Occurrence of Apoptosis_ Secondary Necrosis_ and Cytolysis in

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					Occurrence of Apoptosis, Secondary Necrosis,
and Cytolysis in Eosinophilic Nasal Polyps
Lena Uller, Morgan Andersson, Lennart Greiff, Carl G. A. Persson, and Jonas S. Erjefalt

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

The paradigm states that inflammatory cells disappear from airway                     defect clearance mechanisms (1). Such statements, although of
tissues through apoptosis and phagocytosis. However, cells may                        wide validity, may not yet be fully supported by critical in vivo
also be cleared through primary cytolysis, necrosis secondary to                      research in each of those cases when apoptotic cells have been
apoptosis, or transepithelial migration. This study examines the                      absent in a tissue. It is also of note that macrophages from different
occurrence of apoptosis, secondary necrosis, and cytolysis of eosino-                 sources and at different stages of activation (12) may differ in
phils in human nasal polyps in vivo and blood eosinophils in vitro.                   their ability to engulf apoptotic cells, suggesting the possibility
Eosinophils abounded in subepithelium and in paracellular epithe-
                                                                                      that apoptotic cells would not vanish immediately from all tissues.
lial pathways. Macrophages commonly occurred but without en-
                                                                                      Furthermore, the occurrence of apoptotic eosinophils and neutro-
gulfed eosinophils. Scattered cells, including epithelial cells, were
                                                                                      phils has been reported in human skin (13), indicating that apo-
stained by antibody to the caspase cleavage product of poly(ADP-
ribose) polymerase. Few cells were apoptotic (stained by terminal
                                                                                      ptotic cells in vivo should be well detectible by careful histologic
deoxy RNase nick end labeling). Of more than 3,000 examined                           examination of tissues.
tissue eosinophils, 110 were caspase cleavage positive, but only one                      The eosinophil, with a capacity to release tissue-toxic granule
was apoptotic. Transmission electron microscopy analysis of more                      proteins, is predominant among the infiltrating cells in several
than 500 eosinophils revealed viable and cytolytic eosinophils but                    airway diseases, including asthma, allergic rhinitis, and nasal
not apoptosis, secondary necrosis, or engulfment of eosinophils.                      polyposis (14–16). Based largely on in vitro data, apoptosis is
Plasma cells but neither epithelial cells nor eosinophils exhibited                   considered to account for any clearance of airway tissue eosino-
apoptotic ultrastructural morphology. Eosinophils in vitro exhibited                  phils, not least in steroid-treated patients (17, 18). However, as
different stages of apoptosis, ending with secondary necrosis dis-                    exemplified by several features of the eosinophil, observations
tinct from in vivo eosinophil cytolysis. Our results show that the                    made in vitro may not automatically translate into in vivo (19).
clearance of eosinophils from nasal polyps largely occurs through                     Molecular milieu, cell phenotypes, and cell clearance pathways
nonapoptosis pathways, including cytolysis and paraepithelial mi-                     differ greatly between in vitro and in vivo, as they also may
gration, and they challenge the belief that apoptosis is important                    differ between airway tissue and airway lumen (19). Such differ-
for clearance of eosinophils from respiratory tissues.                                ences may in part explain contradictory reports on the occur-
Keywords: electron microscopy; epithelium; poly(ADP-ribose) polymer-                  rence of eosinophil apoptosis in airway diseases (18–24). Because
ase; terminal deoxy RNase nick end labeling                                           airway eosinophilia is a central feature of animal models of
                                                                                      asthma, there are ample opportunities for studies of the role of
The view that infiltration of leukocytes in diseased airway tissues                    apoptosis for elimination of this tissue leukocyte, yet apoptotic
is counterbalanced by their elimination through apoptosis and                         eosinophils have not been compellingly demonstrated in animal
prompt engulfment by macrophages has developed into a major                           airway tissues, not even under spontaneous or drug-induced
paradigm (1, 2). Work in vitro has generated detailed information                     elimination of the eosinophils (19, 25, 26). Instead, the animal
regarding molecular and pharmacologic regulation of apoptosis                         airway tissue in vivo has obviously been depleted of its eosino-
(3, 4). Human purified blood eosinophils seem particularly prone                       phils by their egression into the airway lumen (19, 26). The
to undergo massive apoptosis, especially when cultured in the                         negative observations in animals underscore the need to obtain
absence of specific growth factors or in the presence of glucocor-                     more data on the occurrence of apoptotic eosinophils in human
ticoids in the cell medium (5–9). Contrasting the numerous in                         airways.
vitro reports, there is in the case of the eosinophil as yet limited                      This study employs surgically removed nasal polyps from nonse-
in vivo data on importance, or even actual occurrence, of apopto-                     lected patients. The polyps are rich in eosinophils and would allow
sis in inflamed airways. However, a paucity of apoptotic cells in                      sufficiently extensive examination of tissue areas to determine the
tissues in vivo has been well accommodated within the realm of                        occurrence of apoptotic eosinophils. We have chosen three differ-
the apoptosis paradigm. It is thus maintained that apoptotic cells                    ent methods claimed to detect different stages of the apoptotic
are removed by scavenger systems so quickly that they “cannot                         process: terminal deoxy RNase nick end labeling (TUNEL) (stains
be detected in tissues at any one time” (10, 11). Furthermore,                        DNA fragmentation occurring at an advanced stage of apoptosis),
any occurrence of significant numbers of apoptotic cells in a                          p85 poly(ADP-ribose) polymerase (PARP) staining (detects a cas-
tissue has been suggested to reflect either massive injury or                          pase-mediated cleavage reaction reflecting early events in apopto-
                                                                                      sis), and transmission electron microscopy (TEM). Importantly, a
                                                                                      clear identification of morphologic characteristics, achievable
                                                                                      exclusively by TEM analysis, is considered crucial for assessment
(Received in original form February 25, 2004; accepted in final form June 29, 2004)   of occurrence of apoptosis of cells in vivo (27). We have also
Supported by Medical Faculty, Lund University, Sweden; the Swedish Medical            looked specifically for eosinophil cell debris occurring in macro-
Research Council; and the Heart and Lung Foundation, Sweden.
                                                                                      phages or other cells, as would be the case if apoptotic eosino-
Correspondence and requests for reprints should be addressed to Lena Uller,           phils had been engulfed in vivo. The present TEM analysis
M.Sc., Department of Physiological Sciences, BMC F10, Lund University, 221 84
                                                                                      further involves the detection of other features of the tissue
Lund, Sweden. E-mail:
                                                                                      eosinophils such as occurrence of primary cytolysis (28) as well as
Am J Respir Crit Care Med Vol 170. pp 742–747, 2004
Originally Published in Press as DOI: 10.1164/rccm.200402-240OC on June 30, 2004      any occurrence of necrosis secondary to apoptosis. To ascertain
Internet address:                                                 detection of different stages of eosinophil apoptosis, including
Uller, Andersson, Greiff, et al.: Occurrence of Eosinophil Apoptosis                                                                            743

