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Published ahead of print on June 30, 2004, doi:10.1164/rccm.200402-240OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 742-747, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200402-240OC

Original Article

Occurrence of Apoptosis, Secondary Necrosis, and Cytolysis in Eosinophilic Nasal Polyps

Lena Uller, Morgan Andersson, Lennart Greiff, Carl G. A. Persson and Jonas S. Erjefält

Department of Physiological Sciences; Department of Otorhinolaryngology, Head and Neck Surgery; and Department of Clinical Pharmacology, Lund University Hospital, Lund, Sweden

Correspondence and requests for reprints should be addressed to Lena Uller, M.Sc., Department of Physiological Sciences, BMC F10, Lund University, 221 84 Lund, Sweden. E-mail: lena.uller{at}mphy.lu.se


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The paradigm states that inflammatory cells disappear from airway tissues through apoptosis and phagocytosis. However, cells may also be cleared through primary cytolysis, necrosis secondary to apoptosis, or transepithelial migration. This study examines the occurrence of apoptosis, secondary necrosis, and cytolysis of eosinophils in human nasal polyps in vivo and blood eosinophils in vitro. Eosinophils abounded in subepithelium and in paracellular epithelial pathways. Macrophages commonly occurred but without engulfed eosinophils. Scattered cells, including epithelial cells, were stained by antibody to the caspase cleavage product of poly(ADP-ribose) polymerase. Few cells were apoptotic (stained by terminal deoxy RNase nick end labeling). Of more than 3,000 examined tissue eosinophils, 110 were caspase cleavage positive, but only one was apoptotic. Transmission electron microscopy analysis of more than 500 eosinophils revealed viable and cytolytic eosinophils but not apoptosis, secondary necrosis, or engulfment of eosinophils. Plasma cells but neither epithelial cells nor eosinophils exhibited apoptotic ultrastructural morphology. Eosinophils in vitro exhibited different stages of apoptosis, ending with secondary necrosis distinct from in vivo eosinophil cytolysis. Our results show that the clearance of eosinophils from nasal polyps largely occurs through nonapoptosis pathways, including cytolysis and paraepithelial migration, and they challenge the belief that apoptosis is important for clearance of eosinophils from respiratory tissues.

Key Words: electron microscopy • epithelium • poly(ADP-ribose) polymerase • terminal deoxy RNase nick end labeling

The view that infiltration of leukocytes in diseased airway tissues is counterbalanced by their elimination through apoptosis and prompt engulfment by macrophages has developed into a major paradigm (1, 2). Work in vitro has generated detailed information regarding molecular and pharmacologic regulation of apoptosis (3, 4). Human purified blood eosinophils seem particularly prone to undergo massive apoptosis, especially when cultured in the absence of specific growth factors or in the presence of glucocorticoids in the cell medium (5–9). Contrasting the numerous in vitro reports, there is in the case of the eosinophil as yet limited in vivo data on importance, or even actual occurrence, of apoptosis in inflamed airways. However, a paucity of apoptotic cells in tissues in vivo has been well accommodated within the realm of the apoptosis paradigm. It is thus maintained that apoptotic cells are removed by scavenger systems so quickly that they "cannot be detected in tissues at any one time" (10, 11). Furthermore, any occurrence of significant numbers of apoptotic cells in a tissue has been suggested to reflect either massive injury or defect clearance mechanisms (1). Such statements, although of wide validity, may not yet be fully supported by critical in vivo research in each of those cases when apoptotic cells have been absent in a tissue. It is also of note that macrophages from different sources and at different stages of activation (12) may differ in their ability to engulf apoptotic cells, suggesting the possibility that apoptotic cells would not vanish immediately from all tissues. Furthermore, the occurrence of apoptotic eosinophils and neutrophils has been reported in human skin (13), indicating that apoptotic cells in vivo should be well detectible by careful histologic examination of tissues.

