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Am J Physiol Lung Cell Mol Physiol 294: L601-L611, 2008. First published January 4, 2008; doi:10.1152/ajplung.00320.2007
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INVITED REVIEW

Apoptosis in the lung: induction, clearance and detection

P. M. Henson and R. M. Tuder

Department of Pediatrics, National Jewish Medical and Research Center, and Division of Pulmonary and Critical Care Medicine, Departments of Medicine and Pathology, University of Colorado at Denver and Health Sciences Center, Denver, Colorado


   ABSTRACT
TOP
 ABSTRACT
PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 
Apoptosis and other forms of programmed cell death are important contributors to lung pathophysiology. In this brief review, we discuss some of the implications of finding apoptotic cells in the lung and methods for their detection. The balance between induction of apoptosis and the normally highly efficient clearance of such cells shows that these are highly dynamic processes and suggests that abnormalities of apoptotic cell clearance may be an alternative explanation for their detection. Because recognition of apoptotic cells by other lung cells has additional effects on inflammation, immunity, and tissue repair, local responses to the dying cells may also have important consequences in addition to the cell death itself.

efferocytosis; inflammation

PRESENTLY, THERE IS A GREAT DEAL of interest in the participation of apoptosis in various forms of lung diseases or, for that matter, in normal lung development, structure, and function. In this short review, we will discuss apoptosis in the lung in the context of its potential role as a process in health and disease and outline methods, with caveats, for its detection. Because most lung diseases have been examined in one way or another for the presence of apoptotic cells, we will not provide a catalog of such studies but rather attempt to address the implications of finding apoptotic cells in the lung for the progression or resolution of disease processes.

Apoptotic cells are usually removed from tissues by a highly efficient recognition and phagocytic clearance mechanism involving either professional phagocytes, macrophages, and dendritic cells or nonprofessional phagocytes, including endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts. We have termed this clearance process efferocytosis (6, 23). Recognition and engulfment of apoptotic cells also modulate local inflammatory and immunologic responses and may therefore contribute to replenishment of normal cells and restoration of tissue structure to maintain homeostasis (22). Disruption of any of these responses to apoptotic cells may contribute to loss of tissue homeostasis, increased inflammation, fibrosis, or even autoimmunity.


   PROGRAMMED CELL DEATH, APOPTOSIS, AND NECROSIS
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 
Programmed cell death (PCD), of which apoptosis is one manifestation, is a mechanism for deletion of unwanted or damaged cells in the maintenance of normal tissue homeostasis. Consequently, it is particularly manifest during development, tissue remodeling, inflammation, and tissue injury. In fact, PCD is occurring all the time throughout most of the mammalian body, particularly evident for the constant clearance (with replacement) of cells within the hematopoetic compartment. The term apoptosis was coined in 1972 by Kerr et al. (34) to describe a particular morphological manifestation of cell death involving condensation of the nucleus. Since that time, the name has often become used superficially to equate with PCD in general. The distinction of PCD is that the cell itself participates in the processes of its death, in contrast with externally induced death, for example, by physical processes such as acid, heat, or chemicals or by the effects of some potent toxins. PCD itself has been divided into a number of categories, including a more strictly defined apoptosis, and also encompasses cell autonomous necrosis, cell death resulting from autophagy, cell senescence, and so-called mitotic catastrophe (see reviews in Refs. 16, 21, 52). The problem is that there are few clear-cut differentiating features for these different forms of cell death. Nuclear condensation, intranucleosomal DNA cleavage, membrane blebbing, cell shrinkage, involvement of caspases, exposure of cell surface phosphatidylserine, and more are all features of classically defined apoptosis. However, exceptions can be found for any one of these, and a number of these features have also been reported during processes, such as frank necrosis, that are not normally considered to be true apoptosis. An additional complexity in attempts to pigeonhole the different processes of PCD is that they also are often not temporally discreet. Thus, if an apoptotic cell is not removed by the normally efficient clearance mechanisms, it undergoes secondary changes, often called secondary necrosis or cytolysis, which eventually result in liberation of cell contents into the environment (13, 35). In the context of this review and the discussion of apoptotic cell detection in the lung, we will focus primarily on caspase-mediated apoptosis. However, as other markers become readily available for different forms of cell death, it may become important to explore these in different lung diseases.

Induction of apoptosis. The mechanisms by which apoptosis is induced and regulated in different cells have received a great deal of investigation (see for example reviews in Refs. 1, 7, 11, 26, 52). In simple terms, extrinsic (from membrane receptor signaling and mediated through caspase 8) or intrinsic (involving the mitochondria and mediated through caspase 9) pathways lead to activation of executioner caspases (caspases 3 and 7) that initiate the multiple changes in the cell, including the alterations to the nucleus. Stimuli for extrinsic pathways involve activation of TNF family receptors, including Fas and TNF receptors themselves (68). Intrinsic pathway activation can result from genotoxic effects, oxidants, ultraviolet and gamma irradiation, and alteration of the homeostatic interactions between pro- and anti-apoptotic Bcl-2 family members (7). Assays to detect active caspases, or their effects, represent some of the methods used to identify apoptotic cells. However, it should be noted that noncaspase modes of PCD (which have also sometimes been called apoptosis) exist (e.g., see Ref. 51). Other changes seen in "apoptosing" cells are alterations to the cell surface. These are critical to recognition of the dying cell, leading both to its removal and to its effects on macrophages and surrounding cells. Some of these changes are outlined in recent reviews (22, 37, 63) and include the exposure of phosphatidylserine on the outer membrane leaflet, which is also used as a marker for apoptosis (see METHODS OF APOPTOTIC CELL DETECTION below).

Apoptosis vs. necrosis. A distinction is often made between apoptosis and necrosis, in terms of mechanism and morphological characteristics of cell death on the one hand and in the tissue response to the dying cell on the other (see Table 1 and Refs. 11, 52). A key separation between these two endpoints is the integrity of the cell structure, i.e., the disruption of cell membrane and loss of cell contents in necrotic cells. However, this also is imprecise, particularly in the context of differentiation between necrosis and apoptosis in tissue sections of pathological specimens. In this setting, although necrotic cells have lost their cellular structure, their outlines may be relatively preserved. On the other side, apoptotic cells may initially retain their plasma membrane integrity, but most cell types do liberate membrane blebs (so-called apoptotic bodies), leading to decreased cell size (11). With time, apoptotic cell membranes become permeable by increasing degrees, although the cell itself may not disintegrate. Accordingly, during apoptosis, each cell type may exhibit a different window of time wherein the membrane remains impermeable and the cell remains essentially intact before undergoing secondary necrosis and loss of contents and structure. This window is thought to be important for effective phagocytic removal of the dying cell before the release of potentially injurious, proinflammatory, and proimmunogenic contents into the tissues. These concepts are intimately linked to the perhaps oversimplified but common concept that apoptotic cells are noninflammatory (actually anti-inflammatory), whereas necrotic cells induce inflammation (and in some models, constitute active "danger signals" to initiate local tissue and immunologic responses) (20, 21). An example of this difference between cells is the relative persistence of intact apoptotic neutrophils compared with that shown in lymphocytes (a more rapid loss of cell integrity after apoptosis; Henson, unpublished observations). A teleological explanation could be that cells such as neutrophils with higher content of proinflammatory and injurious molecules should be rapidly cleared before disruption.


