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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2827-2835
Resting and Cytokine-Stimulated Human Small Airway Epithelial Cells
Recognize and Engulf Apoptotic Eosinophils
By
Garry M. Walsh,
Darren W. Sexton,
Morgan G. Blaylock, and
Catherine M. Convery
From the Department of Medicine & Therapeutics, University of
Aberdeen Medical School, Aberdeen, UK.
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ABSTRACT |
Eosinophils, which are prominent cells in asthmatic inflammation,
undergo apoptosis and are recognized and engulfed by phagocytic macrophages in vitro. We have examined the ability of human small airway epithelial cells (SAEC) to recognize and ingest apoptotic human
eosinophils. Cultured SAEC ingested apoptotic eosinophils but not
freshly isolated eosinophils or opsonized erythrocytes. The ability of
SAEC to ingest apoptotic eosinophils was enhanced by interleukin-1
(IL-1) or tumor necrosis factor (TNF) in a time- and
concentration-dependent fashion. IL-1 was found to be more potent
than TNF and each was optimal at 10 10 mol/L, with a
significant (P < .05) effect observed at 1 hour postcytokine incubation that was maximal at 5 hours. IL-1
stimulation not only increased the number of SAEC engulfing apoptotic
eosinophils, but also enhanced their capacity for ingestion. The amino
sugars glucosamine, n-acetyl glucosamine, and galactosamine
significantly inhibited uptake of apoptotic eosinophils by both resting
and IL-1-stimulated SAEC, in contrast to the parent sugars glucose, galactose, mannose, and fucose. Incubation of apoptotic eosinophils with the tetrapeptide RGDS, but not RGES, significantly inhibited their
uptake by both resting and IL-1-stimulated SAEC, as did monoclonal
antibody against v 3 and CD36. Thus, SAEC recognize apoptotic
eosinophils via lectin- and integrin-dependent mechanisms. These data
demonstrate a novel function for human bronchial epithelial cells that
might represent an important mechanism in the resolution of
eosinophil-induced asthmatic inflammation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
EOSINOPHILS ARE NOW recognized as major
effector cells in the inflammatory process underlying much of the
pathogenesis of asthma and other allergic diseases because of their
ability to release potent mediators, including cytotoxic cationic
proteins, lipid mediators, cytokines, and oxidative
metabolites.1-3 Although many of the complex mechanisms
involved in their accumulation in the asthmatic lung have been
dissected,4,5 the factors responsible for their clearance
are less well understood. Apoptosis or programmed cell death (PCD) is
an ordered and fundamental biological process designed to dispose
safely of surplus, aged, or damaged cells.6,7 Apoptotic
cells are phagocytosed whole or as discrete fragments bound by an
intact membrane, thus ensuring their disposal without inducing
inflammation. Interest in eosinophil PCD has burgeoned,8
due in no small part to the fact that elucidation of the
mechanisms responsible for the induction and control of apoptosis might
enable the development of therapies designed to induce eosinophil PCD,
thereby allowing their rapid and safe removal by phagocytes, thus
preventing their accumulation and limiting their toxic potential.
Eosinophils exhibit the classical changes in morphology associated with
PCD, including cytoplasmic condensation and the internucleosomal cleavage of DNA by endogenous endonucleases. Apoptotic eosinophils are
recognized and ingested as intact cells by autologous macrophages, often before any manifest signs of the morphological changes associated with apoptosis.9 Signals that have been shown to induce
apoptosis in human eosinophils include withdrawal of
viability-enhancing cytokines,10 monoclonal antibody
(MoAb)-dependent ligation of Fas11,12 or
CD69,13 and treatment with glucocorticoids,14 interleukin-4 (IL-4),15 or transforming growth factor (TGF).16 Furthermore, the level of
intracellular protein tyrosine phosphorylation also appears to be an
important factor in determining whether an eosinophil will survive or
undergo PCD.17 Stern et al18 have demonstrated
that monocyte-derived human macrophages recognize and ingest apoptotic
eosinophils via integrin- and sugar/lectin-dependent mechanisms, a
process similar to that described for the neutrophil. Other cells that
are not professional phagocytes, such as fibroblasts,19 smooth muscle cells,20 or dendritic cells,21
have also been shown to have the ability to recognize and ingest
apoptotic cells.
