Burying the Dead

CHEST Translating Basic Research Into Clinical Practice Burying the Dead* The Impact of Failed Apoptotic Cell Removal (Efferocytosis) on Chronic Inflammatory Lung Disease R. William Vandivier, MD; Peter M. Henson, PhD; and Ivor S. Douglas, MD

Apoptosis and the removal of apoptotic cells (termed efferocytosis) are tightly coupled with the regulation of normal lung structure, both in the developing and adult organism. Processes that disrupt or uncouple this balance have the potential to alter normal cell turnover, ultimately resulting in the induction of lung pathology and disease. Apoptotic cells are increased in several chronic inflammatory lung diseases, including cystic fibrosis (CF), non-CF bronchiectasis, COPD, and asthma. While this may well be due to the enhanced induction of apoptosis, increasing data suggest that the clearance of dying cells is also impaired. Because efferocytosis appears to be a key regulatory checkpoint for the innate immune system, the adaptive immune system, and cell proliferation, the failure of this highly conserved process may contribute to disease pathogenesis by impeding both the resolution of inflammation and the maintenance of alveolar integrity. The recognition of impaired efferocytosis as a contributor to chronic inflammation may ultimately direct us toward the identification of new disease biomarkers, as well as novel therapeutic approaches. (CHEST 2006; 129:1673–1682) Key words: asthma; COPD; cystic fibrosis Abbreviations: CF ⫽ cystic fibrosis; CFTR ⫽ cystic fibrosis transmembrane regulator; Gas6 ⫽ growth-arrest-specificfactor 6; GTPase ⫽ guanosine triphosphatase; HGF ⫽ hepatocyte growth factor; IL ⫽ interleukin; LRP ⫽ lipoprotein receptor-related protein; MFG ⫽ milk-fat-globule-factor; PPAR ⫽ peroxidase proliferator-activated receptor; PS ⫽ phosphatidylserine; PSR ⫽ phosphatidylserine receptor; SP ⫽ surfactant protein; TGF ⫽ transforming growth factor; TNF ⫽ tumor necrosis factor; VEGF ⫽ vascular endothelial growth factor

death, removal, and replenishment are inC ellcreasingly recognized as fundamental processes in both the developing and the developed organism. *From the COPD Center (Dr. Vandivier) and the Division of Pulmonary Sciences and Critical Care Medicine (Dr. Douglas), University of Colorado at Denver Health Sciences Center; and the Department of Immunology (Dr. Henson), National Jewish Medical and Research Center, Denver, CO. There are no conflicts of interest for any of the authors. This work was supported by an Atorvastatin Research Award (R.W.V.) sponsored by Pfizer Inc, and by grants from the National Institutes of Health grants HL072018 (R.W.V.), GM61031 and HL68864 (to P.M.H.), and HL070940 (to I.S.D). Manuscript received February 14, 2006; revision accepted March 24, 2006. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: R. William Vandivier, MD, University of Colorado at Denver Health Sciences Center, COPD Center, Division of Pulmonary Sciences and Critical Care Medicine, 4200 E Ninth Ave, Box C272, Denver, CO 80220; e-mail: [email protected] www.chestjournal.org

As with many concepts in biology, the notion that death, removal, and rebirth are integral to the adult organism is far from new. Over 100 years ago, Metchnikoff recognized the importance of the homeostatic removal of dead cells, which he termed physiologic inflammation.1 Whether it is resorption of the tadpole tail or the careful burial of one’s own injured or worn out cells, the processes of cell death, removal, and replenishment are necessary for harmony within the organism. The robust nature of these death/removal/replenishment systems and their far-reaching regulatory effects are perhaps best illustrated by the astonishing observation that ⬎ 1011 circulating neutrophils are eliminated and replaced each day by a process that is so efficient, as to be virtually silent. The exquisite regulation of neutrophil turnover depends, at least in part, on the in situ phagocytosis of apoptotic neutrophils that have migrated into distant organs like the lung, lymph nodes, and terminal ileum.2 Once in CHEST / 129 / 6 / JUNE, 2006

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these organs, macrophages and dendritic cells ingest the dying neutrophils and suppress granulopoesis by decreasing levels of granulocyte colony-stimulating factor.2 Here it is clear that death directly regulates rebirth. In the naı¨ve lung, demonstration of the death/ removal/replenishment cycle has been more elusive. Apoptotic cells are rarely seen in the nondiseased human lung,3 suggesting either low rates of cell death or a rapid removal process. If the natural life span of cells within the intestine or brain is to be a guide for the lung, different cell types will have vastly different life spans. For instance, we would expect that the shortest life span would be found in cells directly interacting with the outside environment (eg, epithelial cells).4 We know that in acute, self-limited, inflammatory diseases of the lung (eg, community-acquired pneumonia and ARDS) the number of observed apoptotic cells remains quite low, in the range of 1 to 2%.5,6 In contrast, apoptotic cells are much more prevalent in a variety of chronic inflammatory diseases of the lung, suggesting (1) enhanced apoptosis, (2) impaired removal, (3) massive death in proportions that overwhelm normal removal systems, or (4) artifactual increases related to the methodology (eg, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling staining).3,7–9 We propose that the increased presence of apoptotic cells in patients with many of these chronic inflammatory lung diseases is largely due to impaired cell removal mechanisms. This may have wideranging pathogenic effects on the regulation of innate and adaptive immunity, protease/antiprotease balance, and cell replenishment, and on the development of fibrosis.

