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(Chest. 2006;129:1673-1682.)
© 2006 American College of Chest Physicians

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

* 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.

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: Bill.Vandivier{at}uchsc.edu

Abstract

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.

Key Words: asthma • COPD • cystic fibrosis

Cell death, removal, and replenishment are increasingly recognized as fundamental processes in both the developing and the developed organism. 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 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 naïve 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%.56 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).3789

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 wide-ranging 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, fluid-filled phagosomes, or "efferosomes" (Fig 1 , top, A). Unlike complement or Fc{gamma} 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 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).12131415


Figure 1
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  		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.

 
"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),111718 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.


Figure 2
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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, ß2-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.)

 
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 receptor tyrosine kinase, {alpha}vß3- and {alpha}vß5-integrins, scavenger receptors, CD44, CD14, and complement receptors 3 and 4 (Fig 2).16 Adenosine triphosphate-binding 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),192122 while other receptors are primarily involved with transducing the engulfment signal (eg, LRP and PS receptors [PSRs]),11212324 hence the so-called tether-and-tickle 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 growth-arrest-specific-factor 6 (Gas6) are soluble bridging molecules that directly link PS on the apoptotic cell surface with their corresponding phagocyte receptors; {alpha}vß3(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.2324 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, ß2-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 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 membrane262728 (P.M. Henson; personal communication; March 2006). Second, while PSR knockout mice (from three different groups) all displayed developmental defects and die perinatally,293031 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.

Signaling Pathways
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).

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 elastase-dependent 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.

Innate Immunity
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)-ß1, prostanoids, peroxidase proliferator-activated receptor (PPAR)-{gamma} (C. Freire de Lima; personal communication; March 2006), and in some cases interleukin (IL)-10 (Fig 3 ).13 Antiinflammatory mediators, such as TGF-ß1, that are released from the phagocyte act in an autocrine/paracrine manner to suppress the production of inflammatory cytokines (eg, tumor necrosis factor [TNF]-{alpha}, IL-6, and IL-1ß), 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-ß1-dependent manner.36 In contrast, the phagocytosis of lysed neutrophils is much less effective at inducing TGF-ß1 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


Figure 3
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  		Figure 3. The efferocytosis hypothesis. Effective efferocytosis promotes the maintenance of normal airway and alveolar structures in both the naïve and inflamed lung (left half of figure). In this model, TGF-ß1 (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 ß-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).

 
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.

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.131638 The suppressive effect of efferocytosis on the adaptive immune system appears to be largely PS-dependent, to include both TGF-ß1-dependent and TGF-ß1-independent mechanisms, and to result in decreased antigen-specific CD4 T cells, antigen-specific IgG, dendritic cell maturation, and IL-12 production.16203839 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 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 ß-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 ß-catenin signaling cascade regulates 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 ß-catenin expression, and impaired postinjury repair, suggesting a pivotal role for postinjury apoptotic clearance in initiating epithelial repair.43

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.44454647 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-ß1 levels on lung fibrosis (whether in idiopathic pulmonary fibrosis, COPD, or CF) are readily apparent. These topics have been thoroughly reviewed elsewhere.1448

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/antiprotease 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-{alpha} (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 patients38 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-{alpha}-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-{alpha} (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 neutrophils) 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.5253 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 naïve 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-{alpha}, and matrix metalloproteinases, and increase PPAR{gamma}, 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

PPAR{gamma} agonists enhance efferocytosis by human alveolar macrophages through the increased expression of CD36.59 Similar to statins, PPAR{gamma} agonists may also increase efferocytosis in the inflamed lung by decreasing oxidant stress, TNF-{alpha}, 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-{alpha} 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 anti-TNF therapies have not yielded positive results in COPD patients.6263 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.

Acknowledgements

The authors thank Dr. Moumita Ghosh for critical review of the manuscript.

Footnotes

Abbreviations: CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane regulator; Gas6 = growth-arrest-specific-factor 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

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).

Received for publication February 14, 2006. Accepted for publication March 24, 2006.

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