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  		Table 1. Comparisons between apoptosis and necrosis

 
Early and late apoptotic cells. A related complexity in terminology is the distinction often seen in the literature between "early" and "late" apoptotic cells. This usually refers to cells in culture rather than in tissues and often reflects different effects on recognition of, and response to, the dying cells by macrophages. Sometimes, the distinction is merely a temporal one, but other investigators have distinguished late apoptotic cells as those that begin to show permeability of their plasma membranes to dyes such as propidium iodide (see below), while still retaining overall integrity of cell structure. Others might consider these as examples of early secondary necrosis. The clear-cut implication of these comments is to draw attention to the need to define the characteristics of the cell undergoing apoptosis or other forms of PCD in any studies of the processes involved or the responses to such cells, certainly in vitro and wherever possible in vivo as well.

Apoptosis and necrosis are dynamic processes, and histological observations serve only as a snapshot. As will be discussed below, a critical issue here is that detection of increased numbers of apoptotic cells in a tissue may indeed mean increased rates of apoptosis; however, it is equally possibly that there is decreased clearance of apoptotic cells or that both of these processes together contribute to increased detection (Fig. 1). Necrotic cells are also usually cleared from tissues (even if the inflammatory and immunologic responses to recognition of necrotic cells differ from those of apoptotic cells), so that detection of necrotic cells in tissues may lead to the same sets of questions. Larger areas of necrosis, for example after ischemia, may suffer from delays in clearance because scavenger cells (usually macrophages) are insufficient in numbers and/or cannot penetrate the damaged tissue. Similar issues of (temporary?) overload of the clearance mechanisms may also explain detection of apoptotic cells.


Figure 1
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  		Fig. 1. Apoptotic neutrophil in the alveolar air space and apoptotic debris in phagosomes within an alveolar macrophage. The apoptotic neutrophil (arrowhead) shows nuclear condensation. The macrophage shows a number of phagosomes (asterisk) with apoptotic material (presumably from ingested neutrophils).

 

   DETECTION OF INCREASED NUMBERS OF APOPTOTIC CELLS IN THE LUNG
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 
Examination of tissues from many different types of lung disease has revealed the presence of apoptotic cells, often in excess of those seen in control "normal" tissue. One example of this is in chronic obstructive pulmonary disease (COPD) (28, 32, 42). These types of observation bring up a number of key questions. One is the issue of appropriate controls. There is a reasonable expectation of increased PCD with age, so matching for this is critical. However, obtaining normal human lung tissue is difficult and emphasizes the need for shared tissue banks. Apoptotic cells have been detected in bronchiolar lavage or induced sputum, which may indeed indicate enhanced PCD and/or decreased clearance. However, the clearance question here may indicate different issues from those seen in the tissues themselves. For example, apoptotic neutrophils in cystic fibrosis may be caught up in the mucus and not be available to macrophages for easy removal (77). The inefficiency of sampling from such sources limits quantitative assessment and again brings up the question of controls. In all cases, it behooves the investigator to include in the examination the presence of apoptotic cells that may be located within the vacuoles of phagocytes. In fact, substantial information can be gleaned from the proportions of free vs. ingested apoptotic cells that can help address the issue of defective clearance (because digestion is the ultimate fate of the ingested cell, the detection of phagocytosed apoptotic cells could also reflect defective digestion). The identity of the cell that is apoptotic will also be critical. Is this a generalized observation; for example, in the alveolus does apoptosis extend to all cells of the alveolar structure or is it limited to only epithelium, endothelium, or interstitial cells? This distinction is likely to be important in determining cause, implications, and effects. The type of cell is often hard to distinguish by light microscopy and requires cell-specific markers that might, unfortunately, be lost during the apoptotic process. Because the form of PCD will also be important for both cause and effect of the dying cells, this too constitutes a question that could reasonably be asked when dead cells are detected. However, this may be very difficult to ascertain in tissues because markers for the different types of cell death are not always specific and, as noted, may for any given cell, depend on time from initiation of the PCD processes.


   METHODS OF APOPTOTIC CELL DETECTION
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 
The list of pathobiological settings associated with apoptosis is growing and includes almost all of the relevant lung diseases, such as acute lung injury (41, 44), emphysema (8, 28), pulmonary hypertension (4, 71), lung cancer, and fibrotic lung processes (40, 72). The documentation of apoptosis in lung cells has been critical in such studies. The lung has more than 40 native cells, highlighting the potential challenge to elucidate the role of apoptosis in a particular lung cell population. The dynamic nature of apoptosis involving complex cellular signaling events taking place within a cell and among different cells of an organ can hardly be captured by a snapshot assay or measurement (Fig. 2). The decision of how best to document apoptosis rests on the understanding of some critical biological and molecular aspects of this process, which can be divided into three stages: 1) initiation or trigger stage, 2) decision or initial stage, and 3) execution stage (75, 76) (Fig. 2). The initiation stage involves signaling pathways related to preparations for the cell to die (76), including altered expression of Bcl-2 family members (Table 2), overexpression of c-myc, or localized hypoxia (61). Although highly relevant to the understanding on how pathological triggers lead to activation of apoptotic process, the identification of these signaling mechanisms does not provide documentation per se that apoptosis is indeed happening or is important for the observed lung pathology. The decision process for the intrinsic pathway involves the interaction between the mitochondria and Bcl-2 family of molecules (76). Progressive depolarization of mitochondrial transmembrane potential and opening of the mitochondrial membrane pore, causing release of apoptosis-inducing factor and cytochrome c, characterize this early stage. Cells may engage the execution phase or have it halted (point of return or potential reversal). Most of the assays aimed at characterizing the decision process can be applied at the isolated cell level and, to a limited extent, to lung tissue sections or fluorescence-activated cell sorting (FACS) preparations (Table 2). The execution stages involve global cellular and molecular alterations, all of which can be used to document the different stages of cell death. These are therefore late features in the apoptotic process. In summary, most of the assays presently available to document lung apoptosis rely on late features involved in the apoptotic process, which are usually of short duration, lasting a few hours (69).


