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: firstname.lastname@example.org
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: www.atsjournals.org 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.
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
TABLE 1. EPITHELIAL AND SUBEPITHELIAL CELLS/0.1 mm2
IN HUMAN NASAL POLYP
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
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