The airway epithelium is primarily made up of ciliated, nonciliated,
and basal cells. Epithelial cell damage resulting in cilial dysfunction
and loss is a major feature of asthma pathogenesis and is thought to be
an important contributor to the development of airway
hyperresponsiveness. Eosinophil-derived mediators, particularly granule-associated basic proteins such as major basic protein (MBP),
have been heavily implicated in this process.22 The
accepted view in asthma pathology, therefore, is that the epithelial
cell is very much the victim of the eosinophil. Examination of induced sputum has provided evidence that the treatment of exacerbations of
asthma with steroids results in the resolution of eosinophilic inflammation by inducing apoptosis in lung eosinophils that were subsequently recognized and phagocytosed by alveolar
macrophages.23 The hypothesis addressed in this study
therefore is that other resident cells in the asthmatic lung also
recognize and phagocytose apoptotic eosinophils and that these might
include bronchial epithelial cells. Thus, we have examined the ability
of resting and cytokine-stimulated human small airway epithelial cells
to recognize and engulf apoptotic eosinophils and investigated which
recognition pathways are important in that process.
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MATERIALS AND METHODS |
Reagents and MoAbs.
Human recombinant human IL-1 (rhIL-1) and recombinant human tumor
necrosis factor (rhTNF) were purchased from R&D Systems (Oxon,
UK). The tetrapeptides RGDS and RGES, monosaccharides, OPD, and bovine
serum albumin (BSA) were supplied by Sigma Ltd (Poole, Dorset, UK). The
specific v3 MoAb 23C6,24 the CD36 MoAb
SMO,25 anticytokeratin peptide 19, anti-intracellular
adhesion molecule-1 (ICAM-1) and isotype-matched negative
controls were all purchased from Serotec (Oxon, UK). The anti-MBP MoAb
BMK-1326 was a kind gift from Prof Redwan Moqbel
(University of Alberta, Edmonton, Alberta, Canada). The polyclonal
antihuman erthrocyte membrane, anti-CD9-phyroerythrin (PE), anti-CD44,
and the anti-CD11b MoAb were from Dako Ltd (High Wycombe, Bucks, UK).
The latter MoAb has been previously shown to block CD11b-dependent
eosinophil adhesion to cultured human microvascular endothelial
cells.27
Small airway epithelial cells (SAEC) culture.
Small airway human bronchial epithelial cells were purchased as frozen
primary cultures from Clonetics Ltd (Walkersville, MD). They were
cultured in the supplier's SAEC basal medium supplemented with
gentamicin, amphotericin-B, bovine pituitary extract, hydrocortisone, human epithelial cell growth factor, epinephrine, transferrin, insulin,
retinoic acid, triiodothyronine, and bovine serum albumin. Although
human SAEC can be maintained in culture for 5 to 6 passages using the
supplier's serum-free medium,28 all experiments presented here were performed with SAEC that had been passaged a maximum of 3 times to ensure no loss of phenotype. These studies used SAEC from 5 different donors and their identity as epithelial cells was guaranteed
by the supplier. However, we further confirmed the epithelial identity
of SAEC that had undergone 2 or 3 passages by their cobblestone
morphology and uniform positive immunostaining with MoAb to the
epithelial marker cytokeratin peptide 19, CD9, CD44, and ICAM-1.
Furthermore, the supplemented basal epithelial medium used for SAEC
culture also inhibits fibroblast growth and fibroblasts do not express
CD9. Moreover, we observed no positive immunostaining with MoAb to von
Willebrand factor or smooth muscle actin (both from Novocastra
Laboratories Ltd, Newcastle-upon-Tyne, UK), ruling out any
contamination of our SAEC cultures by these cell types (data not
shown). Cultures were split at 80% to 90% confluence by trypsin
digestion and subcultured in the same SAEC medium.
Eosinophil isolation and apoptosis induction.
Blood (100 mL) was obtained from normal donors or individuals with a
history of mild allergic disease with an eosinophilia not greater than
0.5 × 106 eosinophils/mL who were not taking any
medication at the time of venesection and who gave informed consent.
Eosinophils were purified under sterile conditions using our
modification of the immunomagnetic-dependent method of Hansel et
al,29 which has been described in detail
elsewhere.30 Briefly, after removal of red blood cells
using dextran sedimentation, the leukocytes were centrifuged on Percoll
gradients (Pharmacia Ltd, Beds, UK) at 700g for 20 minutes at
4°C. Contaminating red blood cells in the granulocyte pellet were
removed by hypotonic shock with ice-cold, sterile, endotoxin-free,
distilled water. Granulocytes were incubated on ice with micromagnetic
beads coated with anti-CD16 for 40 minutes before passage through the
magnetic-activated separation column (Miltenyi Ltd, Bisley, UK). Using
this method, eosinophils with a purity of at least 99% were obtained
with greater than 98% viability as assessed by trypan blue exclusion.