secondary necrosis, we have compared our in vivo observations                of plasma membrane integrity (32). Secondary necrosis of apoptotic
with the ultrastructural features of different stages of eosinophil          eosinophils was distinguished from cytolysis as cells showing condensed
apoptosis emerging in cultured eosinophils in this study.                    dark nucleus and cell membrane rupture.

                                                                             Statistical Analysis
                                                                             All data are mean    SEM. Statistical significance was between mean
Human Nasal Polyp Tissue Material                                            values; the Wilcoxon-Mann-Whitney test was performed using Statview
Nasal polyps were obtained nonselectively from 18 patients undergoing        Software. A value of p less than 0.05 was considered significant.
polypectomy (12 patients were treated with 200 g budesonide daily,
and 6 were untreated). After endoscopic polyp removal, the specimens         RESULTS
were immediately placed in fixative, and different sections of the same
specimen were processed in parallel for histochemical and ultrastruc-        Occurrence and Features of Eosinophils in the Nasal Mucosa
tural analysis. The study was approved by the local ethical committee        Eosinophils were distributed in the epithelium and the subepi-
at Lund University.                                                          thelial layer (Figure 1A). Polyps obtained from patients that had
Detection of Eosinophils                                                     been treated with steroids showed no difference in eosinophil
                                                                             number or distribution as compared with steroid-naive patients
Tissue segments were immersed overnight in Stefaninis fixative (2% para-
                                                                             (p 0.05). All polyps are, therefore, also accounted for as one
formaldehyde and 0.2% picric acid in 0.1-M phosphate buffer, pH 7.2)
and rinsed and frozen in mounting medium (TissueTEK, Sakura Finetek
                                                                             group (Table 1 and Figure 1A). The present TEM analysis (in
EuropeRV; Zoeterwoude, The Netherlands). Eosinophils in polyp tissues        total, we examined 537 eosinophils by TEM) revealed that the
were detected by histochemical visualization of cyanide-resistant eosino-    eosinophils generally exhibited signs of piecemeal degranulation
phil peroxidase (28). Eosinophils were identified by their dark-brown         with partly empty specific granules (Figure 2a). Twenty percent
reaction product and were quantified as numbers of eosinophils/0.1-mm2        of the 537 tissue eosinophils were cytolytic, as shown by incom-
tissue area. At least 150 eosinophils were present in each polyp tissue      plete chromatolysis, cell membrane disruption, and spilling of
section.                                                                     protein-containing free granules (Figures 2b and 2c). None of
                                                                             the tissue eosinophils exhibited morphologic features of apopto-
Detection of Macrophages
                                                                             sis, nor were any epithelial cells identified as apoptotic by the
Macrophages were identified by morphologic criteria and stained using         TEM analysis. Other cells such as plasma cells (Figure 3a) were
a mouse anti-human CD68 monoclonal antibody (28).
                                                                             clearly apoptotic. Eosinophils and neutrophils were occasionally
In Situ Detection of Apoptotic Cells with the TUNEL Technique                seen on paracellular epithelial paths (Figure 2d), and they ap-
                                                                             peared on the airway surface (Figure 2e).
Tissue segments were immersed overnight in buffered 4% paraformal-
dehyde and were dehydrated and embedded in paraffin. Apoptosis was            Macrophages
visualized using the in situ TUNEL technique (26). No staining was
evident in negative control subjects where the Tdt enzyme was omitted.       CD-68 positive cells commonly occurred (Table 1). Double staining
