Cigarette Induce COPD

Mechanisms of cigarette smoke-induced COPD: insights from animal models
Andrew Churg, Manuel Cosio and Joanne L. Wright

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Am J Physiol Lung Cell Mol Physiol 294: L612L631, 2008. First published January 25, 2008; doi:10.1152/ajplung.00390.2007.
Mechanisms of cigarette smoke-induced COPD: insights from animal models

Andrew Churg,1 Manuel Cosio,2 and Joanne L. Wright1
Department 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 smokeinduced animal models of chronic obstructive pulmonary disease support the protease-antiprotease hypothesis of emphysema, although which cells and proteases are the crucial actors remains controversial. Inhibition of either serine or metalloproteases produces signicant protection against emphysema, but inhibition is invariably accompanied by decreases in the inammatory response to cigarette smoke, suggesting that these inhibitors do more than just prevent matrix degradation. Direct anti-inammatory interventions are also effective against the development of emphysema, as are antioxidant strategies; the latter again decrease smokeinduced inammation. There is increasing evidence for autoimmunity, perhaps directed against matrix components, as a driving force in emphysema. There is intriguing but controversial animal model evidence that failure to repair/failure of lung maintenance also plays a role in the pathogenesis of emphysema. Cigarette smoke produces small airway remodeling in laboratory animals, possibly by direct induction of brogenic growth factors in the airway wall, and also produces pulmonary hypertension, at least in part through direct upregulation of vasoactive mediators in the intrapulmonary arteries. Smoke exposure causes goblet cell metaplasia and excess mucus production in the small airways and proximal trachea, but these changes are not good models of either chronic bronchitis or acute exacerbations. Emphysema, small airway remodeling, pulmonary hypertension, and mucus production appear to be at least partially independent processes that may require different therapeutic approaches. animals; emphysema; small airway remodeling; pulmonary hypertension; chronic obstructive pulmonary disease
1
(COPD) is now the fth leading cause of death worldwide (126). A meta-analysis using data from 28 countries suggests that the prevalence of COPD based on spirometric measurements is 9 10% in adults over age 40. It has been estimated that there are 1517 million individuals with COPD in the United States alone (62, 160). By far, the most important risk factor for COPD in the developed 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 there is much less information available about these etiologies. Genetic predisposition seems to play an important role in the development of COPD, and a variety of genetic polymorphisms related to levels of antiproteases (1-antitrypsin, the best established and the most clearly important), metalloproteases, proinammatory and probrotic cytokines, and various antioxidant enzymes and detoxifying enzymes have been linked to COPD (reviewed in Refs. 43, 81, 147, 178). This review will concentrate on mechanistic insights from laboratory animal models of COPD caused by cigarette smoke, both because of the overwhelming role of cigarette
CHRONIC OBSTRUCTIVE PULMONARY DISEASE
Address for reprint requests and other correspondence: A. Churg, Dept. of Pathology, Univ. of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5 (e-mail: achurg@interchange.ubc.ca). L612
smoke as a causative agent, and because of a lack of animal models based on dusts, air pollution particles, and biomass fuels. We will emphasize in vivo models and draw on data from tissue culture and explant studies where these appear directly relevant; for reviews that focus more on in vitro experiments, molecular mechanisms, potential targets, and non-smoke-induced COPD models, see Refs. 7, 8, 18, 40, 41, 99, 156, 197. There are plans to cover, in a future review in this journal, the pros and cons of various different animal models of COPD, including cigarette smoke-induced, elastase-induced, and genetic manipulation-induced models, and so these are not addressed here. Because human COPD really consists of four anatomic lesions (emphysema, small airway remodeling, vascular remodeling with pulmonary hypertension, mucus overproduction and chronic bronchitis) and one functional lesion [acute exacerbation (13, 26)], we have separated our discussion into these broad categories. In some senses, this separation is articial, since many patients have airow limitation because of both emphysema and small airway remodeling, and they may have pulmonary hypertension and chronic bronchitis and develop acute exacerbations as well. However, as will become apparent, the mechanisms behind these anatomic lesions may well be different, so separating them is useful in understanding pathogenesis and in devising therapeutic approaches.
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ANIMAL MODELS OF COPD
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Mechanisms Related to the Protease-Antiprotease Hypothesis of Emphysema Chronic (generally 6 mo or longer) exposure of mice or guinea pigs to cigarette smoke produces lesions that morphologically and physiologically resemble a mild form of centrilobular human emphysema (184) (Fig. 1), and emphysema is the lesion that has received the most study, although the anatomic changes differ from those in humans in a subtle way because these animals do not normally have respiratory bronchioles, the locus of initial destruction in human smokeinduced centrilobular emphysema. The classic theory of emphysema is the protease antiprotease hypothesis. This hypothesis was formulated from the observation that humans decient in 1-antitrypsin (A1AT) developed early emphysema, particularly if they smoked (85), and from the experiments of Gross and colleagues (59) showing that instillation of elastolytic enzymes produced emphysema in experimental animals. These observations lead to the idea that smoke evokes an inammatory cell reaction and that these cells release proteases that overwhelm the antiproteolytic defenses of the lower respiratory tract, leading to matrix destruction and emphysema. Despite a variety of new theories, the proteaseantiprotease hypothesis (now expanded to include metallo and cysteine proteases as well as the original serine proteases) remains the generally accepted basis for the destruction of matrix that leads to emphysema, and is the basis of most experimental smoke exposure models, but exactly what cells/ proteases are crucial to this process is a complex and controversial issue. That smoke evokes an inammatory response in both humans (reviewed in Ref. 165) and animals is clear. Every experimental animal study that has looked at lavage/tissue neutrophils, 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 magnitude of the effect vary (Tables 1, 2, 3), and smoke induces proinammatory cytokine release from cultured macrophages, epithelial cells, and broblasts (90, 195, 198). Most but not all
(68, 97) authors report an increase in gene expression/protein in whole lung/bronchoalveolar lavage (BAL) of chemoattractant and proinammatory 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 hypothesis postulated 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; some reports have shown localization of neutrophils in areas of tissue destruction in human emphysema (39) but others have failed to nd any correlations between neutrophil numbers in tissue sections and the severity of lung destruction (47, 49). Table 1 summarizes interventions related to serine proteases. Shapiro et al. (155) showed that mice lacking neutrophil elastase were 59% protected against emphysema, strong evidence for a role for neutrophil elastase (see below). In acute smoke exposure studies, levels of lavage neutrophils correlated with levels of lavage desmosine, a marker of elastin breakdown, and lavage hydroxyproline, a measure of collagen breakdown (30, 44, 186), and administration of anti-neutrophil antibodies before smoke exposure reduced both neutrophils and matrix breakdown (44). In chronic studies, inhaled (128) or injected (30) A1AT or the synthetic serine elastase inhibitor ZD0892 (186) provided partial protection against emphysema (Table 1). Takubo et al. (163) and Cavarra et al. (24) showed that pallid mice, which are naturally decient in A1AT, developed earlier emphysema than strains with normal A1AT levels. While these reports support a role of the neutrophil/neutrophil elastase in the genesis of emphysema, they produced the surprising result that all interventions decreased the inammatory response and A1AT suppressed smoke-induced elevations of TNF as well (30). There is extensive evidence from alveolar epithelial cell and alveolar macrophage cultures as well as whole mouse and human lung tissue that smoke directly evokes an inammatory response by activating NF-B (112,
Fig. 1. Cigarette smoke-induced emphysema in guinea pigs. Photomicrographs of lung from a control (A) and a 6-mo smoke-exposed (B) animal showing the typical pattern of centrilobular-like emphysema seen in small laboratory animals chronically exposed to smoke. Note that the process of air space enlargement primarily affects alveolar ducts.
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Table 1. Effects of interference with proteases in chronic smoke exposure studies