Figure 2
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  		Fig. 2. Stages of apoptosis: outline of cellular and molecular events that take place in the cytoplasm, nucleus, cell membrane, and mitochondria throughout the process of decision and execution. Efferocytosis leads to removal of apoptotic cells. The final balance between engagement of the apoptotic process and apoptotic cell clearance will dictate the signal of specific assays aimed at identifying and quantifying apoptotic lung cells. PS, phosphatidylserine; {Psi}m, mitochondrial transmembrane potential.

 

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  		Table 2. Comparison of different methods to analyze apoptosis in different experimental samples pertinent to the study of apoptosis in the lung

 
As mentioned above, the value of an endpoint of apoptosis will depend on the rate of apoptosis, the clearance rate of apoptotic cells, and the length of the specific process being probed. For example, cytochrome c release occurs rapidly and is complete by 5 min of an apoptotic stimulus, before the exteriorization of phosphatidylserine and the loss of plasma membrane integrity (24). Further important considerations consist of the specificity of the particular assay, since necrotic and actively transcribing cells share some of the endpoints used for apoptosis. Each of these criteria may be better suited for different cell preparations, i.e., lung sections, dispersed lung cells, or lung cultures. As apparent throughout the present discussion, all of these preparations provide complementary documentation of apoptosis in the lung.

As summarized in Table 1, the characterization of apoptosis can be done morphologically (62), by assessment of cell membrane changes, by documentation of DNA fragmentation, and by measurement of enzyme processes involved in DNA fragmentation and protein degradation. Furthermore, several assays that measure cell and nuclear permeability with cell-impermeable dyes aid significantly in complementing the morphological documentation of apoptosis (Table 2). Examples are shown in Figs. 3 and 4.


Figure 3
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  		Fig. 3. Transferase-mediated dUTP nick end labeling (TUNEL) in alveolar tissue of a normal mouse lung. A: positive control for TUNEL with DNase 1-treated histological section, showing widespread nuclear positivity. B: negative control in which the lung section was processed as in A but with the enzyme terminal deoxynucleotidyl transferase. C: example of TUNEL-positive (green signal), prosurfactant protein C (SpC)-positive type II cell (red signal) (long arrow), in a mouse lung section. Viable SpC-positive cells lack TUNEL positivity (arrowhead), whereas apoptotic non-type II cells can also be detected in the section (short arrow).

 

Figure 4
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  		Fig. 4. A and B: active caspase-3 expression (arrows) detected by immunohistochemistry in a rat lung treated with a combination of an anti-VEGF receptor 1 (MF-1) and receptor 2 (DC101) antibodies, which causes alveolar cell apoptosis and progressive alveolar enlargement. Negative control consisted of rabbit serum in place of anti-caspase 3 antibody, which showed complete lack of reaction with the lung tissue. Note that there is a marked reduction in caspase 3-positive cells in rat IgG-treated rats (B). C–E: colocalization of TUNEL (arrows) (green signal) and endothelial cell (CD34, red; C) or SpC (red; D) of rat lungs treated with DC101 plus MF-1 (as shown in A). Note the marked lack of TUNEL positivity in IgG-treated rat lungs in E (as in B). F and G: colocalization of TUNEL (green, arrow) and smooth muscle cell {alpha}-actin (red) in rats exposed to chronic hypoxia (F) or room air (controls; G). In F, note an apoptotic endothelial cell (arrowhead) and a positive cell outside the vascular smooth muscle cell ring (arrow). H: evidence of apoptosis in rat lungs treated with the VEGF receptor blocker SU5416 for 3, 7, and 21 days vs. vehicle control (reproduced with permission from Ref. 33). I and J: single-stranded DNA immunohistochemistry of a rat lung treated with SU5416 for 3 wk (arrows; I) compared with vehicle control (arrows; J) (reproduced with permission from Ref. 73).

 
Apoptotic cell morphology. Although cell morphology (for example, nuclear condensation) at the light or electron microscopic levels is considered the gold standard of apoptosis (76), it is of limited use in lung tissue sections. Apoptotic cell recognition by light microscopy is difficult in the lung compared with in organs that have a large number of apoptotic cells with high cell turnover, such as lymphoid follicles or gastrointestinal epithelium. Another morphological aspect of an apoptotic cell is the characteristic appearance of cell markers, such as aggregation of partly cleaved keratin 18 and 19 in mesothelial cells (43) and fibroblasts (47). Enhanced accuracy of cell morphology can be achieved by flow cytometric analysis of cultured or dispersed lung cell preparation using forward scattering (measuring size) and side scattering (measuring granularity) (39). These parameters can be easily applied to uniform cell populations, such as in cell culture, and to dispersed lung cells by FACS. However, dispersed lung cells require proper gating to segregate particular cell populations or double labeling with cell-specific markers.

Cell membrane changes. Most assays to measure cell membrane changes rely on functional tests using live cells (generally in culture). Binding exposed on the outer face of the cell membrane allows the recognition of most if not all apoptotic cells throughout the execution stage and potentially throughout the decision process (75). Because phosphatidylserine is normally present on the inner leaflet of the membrane, any disruption of the membrane can lead to annexin V positivity. The assay is therefore usually accompanied by a probe (propidium iodide) or 7-amino actinomycin D for membrane permeability, and true apoptotic cells are taken to be annexin V positive and propidium iodide negative. Unfortunately, although useful, this assay is not completely specific because viable endothelial cells primed by inflammatory stimuli can also transiently expose phosphatidylserine (18). Although annexin V assay has been mostly used for cell cultures and dispersed lung cells by FACS, membrane exteriorization of phosphatidylserine can be potentially monitored in vivo with intravenously administered biotinylated annexin V, as documented with whole chicken embryos (66, 74) and mouse hearts subjected to ischemia-reperfusion (76). A similar approach may be suited to the study of cell membrane alterations in the early and late stages of lung cell apoptosis in vivo.