Efferocytosis: Extracellular and Intracellular Machinery Efferocytosis Because the phagocytosis of apoptotic cells has distinctive morphologic features and unique downstream consequences, deCathelineau and Henson10 and Gardai et al11 coined the unique term efferocytosis (taken from the Latin effero, meaning to take to the grave or to bury). During efferocytosis, large membrane ruffles sprout out from the phagocyte surface and engulf apoptotic cells into large, fluidfilled phagosomes, or “efferosomes” (Fig 1, top, A). Unlike complement or Fc␥ receptor-mediated phagocytosis, efferocytosis and macropinocytosis are morphologically similar, and both are inhibited by amiloride. While efferocytosis was originally thought to be limited to “professional” phagocytes, such as 1674

macrophages and dendritic cells, we now know that most cell types in the body are capable of ingesting apoptotic cells, including epithelial cells and fibroblasts (Fig 1, bottom, B).12–15 “Eat-Me” Signals In order for apoptotic cells to be recognized and ingested, fundamental membrane alterations must occur in a way that differentiates them from viable cells. In short, apoptotic cells must express clear-cut “eat-me” signals (Fig 2, top, A). Surprisingly, little is known about these alterations, but it has become clear that the externalization of phosphatidylserine (PS) is an essential element.13 During apoptosis, PS (which is normally sequestered to the inner membrane leaflet) redistributes to the outer membrane leaflet in a process that is fueled by the inhibition of aminophospholipid translocases and the activation of phospholipid flip-flop.16 This exposure of PS by apoptotic cells is of particular interest, because it induces membrane ruffling and efferocytosis, and it has been implicated in many of the immunomodulatory effects associated with efferocytosis (more below). Calreticulin has also been identified as another eat-me signal on apoptotic cells.11 During apoptosis, calreticulin increases on the cell surface and redistributes into PS-rich patches. These calreticulin/PS-rich patches distribute away from regions rich in CD47 (a newly described “don’t-eat-me” signal on viable cells),11,17,18 ultimately resulting in enhanced efferocytosis. Finally, CD31 (also known as platelet-endothelial cell adhesion molecule-1) is a fascinating molecule with dual roles, functioning as both a don’t-eat-me (detachment) signal on viable leukocytes and as an eat-me facilitator on apoptotic leukocytes.19 In an elegant series of studies, Brown and colleagues19 demonstrated that homophilic ligation of CD31 on viable leukocytes causes them to detach from macrophages. In contrast, the homophilic ligation of CD31 on apoptotic leukocytes enhances their attachment (tethering) to macrophages, the engagement of eat-me signals (eg, PS), and ingestion. It therefore appears that the apoptotic cell membrane is actively reconfigured in a way that enhances recognition, tethering, and removal by the phagocyte. The Phagocytic Synapse Because the interaction between an apoptotic cell and a phagocyte involves an array of receptors and bridging molecules, it has been termed a phagocytic synapse.20 Receptors known to be associated with efferocytosis include the low-density lipoprotein receptor-related protein (LRP [or CD91]), Mer recepTranslating Basic Research Into Clinical Practice

Figure 1. Tether-and-tickle hypothesis for efferocytosis.21 Top, A: in this model, tethering receptors (eg, CD14 and/or CD31) hold the apoptotic cell in close approximation to the apoptotic cells. Tickling receptors (eg, PSRs or LRP) then engage eat-me signals on the apoptotic cell surface, followed by signal transduction, resulting in Rac-1 activation and the extension of membrane ruffles. The apoptotic cell is then ingested into a characteristic fluid-filled efferosome where is it digested. Bottom, B: “nonprofessional phagocytes” ingest apoptotic cells. Fluorescence micrographs demonstrate that nonprofessional phagocytes, such as primary airway epithelial cells (green), are capable of ingesting whole apoptotic Jurkat T cells (red). In these experiments, primary human large airway epithelial cells were cocultured with apoptotic Jurkat T cells for 3 h, washed, and imaged.