Serine Protease Inhibition or Manipulation Report (Ref. no.) Species/Strain Intervention Inammatory Response % Protection Against Emphysema Comments
Wright (186)
Guinea pig
ZD0892 (serine protease inhibitor) A1AT injected (prolastin)
Decreased
45%
Churg (30)
C57Bl/6 mouse
Decreased
67%
Shapiro (155) Pemberton (128) Takubo (163) Cavarra (24)
C57Bl/6 mouse Mouse Pallid mouse Pallid mouse
Neutrophil elastase knockout A1AT inhaled (recombinant human A1AT) A1AT decient A1AT decient
Decreased neutrophils, macrophages Decreased Only CD4 cells signicantly increased in tissue Not reported
59% 72% Early onset emphysema Early onset emphysema
Decreased BAL desmosine, hydroxyproline; treatment for last 2 mo of smoke exposure did not protect against emphysema Decreased BAL desmosine, hydroxyproline; treatment roughly doubled serum A1AT levels and suppressed smoke-mediated increases in serum TNF MMP-12 and neutrophil elastase degrade each others inhibitors Highest dose increased BAL PMN and gave less protection against emphysema Pan-lobular emphysema Pan-lobular emphysema
Metalloprotease and Cysteine Protease Inhibition or Manipulation Report (Ref. no.) Species/Strain Intervention Inammatory Response % Protection Against Emphysema Comments
Hautamaki (63) Shapiro (155) Mahadeva (99) Pemberton (127) Martin (105) Martin (105) Selman (152) Churg (34)
129 mouse C57Bl/6 mouse Mouse Mouse Mouse Mouse Guinea pig Guinea pig
MMP-12 knockout
MMP-9 knockout Broad spectrum MMP inhibitor GM6001 Broad spectrum MMP inhibitor RS113456 Broad spectrum MMP inhibitor RS132908 Broad spectrum MMP inhibitor CP471474 MMP-9/-12 inhibitor AZ11557272 IL-18R knockout
Decreased macrophages but not BAL neutrophils Not reported Decreased Not reported Not reported Decreased Decreased
100%
MMP-12 degrades A1AT and neutrophil elastase degrades TIMP-1 90% average value for 2 highest doses Started after 3 mo of smoke exposure; no increase in mean air space size between 3 and 6 mo Measured as alveolar area at 4 mo; decreased MMP-9 levels at 4 mo Decreased BAL desmosine; strong correlation BAL desmosine with mean air space size; inhibitor also prevents small airway remodeling Decreased apoptosis; decreased cytokines; decreased cathepsin B, S
0% 90% 100% 75% 30% 68%
Kang (78)
Mouse (C57)
Decreased
51%
All studies use 6 mo of smoke exposure, except as noted.
195), and possibly the aryl hydrocarbon receptor (167) and Toll-like receptor-4, at least early on (98). Recent data also suggest that smoke inactivates histone deacetylases, leading to prolonged (and non-steroid-sensitive) inammation (9, 74, 112). TNF production is driven by NF-B and TNF is generally presumed to be a driver of inammatory cell inux in smokers. Thus, a priori, one would expect continuing cigarette smoke exposure to generate a continuing inammatory response, and serine protease inhibitors should provide protection against emphysema without decreasing inammation; suppression of the inammatory response thus implies additional anti-inammatory mechanisms are at work (see below). Interference with/manipulation of metalloproteases. In the last 15 years, there has been an increasing interest in metalloproteases (MMP) as mediators of emphysema. This has stemmed in part from the recognition that a number of metalloproteases,
including MMP-9 and MMP-12 (124), can degrade elastin; in part from 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 uid, alveolar macrophage supernatant, or whole lung tissue from smokers with emphysema compared with those without; and in particular, from the report (63) that mice with a targeted deletion of MMP-12 (MMP-12/) failed to develop emphysema after cigarette smoke exposure. Cigarette smoke causes increased whole lung or alveolar macrophage levels of MMP-2, -9, -12, -13, and -14 in mice (32) and MMP-1 in guinea pigs (151). Table 1 lists experimental studies using genetically targeted mice or MMP inhibitors in smoke exposure models. Several broad conclusions stand out. First, MMP inhibition or deletion can signicantly or even totally abrogate the development of emphysema, indicating a clear role for MMPs in this process. In fact, with broad
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spectrum MMP inhibitors, it is sometimes possible to achieve greater protection against emphysema than with serine elastase inhibitors. Second, the choice of which MMP to target is crucial. Mice lacking MMP-12 are completely protected against emphysema (63, 155), whereas mice lacking MMP-9 show no protection at all (99). This latter observation is of particular interest, since it has been suggested from studies of cultured alveolar human macrophages that MMP-9 is the major mediator of emphysema in humans (143, 144), and some have denied a role for MMP-12 in humans (72). Selman et al. (152) found little protection with a broad spectrum MMP inhibitor in guinea pigs, but we (34) found 70% protection in guinea pigs given a combined MMP-9/-12 inhibitor, AZ11557272, indicating that MMPs are not just conned to murine emphysema models, and lending support to the idea that one or both of these MMPs may be central players in humans. Animals exposed to smoke and AZ11557272 had 70% protection against decreases in airow compared with animals exposed to smoke alone, thus showing that prevention of anatomic changes in animal models confers a corresponding physiological benet. However, as is true of serine proteases, the exact role of MMPs in the pathogenesis of emphysema is unclear, particularly since neutrophils/serine proteases and MMPS appear to interact, and MMP inhibition reduces smoke-induced neutrophil and macrophage inux (29, 34, 128, 155). In an acute smoke model using MMP-12/ mice, we (29) showed that MMP-12 was required for smoke-induced neutrophil inux and neutrophils were required for matrix breakdown. This latter idea was also supported by the nding that the broad spectrum MMP inhibitor RS113456 acutely inhibited neutrophil inux and matrix breakdown (44). But in a 6-mo smoke model, Shapiro et al. (155) found that MMP-12/ mice developed a BAL neutrophilia comparable to that of wildtype animals, implying that proinammatory mechanisms change over time; we have seen a similar late neutrophil recruitment effect in animals treated with A1AT and in TNF receptor (p55 p75/) knockout mice (30, 32). It is of interest in this regard that Stevenson et al. (161), using gene microarrays, recently reported that gene expression patterns in rats change from an early proinammatory to a later pattern of enhanced acquired immunity, although these data must be viewed with great caution, since rats are extremely susceptible to (and the Stevenson paper indeed found) the phenomenon known as particle overload (reviewed in Ref. 119), and particle overload leads to prolonged generation of inammatory, brogenic, and probably mutagenic mediators by alveolar macrophages. Additional evidence for interactions of neutrophils and macrophages comes from Shapiro et al. (155) who showed that neutrophil elastase activates pro-MMP-12 and destroys tissue inhibitor of metalloprotease (TIMP)-1; conversely, MMP-12 degrades A1AT. Thus neutrophil elastase and MMP-12 cooperate to increase each others proteolytic potential. There appear to be a variety of mechanisms by which serine and metalloprotease inhibitors exert anti-inammatory effects. We (186) found that the serine elastase inhibitor ZD0892 inhibited acute smoke-mediated increases in gene expression of the neutrophil/macrophage chemoattractants MIP-2 and MCP-1 in mice. We also observed that alveolar macrophages
from MMP-12/ mice did not increase TNF secretion after smoke exposure, apparently because MMP-12 functions as a form of TNF converting enzyme that liberates active TNF, (Figs. 2 and 3) and lack of TNF appeared to be one reason for a lack of neutrophil inltration in the lungs of MMP-12/ animals (31). As well, A1AT suppresses smoke-mediated increases in TNF release by inhibiting the serine proteases thrombin and plasmin that leak into the air spaces after smoke exposure (31, 35); thrombin and plasmin in turn activate proteinase-activated receptor-1 (PAR-1), thereby causing MMP-12 and TNF release (32, 133) (Fig. 2). This whole set of observations would remove MMP-12 from a matrix destructive role to a signaling role in the context of cigarette smoke exposure, and signaling roles are now recognized as major functions of MMPs (125). An alternate explanation for decreases in inammatory cell inux is that inhibition of matrix destruction by either neutrophil or macrophage-derived proteases decreases the generation of chemotactic matrix fragments (1, 69, 129, 153, 154). Houghton et al. (68) observed that the lavage uid of wild-type mice contained elastin fragments that were chemoattractants for monocytes and that this chemoattractant activity was absent from the lavage of MMP-12/ mice. They suggested that matrix breakdown in emphysema is driven by both neutrophil elastase and MMP-12, with the latter the major player because of the relatively large numbers of monocytes that migrate into the lung after smoke exposure. In practice, MMP-12 may well play both a signaling and a direct matrix destructive role. More recently, Maeno et al. (97) have proposed that this whole process of smoke-driven matrix destruction is initiated by CD8 lymphocyte-mediated production of IFN/IP-10, with resulting neutrophil and macrophage inltration and in-
Fig. 2. Postulated mechanisms of cigarette smoke-induced MMP-12 release. Smoke directly activates Toll-like receptor-4 (TLR4) and probably other (as yet unidentied) cell surface receptors that drive MMP-12 release. In addition, smoke causes leakage of plasma proteins into the alveolar spaces. These include prothrombin and plasminogen, which are converted to thrombin and plasmin by alveolar macrophage-derived tissue factor (TF) and plasminogen activator (PA); thrombin and plasmin in turn activate proteinase-activated receptor-1 (PAR-1), which causes MMP-12 release. IP-10 derived from smoke-evoked T cells also causes increased macrophage production of MMP-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 are similar to ndings reported in human lungs with emphysema by Grumelli et al. (60). Cantor et al. (20, 21) showed that inhaled hyaluronan (HA) can bind to elastin bers in vivo and in vitro and prevents elastolysis by elastases. HA reduced air space enlargement caused by porcine pancreatic elastase (20), provided 100% protection against emphysema in smoke-exposed DBA/2J mice, and reduced smoke-mediated increases in lavage desmosine. These ndings support matrix attack as a fundamental feature of smoke-induced emphysema. Cysteine proteases. Kang et al. (78) showed that smoke induces production of the cysteine proteases cathepsins B and S in mice via a mechanism involving IL-18, and, probably, interferon-. These proteases can degrade matrix components and thus might play a role in emphysema. The same report found greater levels of cathepsins B and S in alveolar macrophages from cigarette smokers compared with controls. IL18R/ mice were signicantly protected against emphysema and had decreases in smoke-induced lavage neutrophils and macrophages as well as a variety of chemokines and cathepsins, along with decreased levels of MMP-12. These data thus present the same mix of possible effectors seen with studies inhibiting/deleting serine and metalloproteases. Anti-inammatory interventions. If the protease-antiprotease hypothesis (i.e., that the smoke-driven inammatory response