DNA fragmentation. This is a characteristic but late feature of apoptosis. However, it should be pointed out that apoptosis can occur in the absence of significant DNA fragmentation, and, conversely, pronounced DNA damage may not represent apoptosis (59, 80). DNA fragmentation, which can be observed morphologically in different organs, is difficult to reliably document in lung sections. The most popular methods to map DNA fragmentation are DNA labeling [transferase-mediated dUTP nick end labeling (TUNEL), in situ end labeling, or DNA nick translation] and DNA laddering. TUNEL, which is based on the incorporation of a labeled deoxyuridine on free 3' DNA ends by terminal deoxynucleotidyl transferase, provides the most sensitivity and specificity compared with the other two labeling methods, in both cell cultures (62) and FACS (56). It offers the advantage of double labeling with cell-specific markers in tissue sections (57), even with prolonged (<12 h) postmortem delays in processing (65) (Fig. 3). However, three important caveats limit its specificity. It detects necrotic cells, which have extensive double-strand breaks and long 3'-prime overhangs (9), live cells with active transcription (36), and active DNA repair (78). This recognized lack of specificity of TUNEL regarding the true detection of apoptosis warrants the use of alternative approaches, such as active caspase 3 detection. Furthermore, nonspecific staining should be controlled by optimization using negative controls, based on omission of the enzyme terminal deoxynucleotidyl transferase and positive controls (DNase 1-treated cells), particularly when targeted for lung sections (Fig. 3) (38, 50, 79). On the other hand, apoptosis-specific single nucleotide 3' overhangs can be labeled in tissue sections by in situ ligation of a labeled 200-bp PCR product (9). This assay works well in lung sections, with a high specificity for apoptosis but somewhat lower sensitivity than TUNEL. Single-stranded DNA antibodies detect breaks revealed in tissue sections subjected to heat denaturation and have strengths similar to that shown with situ-labeled DNA ligation (17, 73).

Early in apoptosis, cells maintain their DNA supranucleosomal order of packaging. Later, degradation of DNA leads to condensation and oligomerization, initially into 50- to 300-kbp fragments and eventually into multimers of ~200 bp (which provides the classical DNA ladder in ethidium bromide-stained gels). In this stage, the DNA forms globules of ~30 nm in diameter (12). DNA laddering detection requires at least 5 x 105 DNA cell equivalents (60). An attractive alternative is the use of linker-based amplification of 200-bp oligomers, which can detect <2% apoptotic cells in a sample (45). This apoptotic-specific assay offers the advantage of only needing small samples of lung and potential quantification using the density of one of the oligomer bands, normalized by an internal control apoptotic DNA (32) (Fig. 4). Furthermore, oligomers can be quantified with an ELISA with histone antibodies (60). On a single cell level, the Comet assay allows the measurement of total DNA and DNA with strand breaks in a given cell (15).

Cell proteolysis. Degradation of cell structures is carried out by a series of cysteine proteases, caspases, particularly caspase 3, and other enzymes, such as proteosomal proteases and calpain. Active caspase 3 is probably involved in most alveolar apoptotic processes (see for example, Ref. 81). Antibodies directed to the cleavage site of the 18-kDa (active) form of caspase 3 have been instrumental in the documentation of apoptosis at the tissue and cell culture levels (70) (Fig. 4). An important consideration in the optimization of immunohistochemical detection of tissue markers is the need for antigen retrieval with heat denaturation in the presence of specific buffers, such as citrate-based solutions or the Borg Decloaker. Although, active caspase 3 immunolocalization provides solid support for apoptosis, the specific retrieval procedures for active caspase 3 immunohistochemistry limit the concomitant immune detection of specific markers of lung cells (55, 57). The localization assays can be further complemented with activity assays performed in tissue lysates (that often measure active caspase 7 as well) (55). Antibodies against specific epitopes on protein targets of caspase 3 provide additional support of apoptosis in the lung, such as lamin (2), poly ADP(ribose) polymerase, and cytokeratin 18 (54).


   WHAT IS NEEDED TO DOCUMENT APOPTOSIS IN THE LUNG
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 
Several of the methods discussed previously are better suited for qualitative analyses, such as those based on electron microscopy. Importantly, most of the assays lend themselves to quantification. However, it is not appropriate to compare multiples of changes among the different assays. These are assays based on different platforms, with particular characteristics concerning the dynamic range and specificity, providing a snapshot of a dynamic process within a heterogeneous tissue (i.e., lung).

Precise quantification of apoptotic endpoints among different cells and compartments within the lung tissue can only be provided by FACS or labeling on tissue sections. FACS combines the advantage of a multistep quantitative approach for a refined delineation of apoptosis in specific (i.e., lymphoid) cells (39, 81). Quantification of apoptotic cells (e.g., using TUNEL or active caspase 3 immunohistochemistry) requires the application of rules of stereology. In a 5-µm section, a positive "cell" in fact represents a positive cellular profile or outline. The statistical chance that a positive cellular profile is identified in a thin histological section is proportional to the size (rather than the number) of a given cell. The accurate number of positive cells expressing a particular cell marker requires the systematic analysis of multilevel planes, distanced at least two-thirds of the height of the cells being counted. Positive profiles counted exclusively in one of a pair of adjacent planes are recorded as a single positive cellular event, i.e., one cell positive for a particular marker. This can be accomplished easily with confocal immunofluorescence. Bright-field signals require a thick section (~20–25 µm in thickness) and imaging through tissue planes obtained along a z-plane (through the thickness of a section). This can be accomplished with a lens with a high numerical aperture (e.g., x60 oil) (31). The results can be normalized by total 4',6-diamidino-2-phenylindole-positive nuclei (in case of single labeling) or cells expressing a specific cell marker (i.e., prosurfactant C).

In summary, no single method can accurately describe and document apoptosis in the lung. A complementary approach based on at least two or more assays probing into different aspects of apoptosis will therefore provide the most compelling evidence of lung cell apoptosis. TUNEL is one of the most used assays and offers the advantage of colocalization imaging with cell-specific markers. Given its important caveats, it is recommended that assessment of active caspase 3 expression (immunohistochemical detection and quantification) or activity assays using lung lysates be used to support the findings obtained with TUNEL. In situ approaches have the advantage of preservation of valuable tissue samples, the evaluation of the identity of a given apoptotic cell, and analysis of localized events, particularly aimed at late in the apoptotic process. FACS of single cells prepared by lung digestion offer the potential for dissection of early and late events in specific lung cells. However, these procedures may induce apoptosis themselves or cause loss of surface markers due to the enzymatic digestion.


   INCREASED APOPTOSIS VS. DECREASED CLEARANCE
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 
The most obvious, and common, interpretation of detecting apoptotic cells in the lung is that it represents increased cell death and demands an assessment of why and how this may be occurring. However, apoptotic cells are also rapidly and efficiently cleared from most tissues so that the net observation at any one point in time (e.g., a tissue section) is a combination of apoptosis induction and apoptotic cell removal (efferocytosis). Because most cells in the body undergo some endogenous rate of turnover, defective clearance could result in the appearance of increased numbers of apoptotic cells in a tissue even in the absence of enhanced cell death processes. The overall efficiency of these clearance processes (see below) have led us to suggest that finding apoptotic cells should automatically generate questions of clearance defects or overload as well as those of increased cell death, or more likely all acting together.