No more than 3% of the freshly isolated eosinophil preparations
displayed any morphological evidence of apoptosis. Flow cytometric
analysis performed as previously described31 demonstrated
that freshly isolated eosinophils did not bind annexin V-fluorescien
isothiocyanate (FITC) and excluded propidium iodide.
Purified eosinophils were washed in RPMI 1640 supplemented with 10%
fetal calf serum, antibiotics, and L-glutamine and resuspended in the
same medium at a concentration of 1 × 106 cells/mL. Cells
were cultured in 1-mL aliquots for 48 hours in a humidified atmosphere
with 5% CO2 in flat-bottom 12-well plates (Life
Technologies, Paisley, UK) that had been previously coated with BSA (1 mg/mL). After aging, eosinophils were assessed for apoptosis by
morphological assessment and binding of annexin V-FITC.30 All apoptotic cells used in the interaction/uptake experiments were
greater than 70% apoptotic and more than 90% viable as judged by
trypan blue exclusion.
Interaction assay.
This was a modification of a previously established
assay.32 Before use, secondary passage SAEC were
trypsinized and seeded into 24-well plates (Life Technologies) at a
concentration of 5 × 105 cells/well and rested for 1 to 2 days before use. At this stage, cells were 80% to 90% confluent in
each well as a whole, but were 95% to 100% confluent in the center of
the well. After a change of medium, SAEC were stimulated with various
concentrations of IL-1 or TNF for 48, 24, 5, 2, or 1 hour.
Apoptotic eosinophils were washed and added to resting or stimulated
SAEC at a final concentration of 1 × 106/well and allowed
to interact for 60 minutes at 37°C, which was found to be the optimum
time for interaction and phagocytosis (Table
1), although we did observe evidence that
by 60 minutes some ingested eosinophils had started to undergo
digestion within the SAEC. Noningested eosinophils were removed by 3 vigorous washes of the SAEC monolayer with phosphate-buffered saline
(PBS) supplemented with 0.02 mol/L EDTA using a 1-mL Gilson pipette,
and the cells were fixed with 2% gluteraldehyde in PBS. Ingested
eosinophils were stained for peroxidase by incubation with
o-phenylenediamine dihydrochloride (OPD) dissolved in PBS with hydrogen
peroxide. Aged eosinophils could be clearly seen as brown cells within
the SAEC. SAEC alone treated with OPD showed no positive peroxidase staining. All experiments were performed in duplicate and at least 200 SAEC were counted using an inverted microscope, and the proportion that
had ingested 1 or more eosinophils was expressed as a percentage. In
some experiments, the number of ingested apoptotic eosinophils within
each resting or cytokine-stimulated SAEC were counted. To confirm that
aged eosinophils were indeed ingested by SAEC and not merely adherent
to them as a result of nonspecific stickiness, trypsin was added to
washed monolayers after the interaction assay, but before the cells
were fixed or stained. Cytospins were prepared from the epithelial cell
suspensions and the cells fixed as before in gluteraldehyde/PBS and
stained with OPD.
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Table 1.
Determination of Optimal Timepoint for Uptake of
Apoptotic Eosinophils by Unstimulated Monolayers of SAEC (n = 4,
±SEM)
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In addition, SAEC were also grown in chamber slides and the following
experiments were performed. SAEC that had ingested apoptotic eosinophils were permeabilized and stained with MoAb BMK-13, which is
specific for the eosinophil granule protein MBP.26 Ingested aged eosinophils were visualized by the addition of antimouse IgG-FITC
before viewing with a fluorescent microscope. Apoptotic eosinophils
were also stained with a CD9 MoAb conjugated to PE (CD9-PE) before
incubation with SAEC that had been stained with anti-CD44 MoAb and
antimouse IgG-FITC before the interaction assay. The 2 cell types were
then visualized with a fluorescent microscope. Interaction experiments
were also performed with human erythrocytes opsonised with an
antierythrocyte membrane polyclonal IgG antibody as previously
described.33
Inhibition assays.