Fig. 3. Postulated mechanisms of matrix attack and emphysema. In this model, smoke causes release of a variety of chemokines from structural cells and alveolar macrophages, leading to an inux of neutrophils (PMN), macrophages, lymphocytes, and dendritic cells. MMP-12 causes conversion of pro-TNF to active TNF, thereby amplifying the inammatory process. Neutrophil elastase and macrophage-derived MMP-12, as well as other proteases, can directly attack the alveolar wall, leading to both matrix destruction and liberation of elastin fragments, which are chemoattractants for macrophages, further amplifying the inammatory response. The scheme shown here encompasses the ndings of Lee et al. (94) that COPD patients have antielastin antibodies, so that protease-derived elastin fragments evoke an autoimmune response, leading to further matrix attack and production of IFN/IP10, which itself increases MMP-12 release by macrophages and also increases matrix destruction. Other matrix fragments or smoke-modied non-matrix proteins might function in a similar fashion. In addition, neutrophil elastase degrades TIMPs and MMP-12 degrades 1-antitrypsin (A1AT), thus removing anti-proteolytic defenses, and oxidants in the smoke may inhibit A1AT and other antiproteases such as SLPI as well. This scheme thus incorporates an innate immune response to smoke, a protease feedback amplication system driven by matrix destruction, and an acquired immune response, also driven by matrix destruction. The relative importance of each process probably changes over time. Modeled from Refs. 31, 38, 88, 92, 97, 155. AJP-Lung Cell Mol Physiol VOL
leads to proteolytic matrix destruction) is correct, one could inhibit smoke-mediated inammatory responses instead of proteases, and a number of studies have directly or indirectly targeted the production and/or signaling of proinammatory molecules (Table 2). TNF is consistently elevated in human smokers (10, 165). In mice, knockout of TNF receptors 1 and 2 greatly reduced inammatory inltrates, emphysema, levels of some MMPs, and gene expression of proinammatory cytokines (28, 32, 46) after smoke exposure; TNF receptor 2 appeared to be the major mediator of the inammatory response (46). Conversely, inducible overexpression of TNF produced an increase in neutrophils, parenchymal B cell nodules, MMP-12, cathepsin K, and emphysema in the absence of smoke (176). There was an extremely strong correlation (R 0.89, P 0.0001) between serum TNF levels and air space size after 6 mo of smoke exposure in guinea pigs (34). While these reports imply an important role for TNF, two studies in humans using TNF antagonists failed to show a benet (10, 134, 172), suggesting that translation of anti-inammatory therapies from animal models to humans is not straightforward. Using a different anti-inammatory approach, Thatcher et al. (166) prevented neutrophil inux by administration of SCH-N, a CXCR2 inhibitor, to mice, with an 50% reduction in parenchymal cells after a 3-day smoke exposure. SCH-N did not decrease smoke-mediated increases in TNF, IL-6, or PGE2, and levels of the neutrophil chemoattractant CXCR2 ligands KC and MIP-2 were considerably elevated. Maes et al. (98) showed that, in mice, Toll-like receptor-4 plays a role in the early but not the chronic inammatory response to cigarette smoke. Phosphodiesterase-4 degrades the anti-inammatory nucleotide cyclic 35-adenosine monophosphate. In C57Bl/6 mice exposed to smoke for 7 mo, Roumilast, a phosphodiesterase-4 inhibitor, provided 100% protection against smoke-induced increases in air space size, reversed smoke-induced loss of lung desmosine, reduced neutrophil and especially macrophage inux, and more than doubled levels of the anti-inammatory cytokine, IL-10 (106). One of the effects of Roumilast is to decrease macrophage production of TNF, and in a human trial, 4 wk of Roumilast decreased blood/sputum TNF, IL-8, and neutrophil elastase, and improved FEV1 (58). IFN appears to drive many proinammatory cytokines via CCR5 (92). IFN/ or CCR5/ mice exposed to smoke for 6 mo were completely protected against emphysema and showed decreased inammatory cells, apoptosis, levels of MIP-1, MIP-1, and RANTES (16, 92). Protection may have been mediated through decreased MMP-12, since transgenic mice overexpressing IFN increased MMP-12 production (92). Administration of the 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase inhibitor, simvastatin (87), to rats completely abolished smoke-induced emphysema and prevented peribronchial and perivascular accumulation of lymphocytes. This report is difcult to interpret: the authors reported a remarkable degree of air space enlargement (80% increase in mean air space size) after a relatively short exposure period (16 wk). Furthermore, statins have been reported to increase macrophage production of MMP-12 (4), which should make emphysema worse. However, there are reports of benecial effects of statins in patients with COPD (102, 159) and reports
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Table 2. Effects of manipulation of the immune/inammatory response in chronic smoke exposure studies
Report (Ref. no.) Species/Strain Intervention Inammatory Response % Protection Against Emphysema Comments
Churg (32)
Mouse (C57Bl/6)
TNF R1 R2/
Decreased BAL macrophages, neutrophils
71%
Dhulst (46) Dhulst (46) Martorana (106) Lee (87)
Mouse (C57) Mouse (C57) Mouse (C57)
TNF R1/ TNF R2/
Increased MMP-2, -9, -12, -13, -14 with smoke exposure; knockout mice: decreased MMP-12, -13, -14; decreased gene expression of TNF, MCP-1, MIP-2 Decreased CD4 and CD8 cells 7-mo smoke exposure; increased IL-10 4-mo smoke exposure; also decreased pulmonary artery pressure; possible technical problems with study Putative protection of elastin bers; possible developmental problems No protection against small airway remodeling; decreased MMP-12, BAL PMN, T lymphocytes, macrophages Decreased apoptosis; MMP-12 probably involved Decreased cytokines, apoptosis Decreased PMN, macrophages, dendritic cells, lymphocytes; no protection against small airway remodeling Some persisting elevation in BAL PMN No T or B lymphocytes; no lymphoid follicles Decreased BAL lymphocytes and dendritic cells at 26 wk No emphysema produced in wild type or knockout
PDE4 inhibitor Roumilast Rat (Sprague- Dawley) Simvastatin
Not affected Decreased BAL neutrophils, macrophages, lymphocytes Decreased Decreased peribronchial lymphoid aggregates Not reported Decreased
None 100% 100% 100%
Cantor (21) Bracke (13)
Mouse (DBA/2) Mouse (C57Bl/6)
Hyaluron aerosol CCR6/
100% 67%
Ma (92) Ma (92) Bracke (16)
Mouse (C57Bl/6) Mouse (C57Bl/6) Mouse (C58Bl/6)
IFN/ CCR5/ CCR5/
Not reported Decreased (primarily decreased macrophages) Decreased
100% 100% 25%
Maeno (97) Maeno (97) DHulst (45) Maes (98)
Mouse (C57Bl/6) Mouse (C57Bl/6) SCID mouse Mouse (C3H/HeJ)
CD8/
Decreased
100% None None None
van der Deen (172) Mouse (FVB)
CD4/ Not decreased No acquired immune Increased neutrophils, response macrophages, dendritic cells TLR4/ Increased BAL PMN macrophages at 26 wk Multi-drug resistance Decreased inammation and protein/ inammatory mediators
All studies use 6 mo of smoke exposure, except as noted.
that statins enhance clearance of apoptotic cells (114), so this type of compound may be worth further investigation. In aggregate, both interventions directed against proteases (serine, cysteine, or metalloproteases) and interventions directed against the development of an inammatory inux provide signicant protection against emphysema in animal models and thus support the protease-antiprotease hypothesis, although the mechanism(s) involved are complex and not entirely clear. Effects of Smoke on Different Mouse Strains: Evidence for a Genetic Propensity to Emphysema As noted above, susceptibility to COPD likely results from multiple genetic and environmental effects. Cavarra et al. (24) showed that mouse strains with differing antioxidant/antiprotease capacity reacted differently to cigarette smoke (see Mechanisms Related to Oxidative Stress in Emphysema). Guerassimov et al. (61) exposed ve different strains of mice to smoke in an attempt to dene genetically susceptible and resistant varieties. The strains were chosen based on differences in the MHC haplotype, a major determinant of the inammatory response 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 and Vandivier (64), it is remarkably easy to induce alveolar enlargement with a very wide variety of manipulations in the mouse, Guerassimov et al. (61) dened emphysema as an increase in Lm along with an increase in lung compliance (Fig. 4). By these criteria, only the AKR strain had signicant emphysema, 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) and susceptible (AKR) strains utilizing expression microarrays. There were striking constitutive differences between the two strains, mainly in the expression of genes that encode for proteins with immune function. The NZW mice had higher constitutive expression of genes that inhibit differentiation and proliferation of T and B cells and protect against apoptosis and T cell activation (I203, CD72, C4, Klra-1, 8, and 13), along with higher levels of several antioxidant genes that were not prominently expressed in AKR mice. In contrast, the constitutive inammatory genes expressed in AKR mice were proin294 APRIL 2008
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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 anesthetized and 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 of 16 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 between 1 and 9 cmH2O. Only the Pallid and AKR/J show a signicant compliance increase. [From Guerassimov et al. (61).]