One of the first demonstrations of how efficient and capacious the apoptotic cell clearance processes could be was in a study by Scott et al. (67). They induced apoptosis of the thymocytes in mice with dexamethasone. Examination of the thymus in wild-type mice yielded scanty apoptotic cells at any time during the subsequent clearance process. On the other hand, dexamethasone caused large numbers of apoptotic thymocytes in mice lacking in Mer, a receptor tyrosine kinase that is involved in recognition and ingestion of apoptotic cells. The rates and extent of thymocyte apoptosis were not different between the different mice. On reflection, we can also infer the tremendous capacity of the clearance processes from the huge ongoing turnover, i.e., removal, of circulating blood cells (for example, >1011 neutrophils per day in humans). Resolution of inflammation is another situation in which apoptosis of the inflammatory cells usually leads to their extremely efficient removal. An example from the lung is the detection of remarkably low numbers of apoptotic cells in lavage analysis of patients with community-acquired pneumonia (10) or in our own studies of apoptosis during resolution of experimental inflammation in mice (30). Even in these circumstances, one must also question whether the apoptotic cell that is detected has already been ingested but not yet digested, for example by a macrophage. By contrast, in some inflammatory lung diseases, larger numbers of apoptotic cells are detectable and have been shown to be associated with local defects in clearance [e.g., cystic fibrosis (77) and COPD (28, 49)].

However, we should note that most of the evidence for very high capacity clearance in vivo has come from deletion of hematopoietic cells. Does this also apply to pulmonary epithelial (or for that matter, endothelial) cells? Apoptosis of cells in an epithelium has been suggested to be accompanied by active extrusion from the monolayer (58); whether this process occurs before or after or whether it accompanies the apoptotic process is so far unclear. This would suggest removal of apoptotic epithelial cells in the alveolar lumen (or via the mucociliary escalator in the lung or into the intestinal lumen in the gut). On the other hand, epithelial cells can function as highly efficient phagocytes for apoptotic cells. An example here is in the postlactation involution of the mammary gland. Forced weaning of mice (also not uncommon in humans) leads to complete removal of the mammary alveolar epithelium in 4 days, a process of apoptosis and ingestion of the apoptotic cells by remaining viable epithelial cells (46). Some of the epithelial cells are extruded from the monolayer and ingested secondarily by the phagocytic epithelial cells, and some appear to be ingested in situ in the epithelium without apparent extrusion. Can these types of clearance mechanisms occur in the lung? How much clearance of apoptotic epithelial cells results from extrusion? How much is carried out by macrophages, and how much by epithelial cells that have become activated (as in the mammary gland) to become more efficient phagocytes? How much clearance of apoptotic cells in the airway lumen (including of inflammatory cells) is carried out by the epithelium? These questions and others still need to be answered. Similar issues and questions could be raised for apoptosis in the alveolar endothelium.

What then is the capacity for cell removal in different compartments of the lung? The emphasis above has been placed on local ingestion by phagocytes of varying types, which we feel is the usual mode of apoptotic cell clearance. However, as noted, clearance via the airways may represent a mode for removal in the lung, and the relative proportions of cells that are removed from the alveolar or airways lumen by each mechanism are very difficult to determine. An additional point here is that pulmonary dendritic cells are also likely candidates for phagocytosis of apoptotic cells, with potential consequences for either suppression or activation of local adaptive (auto)immune responses.


   EFFECTS OF RECOGNITION AND UPTAKE OF APOPTOTIC CELLS IN THE LUNG
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 
Although an immediate implication of finding apoptotic cells in the lung is the effect of losing functional cells from the tissue, the actual process of apoptotic cell recognition also has important secondary consequences. Recognition and uptake of apoptotic cells are themselves noninflammatory and in fact have been shown to lead to an anti-inflammatory and anti-immunogenic environment. These effects are mediated in part by inhibitory intracellular signaling pathways (5) and in part by generation of anti-inflammatory mediators such as transforming growth factor (TGF)-β, IL-10, PGE2, PGI2, and the like (14, 19). This is usually contrasted with the proinflammatory effects induced by necrotic cells. However, it should be noted that most of the contrasts involve artificial experiments using necrotic cell debris induced by excessive heat or freezing and thawing, conditions not usually seen in real life. The stimuli on apoptotic or necrotic cells that induce these contrasting effects are not fully understood. Recognition of exposed phosphatidylserine appears to drive much of the anti-inflammatory and anti-immunogenic effects (14, 19, 29), whereas release of intracellular constituents during necrosis, including from mitochondria, lysosomes, and nucleus, likely all contribute to the subsequent enhanced inflammation. Nevertheless, cell disruption also exposes phosphatidylserine, and, on the other side, apoptosis can lead to exposure of proinflammatory stimuli such as DNA (53) and calreticulin (23). In each case, therefore, a balance between potential suppressors and inducers of inflammation and immunity might be expected, with varying local effects depending on cell type, mode of PCD induction, local environment, and efficacy of clearance. In general, the rapid removal of cells undergoing PCD is thought to limit the generation and/or release of proinflammatory stimuli, and the anti-inflammatory effects are suggested to dominate. Abnormalities of this balance may then be sought in conditions of overexuberant or prolonged inflammation or in examples of autoimmunity.

These anti-inflammatory consequences of apoptotic cell recognition (which by the way can occur from cell contact alone and do not require actual uptake of the apoptotic cell) are thought to play a major role in normal resolution of inflammation, including that in the lung (27, 30, 64). The concept leads inevitably to the question of defects in the process in chronic inflammatory conditions, for example, in cystic fibrosis, asthma or COPD.

Another intriguing consequence of apoptotic cell recognition may be to help initiate repair. Normal tissue homeostasis presumably involves efficient replacement after death of individual cells. Generation of growth factors in response to apoptotic cell recognition could provide a local stimulus for this replacement. Thus the demonstration that apoptotic cells can stimulate the production of VEGF (25) or hepatocyte growth factor (48), growth factors for endothelial and epithelial cells, respectively, is in keeping with this suggestion. Although direct demonstration of this effect in vivo is thus far lacking, one might even extend the concept to ask whether recognition of apoptotic cells might lead to production of chemotactic factors for tissue precursor cells that could also mediate repair of the injured lung by replacement of the apoptotic cells.