tor tyrosine kinase, ␣v␤3- and ␣v␤5-integrins, scavenger receptors, CD44, CD14, and complement receptors 3 and 4 (Fig 2).16 Adenosine triphosphatebinding cassette proteins including ABC-A1, ABC-A7 (A.W. Jehle; personal communication; March 2006), and cystic fibrosis transmembrane regulator (CFTR) also contribute to efferocytosis (unpublished data; March 2006), although perhaps not as true receptors. Some efferocytosis receptors appear to perform primarily a tethering function by holding the apoptotic cell and phagocyte close together (eg, CD14 or CD31),19,21,22 while other receptors are primarily involved with transducing the engulfment signal (eg, LRP and PS receptors [PSRs]),11,21,23,24 hence the so-called tether-andtickle hypothesis (Fig 1, top, A).21 This, however, does not preclude the possibility that some receptors may ultimately be shown to perform both tethering and tickling (signal transduction) functions. Some phagocytic receptors interact directly with apoptotic cells; however, most interactions occur indirectly through bridging proteins (Fig 2). For example, milk-fat-globule-factor (MFG)-E8 and www.chestjournal.org

growth-arrest-specific-factor 6 (Gas6) are soluble bridging molecules that directly link PS on the apoptotic cell surface with their corresponding phagocyte receptors; ␣v␤3(5)-integrins and Mer, respectively. Similarly, collectin family members, including mannose-binding lectin, surfactant protein (SP)-A, SP-D, and the collectin-like C1q, opsonize apoptotic cells with their globular heads, and then form a complex through their collagenous tails with phagocyte calreticulin and LRP.23,24 In this configuration, calreticulin acts in cis with LRP. Apoptotic cell calreticulin may also directly engage phagocyte LRP in a trans configuration, thereby bypassing collectin intermediates.11 Similarly, PS on apoptotic cells may also directly bind to a an unidentified receptors or receptors on the phagocyte surface, effectively bypassing several PS-binding proteins, like MFG-E8, Gas6, thrombospondin, ␤2-glycoprotein-I, protein S, and annexin I.16 Because of controversies, the identity of this PSR deserves special attention. The presumptive PSR was originally identified with a monoclonal antibody (Mab217) that was prepared against macrophages CHEST / 129 / 6 / JUNE, 2006

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Figure 2. Top, A: the phagocytic synapse. Eat-me signals on the apoptotic cell, such as calreticulin (CRT) and PS, initiate their own ingestion either by the direct engagement of receptors on the phagocyte surface (eg, LRP, PSR, CD44, or CD14) or by first binding to soluble bridging proteins. For example, collectins (mannose-binding lectin, SP-A, and SP-D) and the collectin-like C1q bind to an unknown collectin binding site on the apoptotic cell surface via their globular heads. They then engage the LRP/CRT complex on the phagocyte with their collagenous tails, initiating ingestion. Apoptotic cell PS also binds multiple bridging proteins (eg, MFG-E8, thrombospondin [TSP-1], Gas6, ␤2-glycoprotein I, annexin I [AnxI] and protein S), which then engage specific receptors on the phagocyte surface, initiating ingestion. During apoptosis, CD31 switches from a don’t-eat-me (detachment) signal (on viable cells) to a tethering system that allows eat-me signals to engage. Bottom, B: the regulation of efferocytosis. The ingestion of apoptotic cells is governed by a balance between the Rho GTPases, Rac-1 and RhoA, whereby Rac-1 is the principal activator of efferocytosis and RhoA is the principal inhibitor via the downstream effector, Rho kinase. (Please note that this figure in not all-inclusive for eat-me signals, bridging proteins, or efferocytosis receptors.)

that were shown to ingest apoptotic cells through PS-dependent mechanisms. The antibody was then used in a phage display system to identify a gene product that was originally designated as the PSR.25 This designation, however, has been called into question by three lines of evidence. First, the PSR gene product does not appear to be recognized by Mab217, and, moreover, is found in the nucleus and not on the cell membrane26 –28 (P.M. Henson; personal communication; March 2006). Second, while PSR knockout mice (from three different groups) all displayed developmental defects and die perina1676

tally,29 –31 one of these did not exhibit defects in apoptotic cell clearance.31 Third, Mitchell and colleagues28 recently demonstrated that PSR-deficient fibroblasts ingest and respond to apoptotic cells normally. Taken together, these data suggest that the PSR gene product is not a true receptor for PS, although its potential contribution to efferocytosis in vivo by some other mechanism remains to be clarified. On the other hand, the antigen recognized by Mab217 has not yet been characterized and could still act as an efferocytosis receptor, possibly through the recognition of PS. Translating Basic Research Into Clinical Practice

Signaling Pathways

Innate Immunity

The intracellular pathways that transduce the efferocytosis signal were first identified in the nematode Caenorhabditis elegans, with mammalian homologues subsequently identified. In C elegans, two signaling pathways govern corpse removal; both possibly converging at a common effector, ced-10 (mammalian homolog Rac-1).32 The first pathway includes UNC-73, MIG-2, ced-12, ced-5, and ced-2 (representing the mammalian homologues TRIO, RhoG, ELMO, Dock 180, and CrkII, respectively).33 The second pathway includes ced-1, ced-6, and ced-7 (representing the mammalian homologues LRP, GULP, and ABC-A1 or ABC-A7, respectively).33 Once these pathways have been activated, efferocytosis appears to be regulated downstream by the balance of Rho guanosine triphosphatases (GTPases), including Rac-1 and RhoA (Fig 2). Rho GTPases are molecular switches, cycling between inactive (guanosine diphosphate-bound) and active (guanosine triphosphate-bound) configurations. Rac-1 is induced by the PS and CD91 and positively regulates efferocytosis, while RhoA negatively regulates efferocytosis through its downstream effector Rho kinase. Rac-1 induces the formation of cell ruffles, which are characteristic for efferocytosis and macropinocytosis. In contrast, RhoA induces the formation of stress fibers, focal adhesions, and cell spreading. This central point of regulation suggests an exploitable therapeutic target through which efferocytosis could be enhanced by the selective activation of Rac-1 or the inhibition of RhoA/Rho kinase (more below).