ammatory, and several were related to adaptive immunity. With smoke exposure there was a striking difference in gene response: the NZW strain demonstrated decreased expression of 77 of the 82 genes that were signicantly changed in response to smoke, and 44% of the genes attenuated by smoke exposure inuence immune and/or inammatory processes including immunoglobulins, complement, chemokines, cytokines, and other proinammatory factors that enhance the functions 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 genes that were changed by smoke exposure, and 25% of the genes increased after smoking had functions related to the immune response; 13% were proapoptotic (Fig. 5). In another study aimed at investigating the potentiation of inammation by cigarette smoke, Reynolds et al. (137) investigated 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 inammatory cells after smoke exposure. Egr-1 expression was marginally detected by immunochemistry in the lungs of nonsmoking mice, but increased markedly in the susceptible AKR strain after smoke exposure. However, Egr-1 was only minimally induced in the lungs of the resistant NZW. These studies suggest that resistant animals do not increase or actively decrease the proinammatory response to smoke while increasing the antioxidant response, thus preventing matrix breakdown and a potential acquired immune response to
matrix fragments. In contrast, inammation in the susceptible strain is progressive, probably due to ongoing stimulation of innate and adaptive immunity by the expression of genes involved in antigen presentation and T and B cell activation. These ndings support the possibility of an autoimmune process triggered by smoke exposure as a factor important in the maintenance of the inammation and the development of emphysema. Evidence for Acquired Immunity in Animal Models of Smoke-Induced Emphysema An abnormal inammatory response is an important component in the denition of COPD (131). Besides neutrophils and alveolar macrophages, both CD4 (T helper) and CD8 (cytotoxic) T cells are increased in the airways and lung parenchyma of patients with COPD with a predominance of CD8 T cells, and Finkelstein et al. (49) showed that in human lungs, there is a correlation between the number of T lymphocytes/mm3 of lungs and the extent of emphysema. This inltration with T cells, seen in smokers who develop COPD, but not in normal smokers, represents an activation of the adaptive immunity that presumably follows from the initial and then sustained innate immune response characterized by increased numbers of macrophages and neutrophils. The T cells found in patients with COPD are fully activated (60, 146), expressing a large array of Th1 chemokines and cytokines. This inammatory process most likely also involves
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Fig. 5. Overall differences in gene expression proles in mice resistant, NZW, and susceptible, AKR, to the development of emphysema after 6 mo of cigarette smoke exposure. Gene expression proles were investigated in 3 animals per control and exposed groups using Affymetrix Microarray Analysis. Compared with their controls, NZW mice largely downregulated gene expression, whereas AKR mice showed the opposite pattern.
the migration of dendritic cells, since it has been recently reported that T cells in humans with emphysema are being presented with antigens derived from the breakdown of elastin (88). These ndings support a role for autoimmunity in the pathogenesis of COPD. Guerassimov et al. (61) investigated the inammatory response to long-term cigarette smoking in the mice with different susceptibilities to emphysema described in EFFECTS OF SMOKE ON DIFFERENT MOUSE STRAINS: EVIDENCE FOR A GENETIC PROPENSITY TO EMPHYSEMA. To approximate human studies, they used morphometric methods to quantitate the percentage of immunostained inammatory cells in the alveolar walls. After 6 mo of smoking, only the susceptible AKR strain had a orid cellular inammatory inltrate comprising CD4, CD8, and T cells, along with macrophages and neutrophils. Neither the resistant nor the mildly susceptible strains exhibited an inammatory inltrate containing T cells other than some cells in the NZW strain (Fig. 6). Similar results were found by Takubo et al. (163) in the Pallid mouse, a strain that also develops marked emphysema after 6 mo of smoking exposure. Analysis of inammatory chemokines and cytokines in the lungs (Fig. 7) in the three groups with different susceptibilities conrmed that a true Th1 inammatory response, secondary to T cell activation, had developed in the susceptible AKR mice but was not present in the resistant or mildly susceptible strains. These ndings are of interest since they mimic the inammatory changes that smokers develop, and, as in the human
smokers, only the susceptible animals develop the adaptive immune reaction seen in humans. The importance of CD8 T cell inammation in the development of cigarette-induced emphysema in mice has been recently demonstrated by Maeno et al. (97). In contrast to wild-type mice that showed macrophage, neutrophil, and lymphocyte inammation and emphysema after 6 mo of exposure, CD8/ mice had a blunted inammatory response and did not develop emphysema when exposed to long-term cigarette smoke. They showed that the CD8 T cell product IFNinducible protein-10 induces production of MMP-12 that degrades elastin, causing lung destruction and generating elastin fragments that attract more macrophages and probably act as an antigenic stimulus to maintain the adaptive immune reaction bringing more T cells into the lung. It should be noted that some of the literature in this area is contradictory. SCID mice, which completely lack B and T cells, develop emphysema of a degree comparable to that of the wild type (Balb/C), along with an inammatory inux of neutrophils, macrophages, and dendritic cells, and increased levels of MMP-12 (45). However, some workers have found that SCID mice develop hemorrhage after smoke exposure, making emphysema difcult to measure (Shapiro SD, personal communication). As well, reported laboratory to laboratory variations in response to a given mouse strain seem to translate into different molecular responses. For example, two of the authors of this review (Churg and Wright) have consistently found an 35 40% increase in air space size and a smokeinduced elevation in gene and protein expression of TNF and other proinammatory mediators in C57Bl/6 mice (28, 30), but the third author of this paper (Cosio) nds much smaller increases in air space size and has not observed such inammatory responses in C57Bl/6 mice. The mice used by Maeno et al. (97), which developed a CD8 T cell response (see above), were also C57Bl/6 based. It is unclear if these ndings indicate the presence of small genetic differences in the same mouse strain obtained from different sources, or mean that different smoke exposure conditions and smoke exposure systems can force animals of the same notional strain into the type of proinammatory and adaptive immune response just described. Mechanisms Related to Oxidative Stress in Emphysema Cigarette smoke is an extremely concentrated source of reactive oxygen species (ROS) and reactive nitrogen species (130). The inammatory response to smoke potentially augments oxidative stress, since neutrophils and macrophages release ROS, and those from smokers release even greater amounts (17, 66, 91, 95, 96). There is considerable biochemical evidence of oxidative stress in cigarette smokers and greater levels in those with COPD (reviewed in Refs. 14, 95, 96). These changes include increased exhaled H2O2 and 8-isoprostane, decreased plasma antioxidants, and increased plasma and tissue levels of oxidized proteins, various lipid peroxidation products such as 4-hydroxynonenal, as well as protein tyrosine residues/3-nitrotyrosine, indicators of attack by reactive nitrogen species. Antioxidant enzyme levels are also altered. Oxidative attack may inactivate antiproteases such as SLPI (25), and oxidative damage is believed to decrease production of/inactivate some
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Fig. 6. Inammation in the alveolar wall in mouse strains with different susceptibilities to the development of emphysema after 6 mo of cigarette smoke exposure. In these experiments, only the susceptible AKR mice showed a full inammatory prole. PMN, neutrophil; AM, alveolar macrophage; CD4, CD4 T cell; CD8, CD8 T cell. [From Guerassimov et al. (61).]
histone deacetylases (9, 74), leading to a prolonged inammatory response. Evidence of oxidant attack is also seen in animal models. Aoshiba et al. (2) showed that even a 1-h exposure of mice to cigarette smoke resulted in immunochemically detectable 4-hydroxynonenal and 8-hydroxy-2-deoxyguanosine, a marker of oxidative DNA damage, in the airway and alveolar epithelial cells. We (27) found that a 10-min smoke exposure produced lipid peroxidation in rat tracheal explants. Rangasamy et al. (132), using microarrays, showed upregulation of a variety of antioxidant enzymes after smoke exposure of ICR mice. McCusker and Hoidal (109) found increases in superoxide dismutase and catalase expression in alveolar macrophages from smoke-exposed hamsters, whereas Cavarra et al. (25) showed depletion of protein thiols, ascorbic acid, and glutathione, all antioxidant substances, in smoke-exposed C57Bl/6 mice. Interference with the antioxidant response by deletion of Nrf2 (Nrf2/) (71, 132), a redox-sensitive transcription factor that upregulates a variety of detoxication and antioxidant genes, caused increased smoke-induced inammation and earlier and more severe emphysema than in wild-type (ICR or ICR/Balb/c) mice, and Nrf2/ mice had impaired upregulation of antioxidant enzymes (157). As well, Nrf2/ mice were decient in A1AT and secretory leukoprotease inhibitor (SLPI). These ndings link a lack of antioxidant protection to the protease-antiprotease hypothesis (Fig. 3). Conversely, several investigators have acutely increased antioxidant protection (Table 3) with consequent decreases in inammatory cell inux, protein oxidation, inammatory mediators, and airway squamous metaplasia after short-term smoke exposure (5, 116, 158). Rubio et al. (142) found that the antioxidant N-acetyl cysteine ameliorated elastase-induced emphysema in rats. Cavarra et al. (24) reported that ICR mice, which increased antioxidant levels in BAL after smoke exposure, were proAJP-Lung Cell Mol Physiol VOL
