difficultproblem; in this regard, it appears that MMP-12 is consistently

implicated (when looked for) in the murine models and mayrepresent a central chokepoint (Fig. 3). Whether this is true ofhumans is an important question.

One of the puzzling facts about the role of proteases andoxidative stress has always been why only some smokers,mouse or human, develop emphysema while most are spared.Part of the answer probably lies in the genotypic ability ofindividuals to mount an inflammatory/antioxidant response.Recent data on the role of T cells and the adaptive immuneresponse suggest a new paradigm that could partially explainthis question, namely that COPD (at least emphysema) is aconsequence of an autoimmune reaction to antigenic peptidesderived from the breakdown of elastin and perhaps other lungcomponents (37, 38, 98, 97, 100). Thus proteases along withreactive oxygen species would be the initial culprits initiatingthe breakdown of elastic and collagen tissue and producing thenecessary antigenic peptides to potentially trigger the autoim-mune reaction. However, only genetically predisposed hu-mans, and mice, would develop the adaptive immune responsenecessary to enhance and maintain the initial inflammatoryresponse to cigarette smoke and eventually produce disease.

The animal data suggest a variety of therapeutic approachesto human emphysema [indeed, thus far animal models haveaddressed only a few out of many potential targets (40, 41)],but translation to the human setting is not straightforward, asevidenced by the apparent lack of efficacy of anti-TNFtherapy in humans (10, 135, 174) compared with the clear role

of TNF in murine emphysema (32, 176). The reason for thisdiscrepancy is unclear but probably relates in large part to theinability to produce in laboratory animals severe COPD withcigarette smoke; i.e., the smoke-induced animal models arereproducing GOLD Stage 1 and 2 disease, but symptomaticCOPD patients have GOLD 3/4 disease, and thus far no modelreproduces either the anatomic or functional changes or thesmoke-independent progression (136), seen in such patients.This finding implies that the mechanisms driving human earlystage and late stage disease may be quite different and that theanimal models are limited to relatively early stage disease, butthat does not make the animal models useless, since effectiveearly intervention is probably much easier to design thaneffective late stage intervention.

Studies on SAR and PHT suggest that these processes are atleast partially independent of the factors that drive emphysema,although there are clearly areas of overlap, such as the role ofoxidants in SAR and serine elastases in vascular remodeling,and the same is true of increased mucus production and gobletcell metaplasia. This situation implies that a therapeutic ap-proach to human COPD may have to target each anatomiccompartment and a single therapeutic agent may not be able toprevent all of the separate manifestations.

GRANTS

This work was supported by Canadian Institutes of Health Research Grants42539 and 81409.

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