Slides were counterstained with propidium iodide to reveal pyknotic          for CD-68 and eosinophils (Chromotrop -2R and eosinophil peroxi-
nuclei as well as total number of cells. Apoptotic eosinophils were          dase [EPO]) did not reveal any engulfment of eosinophils. As
defined as dual chromotrope-2R and TUNEL-positive cells exhibiting            detected by TEM, macrophages exhibited signs of engulfment of
apoptotic morphology.                                                        cell material, but no specific eosinophil-like features such as specific
                                                                             granules were engulfed (Figure 3c). Similarly, epithelial cells exhib-
Immunocytochemical Staining of PARP,
                                                                             ited engulfed cell material of unidentified origin (Figure 3b).
p85 Fragment-positive Cells
Cryosections (5 m) were washed in phosphate-buffered saline (PBS)            Apoptotic Cells Detected by TUNEL Staining
and incubated with the anti-p85 PARP polyclonal antibody (dilution
1:200; Promega, Madison, WI) overnight at 4 C (29). Sections were rinsed
                                                                             As revealed by the TUNEL method, apoptotic cells were scat-
in PBS and incubated with a secondary antibody (biotinylated goat anti-      tered in the polyp tissue (Figures 1b and 1c and Table 1). No
rabbit; Vector BA; 1,000, dilution 1:200) (Vector Laboratories, Burlin-      difference in the number of apoptotic cells was detected between
game, CA) for 1 hour. After washing in PBS, sections were incubated          steroid-treated and nontreated polyps (p       0.05). Among all of
in alkaline phosphatase-conjugates streptavidin (dilution 1:200; Dako A/S,   the TUNEL-positive cells, only one was an eosinophil, as detected
Glostrup, Denmark) for 45 minutes, rinsed developed using New fuch-          by combined TUNEL and Chromotrope-2R staining (Figure 1b).
sin (Dako) as substrate, and counterstained with Mayer’s hematoxylin.        Thus, of more than 3,000 Chromotrope-2R–stained eosinophils
                                                                             in the present tissues, there was merely a single TUNEL-positive
Eosinophil Apoptosis In Vitro
                                                                             eosinophil. The morphologic features of this cell, originating
Eosinophils were isolated from heparinized blood from healthy individ-       from a patient who did not receive nasal steroids, were not con-
uals using Percoll gradient centrifugation and immunomagnetic deple-
                                                                             clusive as regards apoptosis.
tion of neutrophils using anti-CD16 antibodies (30). Eosinophils were
incubated in cell culture medium without growth factors for 24 hours         PARP, p85-positive Cells
in a humidified chamber at 37 C with 5% CO2. The eosinophils were
then centrifuged and resuspended in a small volume of transmission           Cells stained for the PARP p85 fragment showed a red cytoplasm
electron microscopy (TEM) fixative and kept for 1 hour at room temper-        combined with a light blue nucleus (hematoxylin background
ature. The suspension with fixed cells was centrifuged, and the obtained      staining) (Figures 1d and 1e). PARP-positive cells (Table 1)
pellet was embedded in warm (40–50 C) 3% agarose/PBS. The cell-              included fibroblasts, macrophages, and epithelial cells. PARP-
containing agarose gel was placed in 4% formaldehyde over night and          positive eosinophils (110 cells in total) also occurred (Table 1).
prepared for TEM analysis.
                                                                             Ultrastructural Features of Growth Factor–depleted
                                                                             In Vitro Eosinophils
Samples for TEM were prepared (26). Ultrastructural criteria for eosin-
ophil apoptosis included cell shrinkage, nuclear chromatin condensa-         Sixty percent of the isolated eosinophils showed signs of apopto-
tion, membrane blebbing, and intact cell membrane (31). Eosinophil           sis. At least three different stages of eosinophil apoptosis could
cytolysis was defined as the presence of chromatolysis and the loss           be discerned by the present TEM analysis (Figure 4). An early
744                                                  AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 170 2004