For these experiments, resting or cytokine-stimulated SAEC were
preincubated for 30 minutes at 37°C with MoAb to v3, CD36, isotype-matched controls at a final concentration of 10 µg/mL. Both
untreated and treated SAEC were washed before use. We also assessed the
tetrapeptides RGDS or RGES (final concentration, 2 mmol/L) and
monosaccharides (final concentration, 25 mmol/L) for their effects on
the uptake of apoptotic eosinophils by resting or IL-1-stimulated
SAEC. Aged eosinophils were preincubated for 30 minutes at 37°C with
the tetrapeptides or sugars before their use in the interaction assay.
We did not observe any increase in the uptake of trypan blue by treated
aged eosinophils compared with untreated cells (data not shown). Aged
eosinophils were also treated with a known adhesion blocking MoAb to
CD11b to control for the possibility that adhesion via this receptor
was responsible for apoptotic eosinophil interaction with SAEC.
Measurement of expression of v3 and
CD36 by SAEC.
The expression of the integrins v3 and CD36 by resting and
IL-1-stimulated or TNF-stimulated SAEC were measured using a
specific enzyme-linked immunosorbent assay (ELISA) performed as
previously described.29 Briefly, SAEC were seeded into
96-well flat-bottomed plates and grown to 90% confluence before
stimulation with IL-1 or TNF (final concentration,
1010 mol/L for 5 or 20 hours). The resting or
cytokine-stimulated SAEC were washed, fixed in 2% gluteraldehyde, and
blocked with 0.5% BSA. Specific and control MoAb (10 µg/mL) were
added in triplicate and incubated overnight at 4°C. After several
washes in PBS, the SAEC were treated with rabbit antimouse IgG
conjugated to horseradish peroxidase (HRP) for 30 minutes at room
temperature (RT), washed in PBS, and treated with an
HRP-conjugated goat antirabbit IgG. After development with OPD, the
plate was read on an automated ELISA plate reader at an absorbance of
490 nm.
Statistical analysis.
All data are presented as the mean ± SEM and where n is given this
represents the number of experiments each performed in duplicate.
Statistical analysis was by the unpaired 2-tailed Student's t-test, where a P value of less than .05 was considered significant.
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RESULTS |
Resting and cytokine-stimulated SAEC ingest apoptotic eosinophils.
Figure 1A is a photomicrograph of a
representative experiment that clearly demonstrates uptake of apoptotic
eosinophils by unstimulated SAEC. Several SAEC can be seen to have
ingested apoptotic eosinophils visualized as dark staining
peroxidase-positive cells. Noningested or adherent aged eosinophils
were removed by vigorous washing with PBS/EDTA, as described above. We
confirmed that eosinophils had been ingested by addition of trypsin to
washed SAEC after their interaction with aged eosinophils. Cytospins of
the cell suspension were prepared, fixed, and stained with OPD. Figure 1B clearly shows apoptotic peroxidase-positive eosinophils inside SAEC;
note the absence of eosinophils on other areas of the slide. We further
confirmed our observation by permeabilizing SAEC that had ingested
apoptotic eosinophils and adding a MoAb specific for MBP. Stained
eosinophils were visualized by the addition of antimouse IgG-FITC
before viewing the cells with a fluorescent microscope. Figure 1C
clearly demonstrates the presence of bright green-staining apoptotic
eosinophils inside SAEC. Figure 1D shows an SAEC stained with CD44 MoAb
followed by antimouse IgG-FITC before the addition of apoptotic
eosinophils stained with CD9-PE before the interaction assay. In this
example, 2 apoptotic eosinophils can be seen. The PE-positive
eosinophil is interacting with the SAEC membrane but is not yet
completely engulfed and, therefore, shows red fluorescence. The other
is within a phagosome within the SAEC and is thus surrounded by a
CD44+ membrane and, therefore, exhibits green fluorescence.
We observed no evidence of either interaction with or the ingestion of
freshly prepared nonapoptotic eosinophils or of human erythrocytes
opsonized with IgG by either resting or cytokine-stimulated SAEC (data
not shown). Any adhesive interactions between the freshly prepared eosinophils and the resting or cytokine-stimulated SAEC would have been
disrupted by a combination of the 3 stringent washes together with the
use of 0.02 mol/L EDTA in the washing buffer. Finally, our preliminary
experiments determined that the optimal time-course for the engulfment
of apoptotic eosinophils by SAEC was 60 minutes (Table 1).
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| Fig 1.
Representative photomicrographs (×400) showing
apoptotic eosinophil engulfment by unstimulated (A) SAEC. Eosinophils
can be seen inside SAEC as peroxidase-positive dark staining cells.