tected against emphysema, whereas C57Bl/6 and DBA/2, which did not increase antioxidant levels, developed emphysema; of note, levels of antioxidant protection appeared to be more important than BAL levels of elastase inhibitory capacity in determining the severity of emphysema. Foronjy et al. (52) showed that chronically smoke-exposed transgenic CuZnSOD animals had much smaller increases in neutrophil and macrophage inltration than did wild-type animals, did not show increases in production of a variety of MMPs including MMP12, were protected against generation of lipid peroxidation products, and were 100% protected against the development of emphysema. There was substantial protection against elastaseinduced emphysema as well. These studies thus support a role for oxidant attack in the genesis of smoke-induced emphysema, but it is noteworthy that the immediate mechanism of attack appears to be through increases in inammatory cells, and, probably, decreases in antiprotease protection, thus linking oxidant attack to the protease-antiprotease hypothesis. Mechanisms Related to Repair of Alveolar Structure in Emphysema Retinoids accumulate in the lung during embryogenesis and are essential to alveolar septation; mice lacking retinoid receptors have lower levels of elastin and enlarged alveolar spaces (108). Massaro and Massaro (107) reported that treatment with all-trans retinoic acid reversed the emphysematous changes seen in rats after instillation of elastase, and this was conrmed in rats by Belloni et al. (12). However, reports in other species and with cigarette smoke have been discouraging. March et al. (103) did not nd any protective effects of retinoids against smoke-induced emphysema in either A/J or B6C3F1 mice, and Meshi et al. (110) did not observe any benet in smoke-exposed guinea pigs. A human trial of retinoid therapy did not show any clear improvement in
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Mechanisms Related to Failure to Repair/Failure of Lung Maintenance in Emphysema While there appears to be overwhelming evidence that cigarette smoke-induced damage to the alveolar wall matrix as a result of inammatory cell-derived protease, and, probably, oxidant, attack (but, again, with oxidant-driven increases in inammatory cells) is the driving force behind emphysema, one of the curious features of emphysema is that the alveolar wall largely fails to regenerate new matrix. This is in sharp contradistinction to the small airways and the intrapulmonary arteries, where the response to smoke is a marked increase in matrix and/or structural cells (see Mechanisms of Small Airway Remodeling and Mechanisms of Vascular Remodeling and Pulmonary Hypertension), despite the fact that these anatomic compartments are separated by only a few micrometers. A variety of theories, conveniently grouped as failure to repair/failure of lung maintenance have been advanced to account for this phenomenon (reviewed in Refs. 134, 170, 171). Increases in apoptotic cells, sometimes accompanied by decreases in proliferating cells, have been found in severely emphysematous compared with nonemphysematous human lungs (73, 100, 170, 196). Administration of agents that cause endothelial or epithelial cell apoptosis to animals results in rapid air space enlargement, although, unlike smoke-induced emphysema, this process is also rapidly reversible and is not accompanied by an inammatory response (3, 79). In tissue culture models, smoke exposure interferes with cell proliferation, chemotaxis, and production/remodeling of matrix components by broblasts (23, 134) and depresses the production and activity of lysyl oxidase (53, 86), an enzyme crucial to the formation of stable insoluble elastin and collagen. Fibroblasts from emphysematous human lungs proliferate more slowly than those from nonemphysematous lungs (67, 117) and show increased expression of a variety of senescence-associated markers (118, 169). Only a few animal models have looked at this issue using smoke exposure. Mice lacking senescence-associated marker 30, an anti-aging calcium binding protein, show increased
Fig. 7. Cytokine and chemokine gene expression in mouse strains with different susceptibilities to the development of emphysema. In these experiments, NZW mice tend to downregulate gene expression, C57Bl/6 mice tend not to change expression patterns, and AKR mice tend to upregulate expression. [From Guerassimov et al. (61).]
emphysema (141). But this issue still needs further investigation because the dose of retinoic acid may be crucial. Stinchcombe and Maden (162) have recently observed that retinoic acid will restore alveolar architecture that is disrupted by steroid treatment of newborn mice if a high enough dose is used.
Table 3. Effects of antioxidant manipulation in acute and chronic smoke exposure studies
Report (Ref. no.) Species/Strain Intervention Inammatory Response Effect on Emphysema Comments
Nishikawa (116) Smith (158) Banerjee (5) Cavarra (24)
Guinea pig Rat Guinea pig ICR, C57, DBA/2 mice
rhSOD Catalytic antioxidant AEOL 10150 Black tea (antioxidant) in drinking water ICR mice have increased antioxidant protection, cf. to C57, DBA/2 Nrf2 knockout Nrf2 knockout Tg ZnCuSOD
Decreased Decreased Not reported Not reported
Acute study only Acute study only Acute study only 5% increase in mean air space size in ICR, not signicant; C57 and DBA/2 get emphysema Earlier and more severe Earlier and more severe 100% protection
Decreased IL-8 gene expression, NF-B activation Decreased MIP-2, ICAM-1, squamous metaplasia in airways Decreased oxidation of lung proteins Elastin content decreased in C57 and DBA/2 but not in ICR
Rangasamy (32) Iizuka (71) Foronjy (52)
Mouse (ICR) Mouse (Balb/c) Mouse
Increased Increased Decreased
Decreased A1AT; increased apoptosis Decreased SLPI Protects against smoke-mediated increase in lipid peroxidation
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emphysema when exposed to smoke (148) and also show increased numbers of apoptotic lung structural cells (113). In elastase instillation emphysema, concurrent smoke exposure inhibits new elastin synthesis, whereas with elastase alone, there is brisk production of new protein (121). We have observed, using laser capture microdissection, that the parenchyma initially responds to smoke by upregulation of genes that produce matrix components, but that over time most of these genes are downregulated, suggesting that failure to repair is not a simple yes or no proposition, but a process that changes with increasing exposure (Churg, unpublished data). Kang et al. (78) showed that smoke activates IL-18 and causes apoptosis by direct or IL-18R-mediated activation of a number of caspases. IFN/ mice exposed to smoke for 6 mo had decreased levels of inammatory cells, apoptosis, and emphysema (92). In Nrf2/ mice (132), there was an increase in apoptotic parenchymal cells with smoke exposure. Smoke disrupts lung VEGF signaling in rats, and VEGF appears to be an important trophic mediator of lung endothelial cell survival (79). Tuder et al. (171) have proposed that oxidative damage and apoptosis are the primary events in emphysema and that inammation is secondary, but this idea is contradicted by evidence that smoke directly drives inammation, even in monolayer cultures where smoke causes cytokine release without apoptosis (see Mechanisms Related to the Protease-Antiprotease Hypothesis of Emphysema). Guinea pigs and most genetically intact mice that develop emphysema do not develop increased 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 but not C57Bl/6 mice. The question of whether apoptosis is a primary or secondary event in emphysema (and even whether it occurs in excess) is unresolved and is not straightforward, because apoptosis of epithelial cells (anoikis) may also occur as a result of destruction of the underlying matrix (55). It should also be noted that there is not a total absence of repair in emphysema, since human centrilobular emphysema is associated with increased amounts of collagen (22). Nonetheless, the data suggest that there is ineffective repair in the parenchyma after long-term smoke exposure. Mechanisms of Small Airway Remodeling Small airway remodeling (increases in bronchiolar wall brous tissue, muscle, inammatory cells, and luminal mucus) (SAR) is now recognized as an important cause of airow obstruction in cigarette smokers (65, 122). There are only a few reports that have examined smoke-induced SAR in animals. The changes found are subtle and easily missed on casual examination, and some investigators deny that SAR occurs (104). However, careful morphometric studies from several laboratories have conrmed that there is indeed SAR, largely manifest as increases in airway wall collagen, in both guinea pigs (34) and mice (15, 33, 16) after chronic smoke exposure (Fig. 8). Wright and colleagues (192) reported an increase in thick collagen bers (Fig. 8), an effect that probably increases airway stiffness, in the small airways of guinea pigs exposed to cigarette smoke for 6 mo. They found negative correlations of the amount of thick collagen bers with peak
expiratory ow and FEV0.1/FVC, and positive correlations with airway resistance, thus indicating that smoke-induced airway remodeling in animals is associated with abnormal physiology. In humans, the usual assumption has been that, since emphysema is driven by inammatory 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 of evidence against a primary role of inammation in SAR are reports (15, 16) that mice lacking CCR6 and CCR5 (receptors for various chemoattractant chemokines) had decreased inammation 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 an MMP-9/-12 inhibitor, again evidence that mechanisms other than inammation are important (34). Using laser capture microdissection of small airways (bronchioles) from the lungs of smoke-exposed C57Bl/6 mice, Churg et al. (33) found that smoke persistently upregulated gene expression of type I procollagen and probrotic cytokines, particularly those related to TGF- signaling. With a single acute exposure, elevations in gene expression were seen within 2 h of starting smoke exposure and mostly decreased over 24 h, as opposed to numbers of lavage inammatory cells that increased slowly over 24 h (44), implying that, at least in the short term, upregulation of the brotic response is independent of inammation. In rat tracheal explants, an airway model system that is free of smoke-evoked inammatory cells, a very brief (15-min) exposure to smoke resulted in increases in the same set of genes mentioned above, along with collagen (hydroxyproline) by 24 h, and these increases could be prevented with an inhibitor of TGF- receptor 1 (Churg, unpublished observations) or a TGF- competitor, fetuin (179). The smoke-exposed explants released increased amounts of TGF-1 via an oxidant-driven mechanism (Fig. 9). In short, the animal models suggest that small airway remodeling is driven by direct smoke induction of brogenic growth factors, and the role of inammatory cells is uncertain; rather, the initial driving force appears to be oxidant-mediated activation of TGF-. However, since neutrophils and macrophages release oxidants in response to smoke (17, 66, 91, 95, 96), such cells might potentiate TGF- release and hence potentiate small airway remodeling (Fig. 9). Both B and T lymphocytes are increased in the small airway walls in human COPD (37, 65, 145, 173). Whether they play a direct role in the pathogenesis of SAR or reect colonization of the small airways by infectious agents in patients with advanced COPD is unclear (65). In cigarette smoke-exposed animals, peribronchial lymphoid aggregates increase in number (Fig. 10), but in CCR6/ and CCR5/ mice, a lack of peribronchial lymphoid aggregates with chronic smoke exposure did not protect against SAR (15, 16). Mechanisms of Vascular Remodeling and Pulmonary Hypertension Pulmonary hypertension (PHT) is a relatively common form of cigarette smoke-induced lung disease and develops in 6% of subjects with COPD, but is present in 40% of patients with an FEV1 of less than 1 l (6, 19, 93, 94, 180). PHT is an important complication since it is a signicant predictor of