The eosinophil, with a capacity to release tissue-toxic granule proteins, is predominant among the infiltrating cells in several airway diseases, including asthma, allergic rhinitis, and nasal polyposis (14–16). Based largely on in vitro data, apoptosis is considered to account for any clearance of airway tissue eosinophils, not least in steroid-treated patients (17, 18). However, as exemplified by several features of the eosinophil, observations made in vitro may not automatically translate into in vivo (19). Molecular milieu, cell phenotypes, and cell clearance pathways differ greatly between in vitro and in vivo, as they also may differ between airway tissue and airway lumen (19). Such differences may in part explain contradictory reports on the occurrence of eosinophil apoptosis in airway diseases (18–24). Because airway eosinophilia is a central feature of animal models of asthma, there are ample opportunities for studies of the role of apoptosis for elimination of this tissue leukocyte, yet apoptotic eosinophils have not been compellingly demonstrated in animal airway tissues, not even under spontaneous or drug-induced elimination of the eosinophils (19, 25, 26). Instead, the animal airway tissue in vivo has obviously been depleted of its eosinophils by their egression into the airway lumen (19, 26). The negative observations in animals underscore the need to obtain more data on the occurrence of apoptotic eosinophils in human airways.

This study employs surgically removed nasal polyps from nonselected patients. The polyps are rich in eosinophils and would allow sufficiently extensive examination of tissue areas to determine the occurrence of apoptotic eosinophils. We have chosen three different methods claimed to detect different stages of the apoptotic process: terminal deoxy RNase nick end labeling (TUNEL) (stains DNA fragmentation occurring at an advanced stage of apoptosis), p85 poly(ADP-ribose) polymerase (PARP) staining (detects a caspase-mediated cleavage reaction reflecting early events in apoptosis), and transmission electron microscopy (TEM). Importantly, a clear identification of morphologic characteristics, achievable exclusively by TEM analysis, is considered crucial for assessment of occurrence of apoptosis of cells in vivo (27). We have also looked specifically for eosinophil cell debris occurring in macrophages or other cells, as would be the case if apoptotic eosinophils had been engulfed in vivo. The present TEM analysis further involves the detection of other features of the tissue eosinophils such as occurrence of primary cytolysis (28) as well as any occurrence of necrosis secondary to apoptosis. To ascertain detection of different stages of eosinophil apoptosis, including secondary necrosis, we have compared our in vivo observations with the ultrastructural features of different stages of eosinophil apoptosis emerging in cultured eosinophils in this study.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Nasal Polyp Tissue Material
Nasal polyps were obtained nonselectively from 18 patients undergoing polypectomy (12 patients were treated with 200 µg budesonide daily, and 6 were untreated). After endoscopic polyp removal, the specimens were immediately placed in fixative, and different sections of the same specimen were processed in parallel for histochemical and ultrastructural analysis. The study was approved by the local ethical committee at Lund University.

Detection of Eosinophils
Tissue segments were immersed overnight in Stefaninis fixative (2% paraformaldehyde and 0.2% picric acid in 0.1-M phosphate buffer, pH 7.2) and rinsed and frozen in mounting medium (TissueTEK, Sakura Finetek EuropeRV; Zoeterwoude, The Netherlands). Eosinophils in polyp tissues were detected by histochemical visualization of cyanide-resistant eosinophil peroxidase (28). Eosinophils were identified by their dark-brown reaction product and were quantified as numbers of eosinophils/0.1-mm2 tissue area. At least 150 eosinophils were present in each polyp tissue section.

Detection of Macrophages
Macrophages were identified by morphologic criteria and stained using a mouse anti-human CD68 monoclonal antibody (28).

In Situ Detection of Apoptotic Cells with the TUNEL Technique
Tissue segments were immersed overnight in buffered 4% paraformaldehyde and were dehydrated and embedded in paraffin. Apoptosis was visualized using the in situ TUNEL technique (26). No staining was evident in negative control subjects where the Tdt enzyme was omitted. Slides were counterstained with propidium iodide to reveal pyknotic nuclei as well as total number of cells. Apoptotic eosinophils were defined as dual chromotrope-2R and TUNEL-positive cells exhibiting apoptotic morphology.