However, repair can also represent fibrotic changes in the tissue (as in classical wound healing). Thus the well-documented ability of apoptotic cells to stimulate the production of active TGF-β from responding cells (3, 14) provides yet another consequence of recognition, although here representing a double-edged sword. Apoptotic cells and the phosphatidylserine exposed on their surface have, therefore, to be considered as a candidate for the induction of the profibrogenic TGF-β in the various lung diseases in which local or generalized fibrosis occurs.


   CONCLUSIONS AND IMPLICATIONS
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
GRANTS
 REFERENCES
 
Finding apoptotic cells in lungs under various pathophysiological states is important. However, we suggest that it is now incumbent to further consider in each circumstance the identity of the cells undergoing PCD, the mechanisms of induction and execution, and tissue. On the other hand, despite these various implications, we should note that definitive demonstration of the effects of apoptosis, or its recognition, in lung diseases is extremely difficult. Inhibition of executioner caspases has been used, for example, to block development of emphysematous changes in experimental models of emphysema (e.g., Ref. 33). However, the administration of such agents may not be feasible in longer term pathogenic processes. Furthermore, blocking caspase effects likely allows damaged cells, unable to undergo normal apoptosis and removal, to undergo later nonapoptotic forms of death and cell destruction (necrotic processes), which have their own complex biological effects in the lung. Because the biological response to apoptotic cells is a highly redundant process, involving numerous recognition ligands, receptors, and bridge molecules, it is also not easy to simply manipulate the consequences of apoptosis at this recognition step. Nevertheless, difficult does not mean impossible, and we suggest that there is much to learn from addressing the questions raised, both for understanding pathogenesis and for potential new therapeutic approaches to lung diseases.


   GRANTS
TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-68864, HL-81151, GM-61031, and HL-66554.


   ACKNOWLEDGMENTS
 
The authors thank Shehzin Mozommel and Rheda Girgis for the caspase 3 images and Ann Hoa Le Thi for the TUNEL images.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. M. Henson, Dept. of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (e-mail: hensonp{at}njc.org)


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TOP
 ABSTRACT
 PROGRAMMED CELL DEATH,...
 DETECTION OF INCREASED NUMBERS...
 METHODS OF APOPTOTIC CELL...
 WHAT IS NEEDED TO...
 INCREASED APOPTOSIS VS....
 EFFECTS OF RECOGNITION AND...
 CONCLUSIONS AND IMPLICATIONS
 GRANTS
 REFERENCES
 