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Fig. 8. Small airway remodeling in guinea pigs. Photomicrographs of Picrosirius Redstained small airways (membranous bronchioles) from control (A and B) and 6-mo smokeexposed (C and D) guinea pigs. Note the distinct increase in airway wall collagen. B and D are taken with polarized light; the bright white birefringence indicates that this is thick ber collagen, and increases in thick ber collagen cause airway wall stiffness, leading to increased resistance to airow (see Ref. 192).
mortality, and is a major cause of morbidity, in patients with COPD (36, 168, 189). The mechanism(s) of PHT in smokers is not known. Although it is often stated that PHT arises as a result of loss of vascular bed secondary to emphysematous lung destruction and/or hypoxic vasoconstriction, recent data indicate that this is not true (reviewed in Ref. 75). Guinea pigs exposed to cigarette smoke develop an 25% increase in mean pulmonary artery pressure (191). Using plastic vascular casts in such animals, we showed that PHT is not associated with signicant alveolar capillary
destruction (193, 194). Likewise, although there is a correlation between PHT and PO2, PO2 has not been found to be an independent predictor of pulmonary arterial pressure (149). In guinea pigs, smoke-increased arterial muscularization (Fig. 11) correlates with pulmonary arterial pressure, and interestingly, also correlates with increased mRNA and protein levels of the vasoactive mediators endothelin and VEGF in these vessels (198). Smoke-induced vascular remodeling also appears to be related to matrix reorganization, since in guinea pigs, increases in pulmonary arterial pressure and vascular
Fig. 9. Postulated mechanisms of small airway remodeling. In this model, oxidants in cigarette smoke, and perhaps oxidants released by smoke-evoked inammatory cells, cause activation of latent TGF- on the airway epithelial cell surface and also TGF- bound to matrix, leading to TGF- signaling through Smads and connective tissue growth factor (CTGF), and increased collagen production. In addition, oxidant attack may activate MMP-9, which in turn can activate latent TGF-. Modeled from Refs. 33, 34, 179.
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sis or the Rho-kinase pathway. Figure 12 shows some of the factors that appear to drive PHT in COPD. Mechanisms of Goblet Cell Metaplasia, Mucus Hypersecretion, and Exacerbations In humans, goblet cell metaplasia is a frequent nding in the large and small airways of cigarette smokers (115, 183), as are increases in the number and size of mucus-producing glands in the large airways. Indeed, the amount of intraluminal mucus in the small airways has been shown to represent a major difference between subjects with signicant [GOLD Stage 3 and 4 (48)] COPD compared with subjects with a lesser degree of airow obstruction [GOLD Stage 1 and 2 (48)] (65), and the excess mucus production that denes chronic bronchitis is also thought to be important in the pathogenesis of acute exacerbations of COPD (reviewed in Ref. 13). As opposed to humans, bronchial glands are concentrated in the proximal trachea in rats and mice, whereas in guinea pigs, they are more diffusely distributed but relatively sparse beyond the proximal trachea (57, 181). This anatomic distribution means that it is difcult to produce a model of smokeinduced chronic bronchitis in these animals, and for this reason, the literature tends to emphasize instead goblet cell metaplasia. However, goblet cell metaplasia, mucin production, mucin secretion, and bronchial gland hypertrophy are probably independently regulated processes (reviewed in Ref. 140). There are also a variety of types of mucin (140), and these may not correspond exactly between humans and animals. All of these factors make pathological smoke-induced changes related to mucin production somewhat difcult to model in animals. In the guinea pig model, chronic cigarette smoke exposure induces secretory cell metaplasia of the small non-cartilaginous airway epithelium (185) (Fig. 13), a nding reasonably analogous to that seen in humans, but does not produce the luminal mucous plugs seen in patients with COPD (185). Smoking cessation reduces the degree of metaplasia (187). By contrast, in mice, tobacco smoke has a relatively minor effect, with only a few secretory cells appearing in the small airways (11). Some studies report signicant degrees of goblet cell metaplasia in smoke-exposed rats (84, 138, 139, 177), and this can
Fig. 10. Peribronchiolar lymphoid aggregate in a mouse exposed to smoke for 6 mo. Note the pigmented smokers macrophages (arrows), a nding similar to that seen in human smokers.
muscularization could be reduced by administration of the serine elastase inhibitor ZD0892 (188), and in smoke-exposed mice, there was an early TNF-dependent upregulation of MMP-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 in guinea pigs (34). In rats (164), simvastatin reduced pulmonary arterial pressure, perhaps through suppressing inammation and MMP-9 induction, or perhaps, as in other models of pulmonary hypertension (56), through mechanisms involving increased apopto-
Fig. 11. Vascular remodeling. Smooth muscle actin stains of small intrapulmonary arterial branches from control (A) and animal exposed to smoke for 6 mo (B). Note the marked increase in smooth muscle. Such increases correlate with increased pulmonary arterial pressure.
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Fig. 12. Postulated mechanisms involved in the development of pulmonary hypertension in COPD. Smoke (probably oxidant)-driven increases in vasoproliferative and vasoconstrictive agents (endothelin and VEGF) and TNFdriven increases in MMP activation result in vascular remodeling and endothelial dysfunction. The normally vasorelaxant endothelial nitric oxide synthase (eNOS) pathway is disrupted by smoke-generated oxidants and also by TNF, thus increasing the vasoconstrictive effects and endothelial dysfunction. Vascular remodeling and endothelial dysfunction act in a vicious circle producing pulmonary hypertension. Modeled from Refs. 189, 190, 191.
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 great interest, as exacerbations are a major (poor) prognostic feature in human smokers and are a major health care burden (174). This phenomenon is complicated by a lack of a universally accepted denition (13, 101), and the triggers of exacerbation are not denitively known; bacterial and viral agents are suggested instigators. A recent human model of virus infection using rhinovirus recapitulated the symptoms and signs of an exacerbation (123). Regardless of etiology, there is evidence of upper and lower respiratory tract inammation during exacerbations (70). Since chronic bronchitis is a clinical denition, involving cough and sputum production, it is difcult to establish an animal model as sputum production is difcult to monitor in animals and probably sparse; the lack of diffuse increases in mucus production likewise makes it difcult to make models of acute exacerbations. Potential models of exacerbation include administration of lipopolysaccharide or administration of bacterial or viral agents with or without cigarette smoke. For recent reviews on models of acute exacerbations, the reader is referred to Refs. 54, 80, 1017. Conclusions Taken as a whole, the data on proteases and anti-inammatory agents still support the idea that inammatory cell-derived proteases are the major mediators of emphysema in animal models. However, which cells/proteases are most important in this process remains uncertain, and there are clearly complex interactions among them (Fig. 3). Antioxidant protection is important as well, and oxidative stress is linked to increases in inammation and decreases in antiproteolytic protection, thereby connecting oxidative damage to the protease-antiprotease hypothesis (Fig. 3). As opposed to these various initiating factors, failure of the parenchyma to properly repair also appears to play a role in the genesis of emphysema, but the data
be prevented, or postexposure regression enhanced, with the antioxidant/mucolytic agent N-acetyl cysteine, as well as a variety of nonsteroidal anti-inammatory agents, and steroids (138, 139). In rats, the gastro-protective agent rebamipide (OPC-12759) decreased TNF production and reduced MUC5AC production, reduced inammatory cells in BAL, and inhibited smoke-induced goblet cell metaplasia in the trachea (89). Tracheal goblet cell metaplasia in guinea pigs was induced 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 signicant effect on the minimal goblet cell metaplasia induced by cigarette smoke in the mouse model (106). These ndings suggest that goblet cell metapla-
Fig. 13. Goblet cell metaplasia. Periodic acid Schiff/ diastase-stained sections of small airways from control guinea pig (A) and guinea pig exposed to smoke for 6 mo (B). Note the increased numbers of mucin-containing goblet cells in the epithelium of the smoke-exposed animal.