Immunocytochemical Staining of PARP, p85 Fragment-positive Cells
Cryosections (5 µm) were washed in phosphate-buffered saline (PBS) and incubated with the anti-p85 PARP polyclonal antibody (dilution 1:200; Promega, Madison, WI) overnight at 4°C (29). Sections were rinsed in PBS and incubated with a secondary antibody (biotinylated goat anti-rabbit; Vector BA; 1,000, dilution 1:200) (Vector Laboratories, Burlingame, CA) for 1 hour. After washing in PBS, sections were incubated in alkaline phosphatase-conjugates streptavidin (dilution 1:200; Dako A/S, Glostrup, Denmark) for 45 minutes, rinsed developed using New fuchsin (Dako) as substrate, and counterstained with Mayer's hematoxylin.

Eosinophil Apoptosis In Vitro
Eosinophils were isolated from heparinized blood from healthy individuals using Percoll gradient centrifugation and immunomagnetic depletion of neutrophils using anti-CD16 antibodies (30). Eosinophils were incubated in cell culture medium without growth factors for 24 hours in a humidified chamber at 37°C with 5% CO2. The eosinophils were then centrifuged and resuspended in a small volume of transmission electron microscopy (TEM) fixative and kept for 1 hour at room temperature. The suspension with fixed cells was centrifuged, and the obtained pellet was embedded in warm (40–50°C) 3% agarose/PBS. The cell-containing agarose gel was placed in 4% formaldehyde over night and prepared for TEM analysis.

TEM
Samples for TEM were prepared (26). Ultrastructural criteria for eosinophil apoptosis included cell shrinkage, nuclear chromatin condensation, membrane blebbing, and intact cell membrane (31). Eosinophil cytolysis was defined as the presence of chromatolysis and the loss of plasma membrane integrity (32). Secondary necrosis of apoptotic eosinophils was distinguished from cytolysis as cells showing condensed dark nucleus and cell membrane rupture.

Statistical Analysis
All data are mean ± SEM. Statistical significance was between mean values; the Wilcoxon-Mann-Whitney test was performed using Statview Software. A value of p less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Occurrence and Features of Eosinophils in the Nasal Mucosa
Eosinophils were distributed in the epithelium and the subepithelial layer (Figure 1A). Polyps obtained from patients that had been treated with steroids showed no difference in eosinophil number or distribution as compared with steroid-naive patients (p > 0.05). All polyps are, therefore, also accounted for as one group (Table 1 and Figure 1A). The present TEM analysis (in total, we examined 537 eosinophils by TEM) revealed that the eosinophils generally exhibited signs of piecemeal degranulation with partly empty specific granules (Figure 2a). Twenty percent of the 537 tissue eosinophils were cytolytic, as shown by incomplete chromatolysis, cell membrane disruption, and spilling of protein-containing free granules (Figures 2b and 2c). None of the tissue eosinophils exhibited morphologic features of apoptosis, nor were any epithelial cells identified as apoptotic by the TEM analysis. Other cells such as plasma cells (Figure 3a) were clearly apoptotic. Eosinophils and neutrophils were occasionally seen on paracellular epithelial paths (Figure 2d), and they appeared on the airway surface (Figure 2e).



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  		Figure 1. Representative bright-field micrograph showing eosinophil peroxidase (EPO)-stained eosinophils (brown cells) in the epithelium and the subepithelium of nasal polyps; scale bar = 150 µm (A). Combined chromotrope-2R and terminal deoxy RNase nick end labeling (TUNEL) staining (cells stained dark blue; arrows) revealed a single apoptotic eosinophil (enlarged at insert), scale bar = 80 µm (B). TUNEL-positive cells are distinguished (green cells; arrows) in this fluorescent micrograph where cell nuclei have been counterstained red with propidium iodide; scale bar = 40 µm (C). Typical p85 poly(ADP-ribose) polymerase (PARP) staining of cells (arrows) in the nasal polyp tissue; scale bar = 80 µm (D). Detail of p85 PARP-positive cells (arrows) in the epithelium; scale bar = 40 µm (E).