                                                                                      Figure 1. Representative bright-field micrograph show-
                                                                                      ing eosinophil peroxidase (EPO)-stained eosinophils
                                                                                      (brown cells) in the epithelium and the subepithelium
                                                                                      of nasal polyps; scale bar      150 m (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 in-
                                                                                      sert), scale bar     80 m (B ). TUNEL-positive cells are
                                                                                      distinguished (green cells; arrows) in this fluorescent mi-
                                                                                      crograph where cell nuclei have been counterstained
                                                                                      red with propidium iodide; scale bar 40 m (C ). Typi-
                                                                                      cal p85 poly(ADP-ribose) polymerase (PARP) staining of
                                                                                      cells (arrows) in the nasal polyp tissue; scale bar      80
                                                                                        m (D ). Detail of p85 PARP-positive cells (arrows) in the
                                                                                      epithelium; scale bar     40 m (E ).

stage was characterized by a condensed nucleus with a remaining         that turnover of human airway tissue eosinophils in vivo, irre-
clear distinction between the light euchromatin and dark het-           spective of steroid treatment, largely involves other mechanisms
erochromatin and unaffected nuclear and cellular membranes              than apoptosis. Alternative modes, as also compatible with ob-
(Figure 4b). A more advanced second stage was identified by              servations in this study, include noninjurious egression between
the presence of a fully condensed or dark round pyknotic nucleus        epithelial cells into the airway lumen and proinflammatory disin-
exhibiting a few nuclear membrane blebs (Figure 4c) but with            tegration through cytolysis. We further demonstrate here that
an intact cell membrane. A later third stage, defined as secondary       the eosinophil cytolysis phenomenon that occurs in vivo has
necrosis, was characterized by extensive nuclear membrane bleb-         features that are distinct from the secondary necrosis of apo-
bing, rupture of the cell membrane, and a lucent cytoplasm. The         ptotic eosinophils that commonly occurs in vitro in this study.
apoptotic cell contour remained, whereas the nucleus was finally         These data are of interest with regard to clearance of a major
disintegrated through extensive chromatolysis (Figures 4d and           leukocyte from airway tissues in vivo, and they may highlight
4e). Irrespective of viable state, the in vitro eosinophils exhibited   difficulties regarding translation of concepts from in vitro to
varying degrees of piecemeal degranulation (Figures 4a–4e).             in vivo.
                                                                            Authors employing the TUNEL technique or staining of
DISCUSSION                                                              phosphatidylserine (annexin), without demonstration of apo-
This study has demonstrated that apoptotic eosinophils, as iden-        ptotic morphology, have reported that human diseased airway
tified by TEM and a validated TUNEL technique, are exceed-               tissue, especially nasal polyps, contain numerous apoptotic cells,
ingly rare in human nasal polyp tissues in vivo. The present            including apoptotic eosinophils (19–24). The inconsistency be-
scarcity of apoptotic eosinophils, together with other findings in       tween this prior art and this study likely reflects method differ-
this and previous in vivo studies (19), suggests the possibility        ences. Thus, the annexin staining of histologic sections would
Uller, Andersson, Greiff, et al.: Occurrence of Eosinophil Apoptosis                                                                                  745