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ANIMAL MODELS OF COPD REFERENCES 1. Adair-Kirk TL, Atkinson JJ, Broekelmann TJ, Doi M, Tryggvason K, Miner JH, Mecham RP, Senior RM. A site on laminin alpha 5, AQARSAASKVKVSMKF, induces inammatory cell production of matrix metalloproteinase-9 and chemotaxis. J Immunol 171: 398 406, 2003. 2. Aoshiba K, Koinuma M, Yokohori N, Nagai A. Immunohistochemical evaluation of oxidative stress in murine lungs after cigarette smoke exposure. Inhal Toxicol 15: 1029 1038, 2003. 3. Aoshiba K, Yokohori N, Nagai A. Alveolar wall apoptosis causes lung destruction and emphysematous changes. Am J Respir Cell Mol Biol 28: 555562, 2003. 4. Arikan MC, Shapiro SD, Mariani TJ. Induction of macrophage elastase (MMP-12) gene expression by statins. J Cell Physiol 204: 139 145, 2005. 5. Banerjee S, Maity P, Mukherjee S, Sil AK, Panda K, Chattopadhyay D, Chatterjee IB. Black tea prevents cigarette smoke-induced apoptosis and lung damage. J Inamm 4: 3, 2007. 6. Barbera JA, Peinado VI, Santos S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J 21: 892905, 2003. 7. Barnes PJ, Shapiro SD, Pauwels RA. Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J 22: 672 688, 2003. 8. Barnes PJ. Emerging targets for COPD therapy. Curr Drug Targets Inamm Allergy 4: 675 683, 2005. 9. Barnes PJ. Reduced histone deacetylase in COPD: clinical implications. Chest 129: 151155, 2006. 10. Barnes PJ. Unexpected failure of anti-tumor necrosis factor therapy in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 175: 866 867, 2007. 11. Bartalesi B, Cavarra E, Fineschi S, Lucattelli M, Lunghi B, Martorana PA, Lungarella G. Different lung responses to cigarette smoke in two strains of mice sensitive to oxidants. Eur Respir J 25: 1522, 2005. 12. Belloni PN, Garvin L, Mao CP, Bailey-Healy I, Leaffer D. Effects of all-trans-retinoic acid in promoting alveolar repair. Chest 117: 235S 2341S, 2000. 13. Berge S, Wedzicha JA. COPD exacerbations: denitions and classications. Eur Respir J 21, S41: 46S53S, 2003. 14. Bowler RP, Barnes PJ, Crapo JD. The role of oxidative stress in chronic obstructive pulmonary disease. COPD 1: 255277, 2004. 15. Bracke KR, Dhulst AI, Maes T, Moerloose KB, Demedts IK, Lebecque S, Joos GF, Brusselle GG. Cigarette smoke-induced pulmonary inammation and emphysema are attenuated in CCR6-decient mice. J Immunol 177: 4350 4359, 2006. 16. Bracke KR, Dhulst AI, Maes T, Demedts IK, Moerloose KB, Kuziel WA, Joos GF, Brusselle GG. Cigarette smoke-induced pulmonary inammation, but not airway remodelling, is attenuated in chemokine receptor 5-decient mice. Clin Exp Allergy 37: 14671479, 2007. 17. Bridges RB, Fu MC, Rehm SR. Increased neutrophil myeloperoxidase activity associated with cigarette smoking. Eur J Respir Dis 67: 84 93, 1985. 18. Brusselle GG, Bracke KR, Maes T, Dhulst AI, Moerloose KB, Joos GF, Pauwels RA. Murine models of COPD. Pulm Pharmacol Ther 19: 155165, 2006. 19. Budev MM, Arroliga AC, Wiedemann HP, Matthay RA. Cor pulmonale: an overview. Semin Respir Crit Care Med 24: 233243, 2003. 20. Cantor JO, Shteyngart B, Cerreta JM, Liu M, Armand G, Turino GM. The effect of hyaluronan on elastic ber injury in vitro and elastase-induced airspace enlargement in vivo. Proc Soc Exp Biol Med 225: 6571, 2000. 21. Cantor JO, Cerreta JM, Ochoa M, Ma S, Chow T, Grunig G, Turino GM. Aerosolized hyaluronan limits airspace enlargement in a mouse model of cigarette smoke-induced pulmonary emphysema. Exp Lung Res 31: 417 430, 2005. 22. Cardoso WV, Sekhon HS, Hyde DM, Thurlbeck WM. Collagen and elastin in human pulmonary emphysema. Am Rev Respir Dis 147: 975981, 1993. 23. Carnevali S, Nakamura Y, Mio T, Liu X, Takigawa K, Romberger DJ, Spurzem JR, Rennard SI. Cigarette smoke extract inhibits broblast-mediated collagen gel contraction. Am J Physiol Lung Cell Mol Physiol 274: L591L598, 1998.
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are more controversial and most failure-to-repair models have not used cigarette smoke. This wealth of data in fact produces the interesting conundrum that there are too many potential actors, and sorting out which processes are fundamental to the development of emphysema and which are epiphenomena is a crucial and difcult problem; in this regard, it appears that MMP-12 is consistently implicated (when looked for) in the murine models and may represent a central chokepoint (Fig. 3). Whether this is true of humans is an important question. One of the puzzling facts about the role of proteases and oxidative 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 of individuals to mount an inammatory/antioxidant response. Recent data on the role of T cells and the adaptive immune response suggest a new paradigm that could partially explain this question, namely that COPD (at least emphysema) is a consequence of an autoimmune reaction to antigenic peptides derived from the breakdown of elastin and perhaps other lung components (37, 38, 98, 97, 100). Thus proteases along with reactive oxygen species would be the initial culprits initiating the breakdown of elastic and collagen tissue and producing the necessary antigenic peptides to potentially trigger the autoimmune reaction. However, only genetically predisposed humans, and mice, would develop the adaptive immune response necessary to enhance and maintain the initial inammatory response to cigarette smoke and eventually produce disease. The animal data suggest a variety of therapeutic approaches to human emphysema [indeed, thus far animal models have addressed only a few out of many potential targets (40, 41)], but translation to the human setting is not straightforward, as evidenced by the apparent lack of efcacy of anti-TNF therapy in humans (10, 135, 174) compared with the clear role of TNF in murine emphysema (32, 176). The reason for this discrepancy is unclear but probably relates in large part to the inability to produce in laboratory animals severe COPD with cigarette smoke; i.e., the smoke-induced animal models are reproducing GOLD Stage 1 and 2 disease, but symptomatic COPD patients have GOLD 3/4 disease, and thus far no model reproduces either the anatomic or functional changes or the smoke-independent progression (136), seen in such patients. This nding implies that the mechanisms driving human early stage and late stage disease may be quite different and that the animal models are limited to relatively early stage disease, but that does not make the animal models useless, since effective early intervention is probably much easier to design than effective late stage intervention. Studies on SAR and PHT suggest that these processes are at least partially independent of the factors that drive emphysema, although there are clearly areas of overlap, such as the role of oxidants in SAR and serine elastases in vascular remodeling, and the same is true of increased mucus production and goblet cell metaplasia. This situation implies that a therapeutic approach to human COPD may have to target each anatomic compartment and a single therapeutic agent may not be able to prevent all of the separate manifestations.
GRANTS This work was supported by Canadian Institutes of Health Research Grants 42539 and 81409. AJP-Lung Cell Mol Physiol VOL
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ANIMAL MODELS OF COPD 112. Moodie FM, Marwick JA, Anderson CS, Szulakowski P, Biswas SK, Bauter MR, Kilty I, Rahman I. Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinammatory cytokine release in alveolar epithelial cells. FASEB J 18: 18971899, 2004. 113. Mori H, Nose T, Ishitani K, Kasagi S, Souma S, Akiyoshi T, Kodama Y, Mori T, Kondo M, Sasaki S, Iwase A, Takahashi K, Fukuchi Y, Seyama K. PDE4 inhibitor (GPD-1116) markedly attenuates the development of cigarette smoke-induced emphysema in senescence accelerated mice P1 strain. Am J Physiol Lung Cell Mol Physiol 294: L196 L204, 2008. 114. Morimoto K, Janssen WJ, Fessler MB, McPhillips KA, Borges VM, Bowler RP, Xiao YQ, Kench JA, Henson PM, Vandivier RW. Lovastatin enhances clearance of apoptotic cells (efferocytosis) with implications for chronic obstructive pulmonary disease. J Immunol 176: 76577665, 2006. 115. Mullen JBM, Wright JL, Wiggs BR, Pare PD, Hogg JC. Structure of central airways in current smokers and ex-smokers with and without mucus hypersecretion: relationship to lung function. Thorax 42: 843 848, 1987. 116. Nishikawa M, Kakemizu N, Ito T, Kudo M, Kaneko T, Suzuki M, Udaka N, Ikeda H, Okubo T. Superoxide mediates cigarette smokeinduced inltration of neutrophils into the airways through nuclear factor-kappaB activation and IL-8 mRNA expression in guinea pigs in vivo. Am J Respir Cell Mol Biol 20: 189 198, 1999. 117. Noordhoek JA, Postma DS, Chong LL, Vos JT, Kauffman HF, Timens W, van Straaten JF. Different proliferative capacity of lung broblasts obtained from control subjects and patients with emphysema. Exp Lung Res 29: 291302, 2003. 118. Nyunoya T, Monick MM, Klingelhutz A, Yarovinsky TO, Cagley JR, Hunninghake GW. Cigarette smoke induces cellular senescence. Am J Respir Cell Mol Biol 35: 681 688, 2006. 119. Oberdorster G. Lung particle overload: implications for occupational exposures to particles. Regul Toxicol Pharmacol 21: 123135, 1995. 120. Ohnishi K, Takagi M, Kurokawa Y. Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema. Lab Invest 78: 10771086, 1998. 121. Osman M, Cantor JO, Roffman S, Keller S, Turino GM, Mandl I. Cigarette smoke impairs elastin resynthesis in lungs of hamsters with elastase-induced emphysema. Am Rev Respir Dis 132: 640 643, 1985. 122. Pare PD, Wiggs BR, James A, Hogg JC, Bosken C. The comparative mechanics and morphology of airways in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 143: 1189 1193, 1991. 123. Papi A, Contoli M, Gaetano C, Mallia P, Johnston SL. Models of infection and exacerbations in COPD. Curr Opin Pharm 7: 17, 2007. 124. Parks WC, Shapiro SD. Matrix metalloproteinases in lung biology. Respir Res 2: 10 19, 2001. 125. Parks WC, Wilson CL, Lopez-Boado YS. Matrix metalloproteinases as modulators of inammation and innate immunity. Nat Rev Immunol 4: 617 629, 2004. 126. Pauwels RA, Rabe KF. Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 364: 613 620, 2004. 127. Pemberton PA, Cantwell JS, Kim KM, Sundin DJ, Kobayashi D, Fink JB, Shapiro SD, Barr PJ. An inhaled matrix metalloprotease inhibitor prevents cigarette smoke-induced emphysema in the mouse. COPD 2: 303310, 2005. 128. Pemberton PA, Kobayashi D, Wilk BJ, Henstrand JM, Shapiro SD, Barr PJ. Inhaled recombinant alpha 1-antitrypsin ameliorates cigarette smoke-induced emphysema in the mouse. COPD 3: 101108, 2006. 129. Postlethwaite AE, Kang AH. Collagen-and collagen peptide-induced chemotaxis of human blood monocytes. J Exp Med 143: 1299 1307, 1976. 130. Pryor WA, Stone K. Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann NY Acad Sci 686: 1227, 1993. 131. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, Fukuchi Y, Jenkins C, Rodriguez-Roisin R, van Weel C, Zielinski J. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. GOLD executive summary. Am J Respir Crit Care Med 176: 532555, 2007. 132. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 114: 1248 1259, 2004. AJP-Lung Cell Mol Physiol VOL
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Cigarette Induce COPD