 

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  		TABLE 1. Epithelial and subepithelial cells/0.1 mm2 in human nasal polyp

 


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  		Figure 2. Transmission electron micrographs of polyp tissues demonstrating viable eosinophils with signs of piecemeal degranulation (a), a cytolytic eosinophil exhibiting chromatolysis and an early stage of cell membrane rupture (b), spilling of numerous free eosinophil granules and cell debris (c), eosinophils and neutrophils lining up between columnar epithelial cells (d), and an eosinophil on the epithelial surface (e).

 


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  		Figure 3. Transmission electron micrographs of polyp tissues demonstrating an apoptotic plasma cell (a), an epithelial cell with engulfed cell material (b), and a macrophage containing engulfed cell material and apoptotic bodies (c).

 
Macrophages
CD-68 positive cells commonly occurred (Table 1). Double staining for CD-68 and eosinophils (Chromotrop -2R and eosinophil peroxidase [EPO]) did not reveal any engulfment of eosinophils. As detected by TEM, macrophages exhibited signs of engulfment of cell material, but no specific eosinophil-like features such as specific granules were engulfed (Figure 3c). Similarly, epithelial cells exhibited engulfed cell material of unidentified origin (Figure 3b).

Apoptotic Cells Detected by TUNEL Staining
As revealed by the TUNEL method, apoptotic cells were scattered in the polyp tissue (Figures 1b and 1c and Table 1). No difference in the number of apoptotic cells was detected between steroid-treated and nontreated polyps (p > 0.05). Among all of the TUNEL-positive cells, only one was an eosinophil, as detected by combined TUNEL and Chromotrope-2R staining (Figure 1b). Thus, of more than 3,000 Chromotrope-2R–stained eosinophils in the present tissues, there was merely a single TUNEL-positive eosinophil. The morphologic features of this cell, originating from a patient who did not receive nasal steroids, were not conclusive as regards apoptosis.

PARP, p85-positive Cells
Cells stained for the PARP p85 fragment showed a red cytoplasm combined with a light blue nucleus (hematoxylin background staining) (Figures 1d and 1e). PARP-positive cells (Table 1) included fibroblasts, macrophages, and epithelial cells. PARP-positive eosinophils (110 cells in total) also occurred (Table 1).

Ultrastructural Features of Growth Factor–depleted In Vitro Eosinophils
Sixty percent of the isolated eosinophils showed signs of apoptosis. At least three different stages of eosinophil apoptosis could be discerned by the present TEM analysis (Figure 4). An early stage was characterized by a condensed nucleus with a remaining clear distinction between the light euchromatin and dark heterochromatin and unaffected nuclear and cellular membranes (Figure 4b). A more advanced second stage was identified by the presence of a fully condensed or dark round pyknotic nucleus exhibiting a few nuclear membrane blebs (Figure 4c) but with an intact cell membrane. A later third stage, defined as secondary necrosis, was characterized by extensive nuclear membrane blebbing, rupture of the cell membrane, and a lucent cytoplasm. The apoptotic cell contour remained, whereas the nucleus was finally disintegrated through extensive chromatolysis (Figures 4d and 4e). Irrespective of viable state, the in vitro eosinophils exhibited varying degrees of piecemeal degranulation (Figures 4a–4e).