The interaction of apoptotic cells and phagocytes suppresses the innate immune response and promotes its resolution by actively suppressing inflammatory mediator production through the action of transforming growth factor (TGF)-␤1, prostanoids, peroxidase proliferator-activated receptor (PPAR)-␥ (C. Freire de Lima; personal communication; March 2006), and in some cases interleukin (IL)-10 (Fig 3).13 Antiinflammatory mediators, such as TGF-␤1, that are released from the phagocyte act in an autocrine/paracrine manner to suppress the production of inflammatory cytokines (eg, tumor necrosis factor [TNF]-␣, IL-6, and IL-1␤), chemokines (eg, IL-8 and macrophage inhibitory protein-2), and lipid mediators (eg, thromboxane A2).13 The antiinflammatory effect of efferocytosis can also be seen in vivo where the instillation of apoptotic cells suppresses endotoxin-induced lung inflammation in a TGF-␤1dependent manner.36 In contrast, the phagocytosis of lysed neutrophils is much less effective at inducing TGF-␤1 production and inhibiting inflammatory mediator production. In fact, the ingestion of lysed neutrophils does not suppress the release of macrophage inhibitory protein-2 or IL-8, both of which are neutrophil chemotactic factors.34 Efferocytosis also increases the production of secretory leukoprotease inhibitor, which is an antiprotease with activity against elastase and cathepsin G.37 This may be especially important in suppressing protease/antiprotease imbalance during acute inflammation.

Consequences of Death and Removal Burying the Bodies The most immediate consequence of efferocytosis is the physical removal of apoptotic cells before membrane permeability sets in, thus preventing the release of potentially toxic intracellular contents. This process may be particularly important in the case of neutrophils, because of their large numbers during acute inflammation, their short lifespan, and their internal stores of proteases, inflammatory mediators, and oxidants. This concern seems to be substantiated by in vitro studies34 showing the proinflammatory effect of lysed neutrophils in an elastasedependent manner. It may be, however, that the expeditious removal of all types of apoptotic cells is critical, because caspases 2, 3, and 7 have been shown to be present and active on the surface of apoptotic smooth muscle cells and to exhibit extensive elastolytic activity.35 Therefore, apoptotic cells of all types may be capable of remodeling tissues if not removed swiftly. www.chestjournal.org

Adaptive Immunity The strongest evidence linking efferocytosis with the regulation of adaptive immunity is the association of human and murine autoimmune diseases with the impaired removal of apoptotic cells.13,16,38 The suppressive effect of efferocytosis on the adaptive immune system appears to be largely PS-dependent, to include both TGF-␤1-dependent and TGF-␤1-independent mechanisms, and to result in decreased antigen-specific CD4 T cells, antigen-specific IgG, dendritic cell maturation, and IL-12 production.16,20,38,39 In light of this, it is provocative that the infusion of donor apoptotic lymphocytes in a rat heart transplantation model induced allograft tolerance and was shown to be dependent on intact efferocytosis.40 Proliferation The maintenance of alveolar integrity may be critically dependent on intact efferocytosis mechanisms, because it results in the release of growth CHEST / 129 / 6 / JUNE, 2006

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Figure 3. The efferocytosis hypothesis. Effective efferocytosis promotes the maintenance of normal airway and alveolar structures in both the naı¨ve and inflamed lung (left half of figure). In this model, TGF-␤1 (released by macrophages or epithelial efferocytes) acts in an autocrine/paracrine manner to suppress a variety of inflammatory mediators. Similarly, efferocytosis promotes the release of HGF and VEGF, and the activation of the ␤-catenin pathway, promoting the proliferation of epithelial and endothelial cells, and the maintenance of normal lung structure. In contrast, when efferocytosis is ineffective (eg, in CF or COPD patients), the release of antiinflammatory and progrowth mediators is impaired, resulting in sustained inflammation and inadequate tissue repair (right half of figure).

factors and the activation of signaling molecules involved with the maintenance of the epithelium and endothelium (Fig 3). For example, apoptotic cell ingestion induces the production of hepatocyte growth factor (HGF)41 and vascular endothelial growth factor (VEGF),42 and causes the activation of ␤-catenin (I. S. Douglas, unpublished observation). In this paradigm, efferocytosis may be a key homeostatic regulator by locally inducing proliferation as structural cells die. Activation of the ␤-catenin signaling cascade reg1678

ulates postinjury epithelial repair. This key regulator of cellular integrity controls the transcriptional expression of downstream cell cycle regulators, as well as the interactions between the actin cytoskeleton and cadherin within the adherens junctions. In a sublethal oxidant lung injury model, the pharmacologic inhibition of caspase-dependent apoptosis amplified necrotic injury, reduced ␤-catenin expression, and impaired postinjury repair, suggesting a pivotal role for postinjury apoptotic clearance in initiating epithelial repair.43 Translating Basic Research Into Clinical Practice