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Cigarette Induce COPD

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

Andrew Churg, Manuel Cosio and Joanne L. Wright

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

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1040-0605/08 $8.00 Copyright 2008 the American Physiological Society

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AJP-Lung Cell Mol Physiol VOL

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

ZD0892 (serine protease inhibitor) A1AT injected (prolastin)

Shapiro (155) Pemberton (128) Takubo (163) Cavarra (24)

C57Bl/6 mouse Mouse Pallid mouse Pallid mouse

59% 72% Early onset emphysema Early onset emphysema

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0% 90% 100% 75% 30% 68%

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

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Invited Review L616

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Decreased BAL macrophages, neutrophils

Dhulst (46) Dhulst (46) Martorana (106) Lee (87)

Mouse (C57) Mouse (C57) Mouse (C57)

TNF R1/ TNF R2/

PDE4 inhibitor Roumilast Rat (Sprague- Dawley) Simvastatin

None 100% 100% 100%

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Cantor (21) Bracke (13)

Mouse (DBA/2) Mouse (C57Bl/6)

Hyaluron aerosol CCR6/

Ma (92) Ma (92) Bracke (16)

Mouse (C57Bl/6) Mouse (C57Bl/6) Mouse (C58Bl/6)

IFN/ CCR5/ CCR5/

Not reported Decreased (primarily decreased macrophages) Decreased

100% 100% 25%

Maeno (97) Maeno (97) DHulst (45) Maes (98)

Mouse (C57Bl/6) Mouse (C57Bl/6) SCID mouse Mouse (C3H/HeJ)

100% None None None

van der Deen (172) Mouse (FVB)

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

Invited Review L618

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Invited Review L620

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Nishikawa (116) Smith (158) Banerjee (5) Cavarra (24)

Guinea pig Rat Guinea pig ICR, C57, DBA/2 mice

Decreased Decreased Not reported Not reported

Rangasamy (32) Iizuka (71) Foronjy (52)

Mouse (ICR) Mouse (Balb/c) Mouse

Increased Increased Decreased

AJP-Lung Cell Mol Physiol VOL

294 APRIL 2008

Invited Review L622

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AJP-Lung Cell Mol Physiol VOL

294 APRIL 2008

Invited Review L624

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AJP-Lung Cell Mol Physiol VOL

294 APRIL 2008

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