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  		Figure 4. Transmission electron micrographs of purified blood eosinophils demonstrating a normal in vitro eosinophil (a), an eosinophil at an early stage of apoptosis with typical nuclear chromatin condensation (b), an apoptotic eosinophil with pyknotic nucleus with extensive blebbing at the nuclear envelope (c), apoptotic eosinophils undergoing secondary necrosis with chromatolysis (d), and an eosinophil in which the nucleus is completely lysed in a late phase of secondary necrosis (e).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has demonstrated that apoptotic eosinophils, as identified by TEM and a validated TUNEL technique, are exceedingly rare in human nasal polyp tissues in vivo. The present scarcity of apoptotic eosinophils, together with other findings in this and previous in vivo studies (19), suggests the possibility that turnover of human airway tissue eosinophils in vivo, irrespective of steroid treatment, largely involves other mechanisms than apoptosis. Alternative modes, as also compatible with observations in this study, include noninjurious egression between epithelial cells into the airway lumen and proinflammatory disintegration through cytolysis. We further demonstrate here that the eosinophil cytolysis phenomenon that occurs in vivo has features that are distinct from the secondary necrosis of apoptotic eosinophils that commonly occurs in vitro in this study. These data are of interest with regard to clearance of a major leukocyte from airway tissues in vivo, and they may highlight difficulties regarding translation of concepts from in vitro to in vivo.

Authors employing the TUNEL technique or staining of phosphatidylserine (annexin), without demonstration of apoptotic morphology, have reported that human diseased airway tissue, especially nasal polyps, contain numerous apoptotic cells, including apoptotic eosinophils (19–24). The inconsistency between this prior art and this study likely reflects method differences. Thus, the annexin staining of histologic sections would predictably produce false-positive results because any section-induced cell damage would make the intracellular phosphatidylserine amenable for staining. Also, depending on the concentration and incubation time of involved enzymes and nucleotides, the TUNEL method may fail to stain even truly apoptotic cells or, more commonly, may stain virtually all cells in the tissue irrespective of apoptosis (19, 21).

In this study, our TEM analysis served as a complement to as well as a validation of the present employment of the apoptosis staining techniques. Contrasting the previously reported abundance of apoptotic cells, the conservative use of the TUNEL technique and TEM analysis revealed only a small number of scattered apoptotic cells in the diseased airway tissue. The present single TUNEL-positive eosinophil (of more than 3,000 examined tissue eosinophils) and none assessed by TEM (of more than 500 examined tissue eosinophils) suggest that eosinophil apoptosis rarely occurs in human eosinophilic nasal polyps. This inference is strengthened by the present demonstration by TUNEL and TEM of other apoptotic cells in the polyp tissues; if apoptotic eosinophils had occurred in significant numbers, our methods would have demonstrated this. Furthermore, eosinophil cell debris inside macrophages, or inside other cells, did not occur in the polyp tissues (Figure 3c). Hence, we cannot support the view that eosinophil apoptosis and engulfment regulates the level of eosinophilia in vivo in diseased airway tissue (5, 8, 9, 33).

Airway steroids, through inhibition of growth factors, including interleukin-5 both in vitro (34) and in vivo (35), should theoretically increase the occurrence of eosinophil apoptosis. Thus, the inclusion of steroid-treated patients adds weight to the present negative data. Our results agree with animal studies where apoptotic tissue eosinophils were lacking even during resolution of lung eosinophilia induced by highly effective, systemic steroid doses (26). The antieosinophilic action of steroids in animal airways was explained by a combination of drug-induced inhibition of recruitment of new cells and permission of the elimination of tissue eosinophils into the airway lumen (26). Thus, the intriguing localization of eosinophils and neutrophils in rows between epithelial cells in this study potentially reflects traffic of tissue granulocytes into the airway lumen.