Cells/0.1 mm2                           Epithelial             Subepithelial

Eosinophils                             10         1.5         13.7 2.1
Macrophages                           1.35         0.4          6.3 0.8
TUNEL cells                           0.85         0.2          1.7 0.5
Apoptotic eosinophils                        0                     0.004*
Cytolytic eosinophils                        0.3                   1.9
Total PARP cells                       1.4         0.3          4.7 0.9
PARP eosinophils                       0.2         0.07         0.5 0.1
PARP cytolytic eosinophils                   0                 0.02 0.01
PARP eosinophils                       9.3         1.5         12.2 1.9
PARP cytolytic eosinophils             0.3         0.05         1.9 0.4

 Definition of abbreviations: PARP poly(ADP-ribose) polymerase; TUNEL   ter-
minal deoxy RNase nick end labeling.
 Data expressed as mean value SEM.

predictably produce false-positive results because any section-
induced cell damage would make the intracellular phosphatidylser-
ine 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 irrespec-
tive 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 abun-
dance 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 pres-
ent single TUNEL-positive eosinophil (of more than 3,000 exam-
ined 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                Figure 2. Transmission electron micrographs of polyp tissues demon-
polyp tissues (Figure 3c). Hence, we cannot support the view                   strating viable eosinophils with signs of piecemeal degranulation (a ),
that eosinophil apoptosis and engulfment regulates the level of                a cytolytic eosinophil exhibiting chromatolysis and an early stage of
eosinophilia in vivo in diseased airway tissue (5, 8, 9, 33).                  cell membrane rupture (b ), spilling of numerous free eosinophil granules
    Airway steroids, through inhibition of growth factors, including           and cell debris (c ), eosinophils and neutrophils lining up between colum-
                                                                               nar epithelial cells (d ), and an eosinophil on the epithelial surface (e ).
interleukin-5 both in vitro (34) and in vivo (35), should theoreti-
cally 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 apo-
ptotic tissue eosinophils were lacking even during resolution of               tissues (Figure 4). The vast majority of the present cytolytic
lung eosinophilia induced by highly effective, systemic steroid                eosinophils were PARP negative (Table 1). This finding together
doses (26). The antieosinophilic action of steroids in animal air-             with their ultrastructural features would clearly distinguish cyto-
ways was explained by a combination of drug-induced inhibition                 lytic from apoptotic eosinophils. We have previously demon-
of recruitment of new cells and permission of the elimination of               strated that eosinophil cytolysis occurs independent of prior
tissue eosinophils into the airway lumen (26). Thus, the intriguing            degranulation of the eosinophils (32). These data, in addition,
localization of eosinophils and neutrophils in rows between epi-               indicate that cytolysis of eosinophils is a significant event in
thelial cells in this study potentially reflects traffic of tissue granulo-      its own right that should not be confused with the necrosis of
cytes into the airway lumen.                                                   eosinophils that occurs in vitro secondary to apoptosis and that
    The present ultrastructural characterization of purified,                   may occur also in vivo in airway tissues under the exceptional
growth factor–depleted human blood eosinophils identified early                 conditions caused by anti-Fas treatment of mouse airways (36).
signs of apoptosis involving nuclear bleb formation that would                     The molecular mechanisms involved in apoptosis offer oppor-
not be detectable by common light microscopy, as well as the                   tunities to the development of apoptosis detection methods. In
final stages of apoptosis involving secondary necrosis. Further-                this study we have used an antibody directed against the 85-kD
more, we now demonstrate that secondary necrosis exhibited                     caspase-cleaved fragment (p85) of human poly (ADP-ribose)
structural features distinguishing it from the primary cytolysis               polymerase. Anti-PARP p85 fragment antibody is considered
of eosinophils that commonly occur in diseased human airway                    to be an early marker of apoptosis (29) because cleavage of
746                                                   AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 170 2004