  1. Chipuk JE, Green DR. Dissecting p53-dependent apoptosis. Cell Death Differ 13: 994–1002, 2006.[CrossRef][Web of Science][Medline]
  2. Choe KH, Taraseviciene-Stewart L, Scerbavicius R, Gera L, Tuder RM, Voelkel NF. Methylprednisolone causes matrix metalloproteinase-dependent emphysema in adult rats. Am J Respir Crit Care Med 167: 1516–1521, 2003.[Abstract/Free Full Text]
  3. Clancy RM, Buyon JP. Clearance of apoptotic cells: TGF-β in the balance between inflammation and fibrosis. J Leukoc Biol 74: 959–960, 2003.[Free Full Text]
  4. Cowan KN, Heilbut A, Humpl T, Lam C, Ito S, Rabinovitch M. Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor. Nat Med 6: 698–702, 2000.[CrossRef][Web of Science][Medline]
  5. Cvetanovic M, Mitchell JE, Patel V, Avner BS, Su Y, van der Saag PT, Witte PL, Fiore S, Levine JS, Ucker DS. Specific recognition of apoptotic cells reveals a ubiquitous and unconventional innate immunity. J Biol Chem 281: 20055–20067, 2006.[Abstract/Free Full Text]
  6. DeCathelineau AM, Henson PM. The final step in programmed cell death: phagocytes carry apoptotic cells to the grave. Essays Biochem 39: 105–117, 2003.[Web of Science][Medline]
  7. Delivani P, Martin SJ. Mitochondrial membrane remodeling in apoptosis: an inside story. Cell Death Differ 13: 2007–2010, 2006.[CrossRef][Web of Science][Medline]
  8. Demedts IK, Demoor T, Bracke KR, Joos GF, Brusselle GG. Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema. Respir Res 7: 53, 2006.[CrossRef][Medline]
  9. Didenko VV, Hornsby PJ. Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis. J Cell Biol 135: 1369–1376, 1996.[Abstract/Free Full Text]
  10. Droemann D, Aries SP, Hansen F, Moellers M, Braun J, Katus HA, Dalhoff K. Decreased apoptosis and increased activation of alveolar neutrophils in bacterial pneumonia. Chest 117: 1679–1684, 2000.[CrossRef][Web of Science][Medline]
  11. Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 16: 663–669, 2004.[CrossRef][Web of Science][Medline]
  12. Erenpreisa J, Freivalds T, Roach H, Alston R. Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes. Histochem Cell Biol 108: 67–75, 1997.[CrossRef][Web of Science][Medline]
  13. Erjefalt J. Transepithelial migration, necrosis and apoptosis as silent and pro-inflammatory fates of airway granulocytes. Curr Drug Targets Inflamm Allergy 4: 425–431, 2005.[CrossRef][Medline]
  14. Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-β, PGE2, and PAF. J Clin Invest 101: 890–898, 1998.[Web of Science][Medline]
  15. Fairbairn DW, Olive PL, O'Neill KL. The comet assay: a comprehensive review. Mutat Res 339: 37–59, 1995.[Web of Science][Medline]
  16. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun 73: 1907–1916, 2005.[Free Full Text]
  17. Frankfurt OS, Robb JA, Sugarbaker EV, Villa L. Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp Cell Res 226: 387–397, 1996.[CrossRef][Web of Science][Medline]
  18. Frasch SC, Henson PM, Nagaosa K, Fessler MB, Borregaard N, Bratton DL. Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils. J Biol Chem 279: 17625–17633, 2004.[Abstract/Free Full Text]
  19. Freire-de-Lima CG, Xiao YQ, Gardai SJ, Bratton DL, Schiemann WP, Henson PM. Apoptotic cells, through transforming growth factor-beta, coordinately induce anti-inflammatory and suppress pro-inflammatory eicosanoid and NO synthesis in murine macrophages. J Biol Chem 281: 38376–38384, 2006.[Abstract/Free Full Text]
  20. Gallucci S, Matzinger P. Danger signals: SOS to the immune system. Curr Opin Immunol 13: 114–119, 2001.[CrossRef][Web of Science][Medline]
  21. Galluzzi L, Maiuri MC, Vitale I, Zischka H, Castedo M, Zitvogel L, Kroemer G. Cell death modalities: classification and pathophysiological implications. Cell Death Differ 14: 1237–1243, 2007.[CrossRef][Medline]
  22. Gardai SJ, Bratton DL, Ogden CA, Henson PM. Recognition ligands on apoptotic cells: a perspective. J Leukoc Biol 79: 896–903, 2006.[Abstract/Free Full Text]
  23. Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE, Bratton DL, Oldenborg PA, Michalak M, Henson PM. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 123: 321–334, 2005.[CrossRef][Web of Science][Medline]
  24. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2: 156–162, 2000.[CrossRef][Web of Science][Medline]
  25. Golpon HA, Fadok VA, Taraseviciene-Stewart L, Scerbavicius R, Sauer C, Welte T, Henson PM, Voelkel NF. Life after corpse engulfment: phagocytosis of apoptotic cells leads to VEGF secretion and cell growth. FASEB J 18: 1716–1718, 2004.[Abstract/Free Full Text]
  26. Green DR. Apoptotic pathways: ten minutes to dead. Cell 121: 671–674, 2005.[CrossRef][Web of Science][Medline]
  27. Haslett C. Granulocyte apoptosis and its role in the resolution and control of lung inflammation. Am J Respir Crit Care Med 160: S5–11, 1999.[Abstract/Free Full Text]
  28. Hodge S, Hodge G, Holmes M, Reynolds PN. Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation. Eur Respir J 25: 447–454, 2005.[Abstract/Free Full Text]
  29. Hoffmann PR, Kench JA, Vondracek A, Kruk E, Daleke DL, Jordan M, Marrack P, Henson PM, Fadok VA. Interaction between phosphatidylserine and the phosphatidylserine receptor inhibits immune responses in vivo. J Immunol 174: 1393–1404, 2005.[Abstract/Free Full Text]
  30. Huynh ML, Fadok VA, Henson PM. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation. J Clin Invest 109: 41–50, 2002.[CrossRef][Web of Science][Medline]
  31. Hyde DM, Harkema JR, Tyler NK, Plopper CG. Design-based sampling and quantitation of the respiratory airways. Toxicol Pathol 34: 286–295, 2006.[Abstract/Free Full Text]
  32. Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC, Voelkel NF. 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 163: 737–744, 2001.[Abstract/Free Full Text]
  33. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, Voelkel NF. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 106: 1311–1319, 2000.[Web of Science][Medline]
  34. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257, 1972.[Web of Science][Medline]
  35. Kirkham P. Oxidative stress and macrophage function: a failure to resolve the inflammatory response. Biochem Soc Trans 35: 284–287, 2007.[CrossRef][Web of Science][Medline]
  36. Kockx MM, Muhring J, Knaapen MW, de Meyer GR. RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am J Pathol 152: 885–888, 1998.[Abstract]
  37. Krysko DV, D'Herde K, Vandenabeele P. Clearance of apoptotic and necrotic cells and its immunological consequences. Apoptosis 11: 1709–1726, 2006.[CrossRef][Web of Science][Medline]
  38. Labat-Moleur F, Guillermet C, Lorimier P, Robert C, Lantuejoul S, Brambilla E, Negoescu A. TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement. J Histochem Cytochem 46: 327–334, 1998.[Abstract/Free Full Text]
  39. Lecoeur H, Ledru E, Prevost MC, Gougeon ML. Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods. J Immunol Methods 209: 111–123, 1997.[CrossRef][Web of Science][Medline]
  40. Lee CG, Cho SJ, Kang MJ, Chapoval SP, Lee PJ, Noble PW, Yehualaeshet T, Lu B, Flavell RA, Milbrandt J, Homer RJ, Elias JA. Early growth response gene 1-mediated apoptosis is essential for transforming growth factor β1-induced pulmonary fibrosis. J Exp Med 200: 377–389, 2004.[Abstract/Free Full Text]
  41. Ma B, Liu W, Homer RJ, Lee PJ, Coyle AJ, Lora JM, Lee CG, Elias JA. Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodeling. J Immunol 176: 4968–4978, 2006.[Abstract/Free Full Text]
  42. Majo J, Ghezzo H, Cosio MG. Lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema. Eur Respir J 17: 946–953, 2001.[Abstract/Free Full Text]
  43. Marchi E, Liu W, Broaddus VC. Mesothelial cell apoptosis is confirmed in vivo by morphological change in cytokeratin distribution. Am J Physiol Lung Cell Mol Physiol 278: L528–L535, 2000.[Abstract/Free Full Text]