The present ultrastructural characterization of purified, growth factor–depleted human blood eosinophils identified early signs of apoptosis involving nuclear bleb formation that would not be detectable by common light microscopy, as well as the final stages of apoptosis involving secondary necrosis. Furthermore, we now demonstrate that secondary necrosis exhibited structural features distinguishing it from the primary cytolysis of eosinophils that commonly occur in diseased human airway tissues (Figure 4). The vast majority of the present cytolytic eosinophils were PARP negative (Table 1). This finding together with their ultrastructural features would clearly distinguish cytolytic from apoptotic eosinophils. We have previously demonstrated that eosinophil cytolysis occurs independent of prior degranulation of the eosinophils (32). These data, in addition, indicate that cytolysis of eosinophils is a significant event in its own right that should not be confused with the necrosis of eosinophils that occurs in vitro secondary to apoptosis and that may occur also in vivo in airway tissues under the exceptional conditions caused by anti-Fas treatment of mouse airways (36).

The molecular mechanisms involved in apoptosis offer opportunities to the development of apoptosis detection methods. In this study we have used an antibody directed against the 85-kD caspase-cleaved fragment (p85) of human poly (ADP-ribose) polymerase. Anti-PARP p85 fragment antibody is considered to be an early marker of apoptosis (29) because cleavage of PARP occurs before DNA fragmentation. However, other cellular repair proteins than PARP may operate, and it has not been determined in detail that PARP p85-positive cells always are or become apoptotic cells. We demonstrated PARP p85-positive staining in both the epithelium and subepithelium in the present nasal polyp tissue involving fibroblasts, epithelial cells, and eosinophils. The total number of PARP p85-positive cells was markedly higher than the TUNEL-positive cells in this study. As also observed in the present nasal polyps, previous work has demonstrated PARP-positive epithelial cells in human airway disease, including asthma (29). The polyp tissue as a model for human chronic airway inflammation may, in fact, exhibit a range of interesting similarities to asthmatic airway mucosa (37). The occurrence of apoptotic epithelial cells, however, could not be confirmed by the present TEM analysis, nor have other workers produced compelling confirmation of apoptotic epithelial cell morphology in human airways in vivo (29). It is possible that epithelial cells, too, are eliminated by entering the airway lumen. Epithelial cells may thus detach without prior apoptosis (29) and be found intact (even with beating cilia) in asthmatic sputa (38). Further work is warranted to define what cell features may be associated with PARP positivity. Currently, it cannot be excluded that the PARP-positive cells are at an early stage of apoptosis, but then nothing is known regarding the extent to which the PARP-positive cells, such as the present PARP-positive eosinophils, actually proceed into a true stage of apoptosis with distinct morphologic features. There was no indication in this study that PARP-positive eosinophils were moving toward the airway lumen (Table 1). Thus, our findings do not support the possibility that PARP-positive eosinophils are designated for transepithelial migration to contribute to the pool of apoptotic eosinophils that actually occur in airway luminal liquids in animals and humans (25, 26, 39, 40).

In summary, we have demonstrated that major features of eosinophils in culture, including apoptosis and secondary necrosis, may not occur to significant extents in vivo in human eosinophilic airway tissues such as nasal polyps, yet other cells scattered in the tissue clearly are both apoptotic and unengulfed by macrophages or other neighbor cells. We also demonstrate that the cytolysis of eosinophils, occurring in vivo, is of a primary nature distinct from the secondary necrosis phenomenon. Additionally, granulocytes occur between columnar epithelial cells and on the mucosal surface. We conclude that local turnover of tissue eosinophils in diseased airways occurs through other pathways than apoptosis. These pathways would include "silent" egression into the lumen and "proinflammatory" disintegration through primary cytolysis.


   Acknowledgments
 
The authors thank Dr. Eric Carlemalm, Electron Microscopy Unit, Lund University, for expert assistance with processing of TEM figures and Monika Malm-Erjefält for technical assistance regarding the purification of blood eosinophils.


   FOOTNOTES
 
Supported by Medical Faculty, Lund University, Sweden; the Swedish Medical Research Council; and the Heart and Lung Foundation, Sweden.

Conflict of Interest Statement: L.U. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; L.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.G.A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.S.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 25, 2004; accepted in final form June 29, 2004


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 ABSTRACT
 METHODS
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 DISCUSSION
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