Efferocytosis also induces the secretion of VEGF by both epithelial cells and macrophages, which enhances the proliferation of pulmonary microvascular endothelial cells, and protects both endothelial and epithelial cells against ultraviolet-induced apoptosis.42 Similarly, apoptotic cell ingestion in vitro and in vivo increases HGF release from both alveolar macrophages and airway epithelial cells.41 The importance of this response is suggested by the well-known ability of HGF to enhance epithelial cell proliferation and to facilitate lung regeneration. Chronic Inflammatory Diseases of the Lung Impaired efferocytosis is associated with a variety of chronic lung diseases, including cystic fibrosis (CF), non-CF bronchiectasis, COPD, asthma, and idiopathic pulmonary fibrosis (H.R. Collard; personal communication; March 2006), and indeed, it may play a role in their pathogenesis. While we will focus on the effect of impaired efferocytosis on the development of lung disease, we have no reason to presume that this mechanism is exclusive to the lung. In fact, defective efferocytosis has been demonstrated in patients with rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, and atherosclerosis.44 – 47 We will also not discuss the potential impact of persistent apoptotic cells on the development of fibrosis, but the consequences of continuous PS signaling and chronically high TGF-␤1 levels on lung fibrosis (whether in idiopathic pulmonary fibrosis, COPD, or CF) are readily apparent. These topics have been thoroughly reviewed elsewhere.14,48 CF Apoptotic cells are increased in the sputum and tissues of patients with CF.8 While this does not exclude the possibility that apoptosis is increased, it does suggest that normal clearance mechanisms are defective; this could be due to (1) the CF lung environment or (2) mutated CFTR. Our studies have demonstrated that high airway elastase activity in CF patients suppresses efferocytosis by acting on both the phagocyte and the apoptotic cell. Neutrophil elastase inhibits PS-mediated ingestion, presumably by cleaving an unidentified PSR and/or the antigenic target of Mab217. In addition, elastase cleaves calreticulin and LRP on the phagocyte surface (unpublished observations), suggesting additional modes of action. How elastase exerts its effect on apoptotic cells has not been elucidated, but one possibility is the degradation of the eat-me signal, calreticulin. The protease/antiprowww.chestjournal.org

tease imbalance, therefore, may be a key factor driving impaired efferocytosis in CF patients. Other probable environmental factors include the following: (1) low levels of SP-A, SP-D, lipoxins, and annexin I; (2) high levels of TNF-␣ (K.A. McPhillips; personal communication; March 2006; and V.M. Borges; personal communication; March 2006); and (3) the mechanical effect of thick, tenacious mucus, which could effectively separate predator from prey. More recently, we have found that CFTR deficiency directly impairs efferocytosis by epithelial cells (unpublished data), an effect that may be related to its membership in the ABC transporter superfamily (such as ced-7, ABC-A1, and ABC-A7). CFTR deficiency suppresses efferocytosis by increasing the expression of active RhoA in airway epithelial cells, thereby altering the Rac-1/RhoA balance against effective efferocytosis. CFTR deficiency also alters the epithelial response to apoptotic cells, resulting in the enhanced release of the neutrophil chemoattractant IL-8. COPD Apoptotic cells are increased in the lungs of COPD patients3,8 and in animal models of COPD. Increased numbers of apoptotic cells have even been found in the skeletal muscles of COPD patients, raising the exciting possibility that dysregulated cell death or efferocytosis may be systemic,49 and therefore may serve as a disease biomarker. Hodge et al50 were the first to report defective efferocytosis by alveolar macrophages taken from COPD patients. When we examined BAL fluid from COPD patients, we found increased numbers of apoptotic cells and decreased numbers of apoptotic cell ingestions, which is consistent with an efferocytosis defect (W.J. Janssen; unpublished data; March 2006). Efferocytosis is impaired in several COPD animal models, including SP-D knockout mice24 TNF-␣overexpressing mice (K. Morimoto; unpublished data; March 2006) and mice treated with the VEGF inhibitor SU5416 (unpublished observation). Cigarette smoke also suppresses efferocytosis in vitro and in vivo51 (unpublished observations), suggesting a role for impaired efferocytosis in the early pathogenesis of COPD. In support of this notion, the intratracheal instillation of apoptotic Jurkat T cells in the presence of TNF-␣ (an inhibitor of efferocytosis) results in the development of a transient emphysema-like injury pattern (V.M. Borges; unpublished data; March 2006). The mechanism for this direct effect of uncleared T cells (as opposed to neutroCHEST / 129 / 6 / JUNE, 2006

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phils) on emphysema is not yet clear, but could conceivably be due to the elastolytic effect of caspases.35