                                           Figure 3. Transmission elec-
                                           tron micrographs of polyp
                                           tissues demonstrating an        Figure 4. Transmission electron micrographs of purified blood eosino-
                                           apoptotic plasma cell (a),      phils demonstrating a normal in vitro eosinophil (a ), an eosinophil at
                                           an epithelial cell with en-     an early stage of apoptosis with typical nuclear chromatin condensation
                                           gulfed cell material (b), and   (b ), an apoptotic eosinophil with pyknotic nucleus with extensive bleb-
                                           a macrophage containing         bing at the nuclear envelope (c ), apoptotic eosinophils undergoing
                                           engulfed cell material and      secondary necrosis with chromatolysis (d ), and an eosinophil in which
                                           apoptotic bodies (c).           the nucleus is completely lysed in a late phase of secondary necrosis (e ).

                                                                           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 eosino-
                                                                           phils, 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 mi-
                                                                           gration to contribute to the pool of apoptotic eosinophils that
                                                                           actually occur in airway luminal liquids in animals and humans
                                                                           (25, 26, 39, 40).
PARP occurs before DNA fragmentation. However, other cellu-                    In summary, we have demonstrated that major features of
lar repair proteins than PARP may operate, and it has not been             eosinophils in culture, including apoptosis and secondary necro-
determined in detail that PARP p85-positive cells always are or            sis, may not occur to significant extents in vivo in human eosino-
become apoptotic cells. We demonstrated PARP p85-positive                  philic airway tissues such as nasal polyps, yet other cells scattered
staining in both the epithelium and subepithelium in the present           in the tissue clearly are both apoptotic and unengulfed by macro-
nasal polyp tissue involving fibroblasts, epithelial cells, and eosin-      phages or other neighbor cells. We also demonstrate that the
ophils. The total number of PARP p85-positive cells was mark-              cytolysis of eosinophils, occurring in vivo, is of a primary nature
edly higher than the TUNEL-positive cells in this study. As                distinct from the secondary necrosis phenomenon. Additionally,
also observed in the present nasal polyps, previous work has               granulocytes occur between columnar epithelial cells and on
demonstrated PARP-positive epithelial cells in human airway                the mucosal surface. We conclude that local turnover of tissue
disease, including asthma (29). The polyp tissue as a model for            eosinophils in diseased airways occurs through other pathways
human chronic airway inflammation may, in fact, exhibit a range             than apoptosis. These pathways would include “silent” egression
of interesting similarities to asthmatic airway mucosa (37). The           into the lumen and “proinflammatory” disintegration through
occurrence of apoptotic epithelial cells, however, could not be            primary cytolysis.
Uller, Andersson, Greiff, et al.: Occurrence of Eosinophil Apoptosis                                                                                                   747

Conflict of Interest Statement : L.U. does not have a financial relationship with a              of Bcl-2, Fas, and Fas ligand in bronchial biopsies from asthmatics.
commercial entity that has an interest in the subject of this manuscript; M.A. does              Am J Respir Cell Mol Biol 1998;19:747–757.
not have a financial relationship with a commercial entity that has an interest in      21.   Davidsson A, Anderson T, Hellquist HB. Apoptosis and phagocytosis
the subject of this manuscript; L.G. does not have a financial relationship with a               of tissue-dwelling eosinophils in sinonasal polyps. Laryngoscope 2000;
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   22.   Vignola AM, Chanez P, Chiappara G, Siena L, Merendino A, Reina C,
a commercial entity that has an interest in the subject of this manuscript.                      Gagliardo R, Profita M, Bousquet J, Bonsignore G. Evaluation of
                                                                                                 apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal
Acknowledgment : The authors thank Dr. Eric Carlemalm, Electron Microscopy                       biopsy specimens of patients with asthma and chronic bronchitis.
Unit, Lund University, for expert assistance with processing of TEM figures and                  J Allergy Clin Immunol 1999;103:563–573.
Monika Malm-Erjefalt for technical assistance regarding the purification of blood
                  ¨                                                                     23.   Fan GK, Itoh T, Imanaka M, Fujieda S, Takenaka H. Eosinophilic apo-
eosinophils.                                                                                     ptosis in sinus mucosa: relationship to tissue eosinophilia and its resolu-
                                                                                                 tion in allergic sinusitis. J Allergy Clin Immunol 2000;106:551–558.
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