  44. Martin TR, Hagimoto N, Nakamura M, Matute-Bello G. Apoptosis and epithelial injury in the lungs. Proc Am Thorac Soc 2: 214–220, 2005.[Abstract/Free Full Text]
  45. McLachlan CS, Yin JL, Driussi C, Jusuf PR, Hambly B, McGuire MA. A semi-quantitative PCR method for the detection of low levels of apoptotic DNA fragmentation in a heart failure model. Jpn J Physiol 50: 281–284, 2000.[CrossRef][Web of Science][Medline]
  46. Monks J, Rosner D, Geske FJ, Lehman L, Hanson L, Neville MC, Fadok VA. Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release. Cell Death Differ 12: 107–114, 2005.[CrossRef][Web of Science][Medline]
  47. Moodley YP, Caterina P, Scaffidi AK, Misso NL, Papadimitriou JM, McAnulty RJ, Laurent GJ, Thompson PJ, Knight DA. Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis. J Pathol 202: 486–495, 2004.[CrossRef][Web of Science][Medline]
  48. Morimoto K, Amano H, Sonoda F, Baba M, Senba M, Yoshimine H, Yamamoto H, Ii T, Oishi K, Nagatake T. Alveolar macrophages that phagocytose apoptotic neutrophils produce hepatocyte growth factor during bacterial pneumonia in mice. Am J Respir Cell Mol Biol 24: 608–615, 2001.[Abstract/Free Full Text]
  49. 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: 7657–7665, 2006.[Abstract/Free Full Text]
  50. Negoescu A, Lorimier P, Labat-Moleur F, Drouet C, Robert C, Guillermet C, Brambilla C, Brambilla E. In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations. J Histochem Cytochem 44: 959–968, 1996.[Abstract]
  51. Niikura Y, Dixit A, Scott R, Perkins G, Kitagawa K. BUB1 mediation of caspase-independent mitotic death determines cell fate. J Cell Biol 178: 283–296, 2007.[Abstract/Free Full Text]
  52. Okada H, Mak TW. Pathways of apoptotic and nonapoptotic death in tumour cells. Nat Rev Cancer 4: 592–603, 2004.[CrossRef][Web of Science][Medline]
  53. Palaniyar N, Nadesalingam J, Clark H, Shih MJ, Dodds AW, Reid KB. Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin. J Biol Chem 279: 32728–32736, 2004.[Abstract/Free Full Text]
  54. Perl M, Chung CS, Perl U, Lomas-Neira J, de Paepe M, Cioffi WG, Ayala A. Fas induced pulmonary apoptosis and inflammation during indirect acute lung injury. Am J Respir Crit Care Med 176: 591–601, 2007.[Abstract/Free Full Text]
  55. Petrache I, Natarajan V, Zhen L, Medler TR, Richter AT, Cho C, Hubbard WC, Berdyshev EV, Tuder RM. Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice. Nat Med 11: 491–498, 2005.[CrossRef][Web of Science][Medline]
  56. Piqueras B, Autran B, Debre P, Gorochov G. Detection of apoptosis at the single-cell level by direct incorporation of fluorescein-dUTP in DNA strand breaks. Biotechniques 20: 634–640, 1996.[Web of Science][Medline]
  57. 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.[CrossRef][Web of Science][Medline]
  58. Rosenblatt J, Raff MC, Cramer LP. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr Biol 11: 1847–1857, 2001.[CrossRef][Web of Science][Medline]
  59. Sakahira H, Enari M, Ohsawa Y, Uchiyama Y, Nagata S. Apoptotic nuclear morphological change without DNA fragmentation. Curr Biol 9: 543–546, 1999.[CrossRef][Web of Science][Medline]
  60. Salgame P, Varadhachary AS, Primiano LL, Fincke JE, Muller S, Monestier M. An ELISA for detection of apoptosis. Nucleic Acids Res 25: 680–681, 1997.[Abstract/Free Full Text]
  61. Sano M, Minamino T, Toko H, Miyauchi H, Orimo M, Qin Y, Akazawa H, Tateno K, Kayama Y, Harada M, Shimizu I, Asahara T, Hamada H, Tomita S, Molkentin JD, Zou Y, Komuro I. p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature 446: 444–448, 2007.[CrossRef][Medline]
  62. Saraste A. Morphologic criteria and detection of apoptosis. Herz 24: 189–195, 1999.[Web of Science][Medline]
  63. Savill J, Dransfield I, Gregory C, Haslett C. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2: 965–975, 2002.[CrossRef][Web of Science][Medline]
  64. Savill J, Gregory C, Haslett C. Cell biology. Eat me or die. Science 302: 1516–1517, 2003.[Abstract/Free Full Text]
  65. Schallock K, Schulz-Schaeffer WJ, Giese A, Kretzschmar HA. Postmortem delay and temperature conditions affect the in situ end-labeling (ISEL) assay in brain tissue of mice. Clin Neuropathol 16: 133–136, 1997.[Web of Science][Medline]
  66. Schrevens A, Van Nassauw L, Harrisson F. Histochemical demonstration of apoptotic cells in the chicken embryo using annexin V. Histochem J 30: 917–922, 1998.[CrossRef][Web of Science][Medline]
  67. Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, Earp HS, Matsushima GK. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411: 207–211, 2001.[CrossRef][Medline]
  68. Shen HM, Pervaiz S. TNF receptor superfamily-induced cell death: redox-dependent execution. FASEB J 20: 1589–1598, 2006.[Abstract/Free Full Text]
  69. Sloop GD, Roa JC, Delgado AG, Balart JT, Hines MO, 3rd, Hill JM. Histologic sectioning produces TUNEL reactivity. A potential cause of false-positive staining. Arch Pathol Lab Med 123: 529–532, 1999.[Web of Science][Medline]
  70. Srinivasan A, Roth KA, Sayers RO, Shindler KS, Wong AM, Fritz LC, Tomaselli KJ. In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Differ 5: 1004–1016, 1998.[CrossRef][Web of Science][Medline]
  71. Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, Mc Mahon G, Waltenberger J, Voelkel NF, Tuder RM. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J 15: 427–438, 2001.[Abstract/Free Full Text]
  72. Thannickal VJ, Horowitz JC. Evolving concepts of apoptosis in idiopathic pulmonary fibrosis. Proc Am Thorac Soc 3: 350–356, 2006.[Abstract/Free Full Text]
  73. Tuder RM, Zhen L, Cho CY, Taraseviciene-Stewart L, Kasahara Y, Salvemini D, Voelkel NF, Flores SC. Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade. Am J Respir Cell Mol Biol 29: 88–97, 2003.[Abstract/Free Full Text]
  74. Van den Eijnde SM, Luijsterburg AJ, Boshart L, De Zeeuw CI, van Dierendonck JH, Reutelingsperger CP, Vermeij-Keers C. In situ detection of apoptosis during embryogenesis with annexin V: from whole mount to ultrastructure. Cytometry 29: 313–320, 1997.[CrossRef][Web of Science][Medline]
  75. Van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31: 1–9, 1998.[CrossRef][Web of Science][Medline]
  76. Van Heerde WL, Robert-Offerman S, Dumont E, Hofstra L, Doevendans PA, Smits JF, Daemen MJ, Reutelingsperger CP. Markers of apoptosis in cardiovascular tissues: focus on annexin V. Cardiovasc Res 45: 549–559, 2000.[Abstract/Free Full Text]
  77. Vandivier RW, Fadok VA, Hoffmann PR, Bratton DL, Penvari C, Brown KK, Brain JD, Accurso FJ, Henson PM. Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. J Clin Invest 109: 661–670, 2002.[CrossRef][Web of Science][Medline]
  78. Wang H, Liu X, Umino T, Skold CM, Zhu Y, Kohyama T, Spurzem JR, Romberger DJ, Rennard SI. Cigarette smoke inhibits human bronchial epithelial cell repair processes. Am J Respir Cell Mol Biol 25: 772–779, 2001.[Abstract/Free Full Text]
  79. Wolvekamp MC, Darby IA, Fuller PJ. Cautionary note on the use of end-labelling DNA fragments for detection of apoptosis. Pathology 30: 267–271, 1998.[CrossRef][Web of Science][Medline]
  80. Zamai L, Falcieri E, Marhefka G, Vitale M. Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation. Cytometry 23: 303–311, 1996.[CrossRef][Web of Science][Medline]
  81. Zheng T, Kang MJ, Crothers K, Zhu Z, Liu W, Lee CG, Rabach LA, Chapman HA, Homer RJ, Aldous D, De Sanctis GT, Underwood S, Graupe M, Flavell RA, Schmidt JA, Elias JA. Role of cathepsin S-dependent epithelial cell apoptosis in IFN-{gamma}-induced alveolar remodeling and pulmonary emphysema. J Immunol 174: 8106–8115, 2005.[Abstract/Free Full Text]



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