Therapeutic Implications In patients with chronic inflammatory lung disease, ineffective efferocytosis may lead to the accumulation of apoptotic cells and the impaired regulation of the inflammatory response, and ultimately may suggest a new therapeutic target. While at this moment the demonstration of a direct role for impaired efferocytosis in disease pathogenesis is in its infancy, we think that it is reasonable to speculate on potential therapeutic approaches. Compounds that alter the Rac-1/RhoA balance, by either increasing the level of active Rac-1 (preferable) or decreasing the levels and/or activity of RhoA/ Rho kinase, would be potential candidates for use in therapy. Because Rac-1 and RhoA are integral to biological processes as fundamental as cell movement, phagocytosis, and actin cytoskeletal organization, concerns about collateral effects would be paramount. As it turns out, glucocorticoids increase efferocytosis in vitro by increasing active Rac-1, and they are already used in all of the mentioned chronic lung inflammatory diseases, particularly asthma.52,53 Lipoxin A4 is another candidate for modulating the Rac-1/RhoA balance. Lipoxin enhances efferocytosis in vitro54 and suppresses the inflammatory response, and its levels are low in CF patients.55 Even though lipoxin activates both Rac-2 and RhoA, its positive effect on efferocytosis suggests that the ultimate balance favors Rac activation. Statins are 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitors with potent antiinflammatory effects, largely due to their ability to inhibit the prenylation of Rho GTPases, including Rac-1 and RhoA. Therefore, statins act primarily by decreasing membrane localization and hence the effectiveness of Rho GTPases. We have found that lovastatin enhances efferocytosis in vitro in the naı¨ve murine lung and in alveolar macrophages taken from COPD patients.56 This effect of lovastatin stems from its disproportionate effect on suppressing the prenylation of RhoA over Rac-1. Statins also suppress oxidant stress, TNF-␣, and matrix metalloproteinases, and increase PPAR␥, all of which may further enhance efferocytosis in the inflamed lung. Statins also prevent cigarette smoke-induced emphysema and pulmonary hypertension in rats.57 Clinically, new data suggest that statins decrease COPD-related hospitalizations and all-cause mortality, indicating that they may have an important future role in the treatment of COPD.58 1680

PPAR␥ agonists enhance efferocytosis by human alveolar macrophages through the increased expression of CD36.59 Similar to statins, PPAR␥ agonists may also increase efferocytosis in the inflamed lung by decreasing oxidant stress, TNF-␣, and matrix metalloproteinases. Macrolide antibiotics have wide-ranging antiinflammatory effects that include enhanced efferocytosis.60 The therapeutic potential of these drugs has already been recognized, as is demonstrated by their use in patients with CF, asthma, and panbronchiolitis.61 Studies61 have also suggested a therapeutic role for macrolides in the treatment of COPD, which has resulted in a multicenter clinical trial sponsored by the National Institutes of Health COPD Clinical Research Network. Finally, because proteolytic activity, oxidant stress, and TNF-␣ all suppress efferocytosis, and are thought to be significant contributors to chronic lung inflammation, we would expect that any therapy directed toward these imbalances may have potential beneficial effects on both efferocytosis and clinical outcomes. To date, however, antioxidant and antiTNF therapies have not yielded positive results in COPD patients.62,63 But, it may be too early to draw firm conclusions. For example, early studies64 of anti-TNF therapy in patients with mild asthma did not improve outcomes, whereas later studies65 in refractory asthma suggest a role for this approach. Conclusion Cell death, removal, and replenishment are essential both to maintain homeostasis in the nondiseased organism and to appropriately regulate inflammation and tissue repair during disease. Chronic inflammation in the lung appears to be associated with the delayed removal of dying cells, which may directly impact the natural ability of the injured organism to shut down inflammation and initiate tissue repair. Further investigations will be necessary to determine whether impaired efferocytosis directly impacts disease pathogenesis (eg, the development of emphysema) and whether novel biomarkers can be identified based on these mechanisms. Ultimately, the recognition of impaired efferocytosis as a mechanism of disease may allow a new therapeutic approach based on fundamental processes that have remained highly conserved across the metazoa. ACKNOWLEDGMENT: The authors thank Dr. Moumita Ghosh for critical review of the manuscript.

References 1 Tauber AI. Metchnikoff and the phagocytosis theory. Nat Rev Mol Cell Biol 2003; 4:897–901 Translating Basic Research Into Clinical Practice

2 Stark MA, Huo Y, Burcin TL, et al. Phagocytosis of apoptotic neutrophils regulates granulopoiesis via IL-23 and IL-17. Immunity 2005; 22:285–294 3 Kasahara Y, Tuder RM, Cool CD, et al. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med 2001; 163:737–744 4 Spalding KL, Bhardwaj RD, Buchholz BA, et al. Retrospective birth dating of cells in humans. Cell 2005; 122:133–143 5 Droemann D, Aries SP, Hansen F, et al. Decreased apoptosis and increased activation of alveolar neutrophils in bacterial pneumonia. Chest 2000; 117:1679 –1684 6 Matute-Bello G, Liles WC, Radella F 2nd, et al. Neutrophil apoptosis in the acute respiratory distress syndrome. Am J Respir Crit Care Med 1997; 156:1969 –1977 7 Hodge S, Hodge G, Holmes M, et al. Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation. Eur Respir J 2005; 25:447– 454 8 Vandivier RW, Fadok VA, Hoffmann PR, et al. Elastasemediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. J Clin Invest 2002; 109:661– 670 9 Hansen PR, Holm AM, Svendsen UG, et al. Apoptosis and formation of peroxynitrite in the lungs of patients with obliterative bronchiolitis. J Heart Lung Transplant 2000; 19:160 –166 10 deCathelineau AM, Henson PM. The final step in programmed cell death: phagocytes carry apoptotic cells to the grave. Essays Biochem 2003; 39:105–117 11 Gardai SJ, McPhillips KA, Frasch SC, et al. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 2005; 123:321–334 12 Walsh GM, Sexton DW, Blaylock MG, et al. Resting and cytokine-stimulated human small airway epithelial cells recognize and engulf apoptotic eosinophils. Blood 1999; 94: 2827–2835 13 Henson PM, Bratton DL, Fadok VA. Apoptotic cell removal. Curr Biol 2001; 11:R795–R805 14 Henson PM. Possible roles for apoptosis and apoptotic cell recognition in inflammation and fibrosis. Am J Respir Cell Mol Biol 2003; 29:S70 –S76 15 Hu B, Sonstein J, Christensen PJ, et al. Deficient in vitro and in vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages. J Immunol 2000; 165:2124 –2133 16 Lauber K, Blumenthal SG, Waibel M, et al. Clearance of apoptotic cells: getting rid of the corpses. Mol Cell 2004; 14:277–287 17 Gresham HD, Dale BM, Potter JW, et al. Negative regulation of phagocytosis in murine macrophages by the Src kinase family member, Fgr. J Exp Med 2000; 191:515–528 18 Okazawa H, Motegi S, Ohyama N, et al. Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system. J Immunol 2005; 174:2004 –2011 19 Brown S, Heinisch I, Ross E, et al. Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature 2002; 418:200 –203 20 Stuart LM, Ezekowitz RA. Phagocytosis: elegant complexity. Immunity 2005; 22:539 –550 21 Hoffmann PR, deCathelineau AM, Ogden CA, et al. Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells. J Cell Biol 2001; 155:649 – 660 22 Devitt A, Parker KG, Ogden CA, et al. Persistence of apoptotic cells without autoimmune disease or inflammation in CD14-/- mice. J Cell Biol 2004; 167:1161–1170 23 Ogden CA, deCathelineau A, Hoffmann PR, et al. C1q and www.chestjournal.org

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mannose binding lectin engagement of cell surface calreticulin and cd91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 2001; 194:781–796 Vandivier RW, Ogden CA, Fadok VA, et al. Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex. J Immunol 2002; 169:3978 –3986 Fadok VA, Bratton DL, Rose DM, et al. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 2000; 405:85–90 Cikala M, Alexandrova O, David CN, et al. The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity. BMC Cell Biol 2004; 5:26 Cui P, Qin B, Liu N, et al. Nuclear localization of the phosphatidylserine receptor protein via multiple nuclear localization signals. Exp Cell Res 2004; 293:154 –163 Mitchell JE, Cvetanovic M, Tibrewal N, et al. The presumptive phosphatidylserine receptor is dispensable for innate anti-inflammatory recognition and clearance of apoptotic cells. J Biol Chem 2006; 281:5718 –5725 Li MO, Sarkisian MR, Mehal WZ, et al. Phosphatidylserine receptor is required for clearance of apoptotic cells. Science 2003; 302:1560 –1563 Kunisaki Y, Masuko S, Noda M, et al. Defective fetal liver erythropoiesis and T lymphopoiesis in mice lacking the phosphatidylserine receptor. Blood 2004; 103:3362–3364 Bose J, Gruber AD, Helming L, et al. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal. J Biol 2004; 3:15 Kinchen JM, Cabello J, Klingele D, et al. Two pathways converge at CED-10 to mediate actin rearrangement and corpse removal in C. elegans. Nature 2005; 434:93–99 deBakker CD, Haney LB, Kinchen JM, et al. Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO. Curr Biol 2004; 14:2208 –2216 Fadok VA, Bratton DL, Guthrie L, et al. Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases. J Immunol 2001; 166:6847– 6854 Cowan KN, Leung WC, Mar C, et al. Caspases from apoptotic myocytes degrade extracellular matrix: a novel remodeling paradigm. FASEB J 2005; 19:1848 –1850 Huynh ML, Fadok VA, Henson PM. Phosphatidylserinedependent ingestion of apoptotic cells promotes TGF-␤1 secretion and the resolution of inflammation. J Clin Invest 2002; 109:41–50 Odaka C, Mizuochi T, Yang J, et al. Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response. J Immunol 2003; 171:1507–1514 Henson PM. Fingering IL-12 with apoptotic cells. Immunity 2004; 21:604 – 606 Kim S, Elkon KB, Ma X. Transcriptional suppression of interleukin-12 gene expression following phagocytosis of apoptotic cells. Immunity 2004; 21:643– 653 Sun E, Gao Y, Chen J, et al. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient. Cell Death Differ 2004; 11:1258 –1264 Morimoto K, Amano H, Sonoda F, et al. Alveolar macrophages that phagocytose apoptotic neutrophils produce hepatocyte growth factor during bacterial pneumonia in mice. Am J Respir Cell Mol Biol 2001; 24:608 – 615 Golpon HA, Fadok VA, Taraseviciene-Stewart L, et al. Life after corpse engulfment: phagocytosis of apoptotic cells leads to VEGF secretion and cell growth. FASEB J 2004; 18:1716 – 1718 CHEST / 129 / 6 / JUNE, 2006

1681

43 Douglas IS, Diaz Del Valle F, Winn RA, et al. Beta-catenin in the fibroproliferative response to acute lung injury. Am J Respir Cell Mol Biol 2006; 34:274 –285 44 Botto M, Dell’Agnola C, Bygrave AE, et al. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 1998; 19:56 –59 45 Baumann I, Kolowos W, Voll RE, et al. Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus. Arthritis Rheum 2002; 46:191–201 46 Firestein GS, Yeo M, Zvaifler NJ. Apoptosis in rheumatoid arthritis synovium. J Clin Invest 1995; 96:1631–1638 47 Schrijvers DM, De Meyer GR, Kockx MM, et al. Phagocytosis of apoptotic cells by macrophages is impaired in atherosclerosis. Arterioscler Thromb Vasc Biol 2005; 25:1256 –1261 48 Li X, Shu R, Filippatos G, et al. Apoptosis in lung injury and remodeling. J Appl Physiol 2004; 97:1535–1542 49 Agusti AG, Sauleda J, Miralles C, et al. Skeletal muscle apoptosis and weight loss in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2002; 166:485– 489 50 Hodge S, Hodge G, Scicchitano R, et al. Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells. Immunol Cell Biol 2003; 81:289 –296 51 Kirkham PA, Spooner G, Rahman I, et al. Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products. Biochem Biophys Res Commun 2004; 318: 32–37 52 Huynh ML, Malcolm KC, Kotaru C, et al. Defective apoptotic cell phagocytosis attenuates prostaglandin e2 and 15hydroxyeicosatetraenoic acid in severe asthma alveolar macrophages. Am J Respir Crit Care Med 2005; 172:972–979 53 Giles KM, Ross K, Rossi AG, et al. Glucocorticoid augmentation of macrophage capacity for phagocytosis of apoptotic cells is associated with reduced p130Cas expression, loss of paxillin/pyk2 phosphorylation, and high levels of active Rac. J Immunol 2001; 167:976 –986 54 Godson C, Mitchell S, Harvey K, et al. Cutting edge: lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. J Immunol 2000; 164:1663–1667

1682

55 Karp CL, Flick LM, Park KW, et al. Defective lipoxinmediated anti-inflammatory activity in the cystic fibrosis airway. Nat Immunol 2004; 5:388 –392 56 Morimoto K, Janssen WJ, Fessler MB, et al. Lovastatin enhances clearance of apoptotic cells (efferocytosis) with implications for chronic obstructive pulmonary disease. J Immunol 2006; (in press) 57 Lee JH, Lee DS, Kim EK, et al. Simvastatin inhibits cigarette smoking-induced emphysema and pulmonary hypertension in rat lungs. Am J Respir Crit Care Med 2005; 172:987–993 58 Mancini GBJ, Etminan M, Zhang B, et al. Reduction of morbidity and mortality by statins, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers in patients with chronic obstructive pulmonary disease. J Am Coll Cardiol 2006 (in press) 59 Asada K, Sasaki S, Suda T, et al. Antiinflammatory roles of peroxisome proliferator-activated receptor gamma in human alveolar macrophages. Am J Respir Crit Care Med 2004; 169:195–200 60 Yamaryo T, Oishi K, Yoshimine H, et al. Fourteen-member macrolides promote the phosphatidylserine receptor-dependent phagocytosis of apoptotic neutrophils by alveolar macrophages. Antimicrob Agents Chemother 2003; 47:48 –53 61 Rubin BK, Henke MO. Immunomodulatory activity and effectiveness of macrolides in chronic airway disease. Chest 2004; 125:70S–78S 62 Decramer M, Rutten-van Molken M, Dekhuijzen PN, et al. Effects of N-acetylcysteine on outcomes in chronic obstructive pulmonary disease (Bronchitis Randomized on NAC Cost-Utility Study, BRONCUS): a randomised placebo-controlled trial. Lancet 2005; 365:1552–1560 63 van der Vaart H, Koeter GH, Postma DS, et al. First study of infliximab treatment in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2005; 172: 465– 469 64 Rouhani FN, Meitin CA, Kaler M, et al. Effect of tumor necrosis factor antagonism on allergen-mediated asthmatic airway inflammation. Respir Med 2005; 99:1175–1182 65 Berry MA, Hargadon B, Shelley M, et al. Evidence of a role of tumor necrosis factor alpha in refractory asthma. N Engl J Med 2006; 354:697–708

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