Cytospins were also prepared from SAEC that had ingested aged
eosinophils and were removed from their wells by trypsin digestion.
Under these conditions (B), OPD-stained apoptotic eosinophils can be
seen inside the SAEC, with no eosinophils present on the portions of
the slide from which epithelial cells were absent. (C) Representative
fluorescent micrograph (×1,000) showing SAEC permeabilized before
ingested eosinophils were stained with the anti-MBP MoAb BMK-13,
followed by antimouse IgG-FITC. Bright green staining ingested
eosinophils can be clearly seen inside the SAEC. (D) Apoptotic
eosinophils were also stained with a CD9 MoAb conjugated to PE (CD9-PE)
and allowed to interact with SAEC that had been stained with anti-CD44
MoAb and antimouse IgG-FITC. Both cell types were immunostained before
the interaction assay (30 minutes) and visualized with a fluorescent
microscope. The epithelial cell displays CD44+ green
fluorescence. Two apoptotic eosinophils can be seen, both of which were
stained with CD9-PE before the interaction assay. The PE-positive
eosinophil (top) is interacting with the SAEC membrane but is not yet
completely engulfed and therefore shows red fluorescence. The other
(bottom) is within a phagosome inside the SAEC and is thus surrounded
by a CD44+ membrane and therefore exhibits green
fluorescence.
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Having made the initial observation that SAEC are indeed capable of
ingesting apoptotic eosinophils, the effect on this process of
stimulation of SAEC with the proinflammatory cytokine IL-1 was
examined. Stimulation with IL-1 resulted in a marked enhancement of
the ability of SAEC to ingest apoptotic eosinophils (Fig
2A). The proportion of SAEC that ingested
eosinophils was enhanced by IL-1 stimulation, as was the ability of
individual SAEC to ingest multiples of aged cells (Fig 2B).
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| Fig 2.
(A) Representative experiment demonstrating that IL-1
stimulation (1010 mol/L) increased the percentage of
SAEC that ingested apoptotic eosinophils; (B) together with their
capacity to ingest multiples of aged eosinophils. Each bar represents
the mean ± SEM of 4 separate experiments in which the number of
ingested aged eosinophils inside 200 resting or cytokine-stimulated
SAEC were counted.
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Dose-response and kinetics of stimulation of SAEC with
IL-1 or TNF.
We next assessed the effects on uptake of aged eosinophils by SAEC
after stimulation with a dose-range of IL-1 or TNF. Figure 3A shows the effect on the uptake of
apoptotic eosinophils by resting SAEC after prestimulation with
increasing concentrations of IL-1 or TNF. The maximal increase in
the number of SAEC ingesting apoptotic eosinophils was seen with
IL-1 at a concentration of 1010 mol/L, and this
represented an approximate doubling of the numbers of stimulated SAEC
capable of ingesting apoptotic eosinophils. Kinetic studies showed that
a significant (P < .05) increase in uptake of apoptotic
eosinophils by SAEC was observed at 1 hour post-IL-1 incubation, an
effect that essentially had plateaued at 5 hours (Fig 3B). TNF
stimulation of SAEC resulted in a more modest, but statistically
significant (P < .05), increase in the ability of SAEC to
ingest apoptotic eosinophils (Fig 3A). Again, the optimal cytokine
concentration was 1010 mol/L, with the effect plateauing
at 2 to 5 hours poststimulation (Fig 3B). In all subsequent interaction
experiments that used cytokine-stimulated SAEC, IL-1 was used at a
concentration of 1010 mol/L for 24 hours.
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| Fig 3.
(A) The effect of increasing concentrations of ( )
IL-1 and ( ) TNF on the uptake of aged human eosinophils by
SAEC. Each point represents the mean ± SEM of at least 6 experiments.
(B) A time course of the effect of stimulation of SAEC with IL-1 and
TNF (1010 mol/L final concentration in each case) on
the engulfment of apoptotic eosinophils. In each case, each point
represents the mean ± SEM of at least 4 experiments
(*P < .05, **P < .001,
***P < .0005).
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The receptors involved in apoptotic eosinophil recognition and
engulfment by SAEC.
We assessed the effect of amino sugars to confirm that phagocytosis of
aged eosinophils by resting and cytokine-stimulated SAEC was a specific
receptor-mediated process as described in other phagocytic cells,
including macrophages.18 Preincubation of apoptotic
eosinophils with the amino sugars glucosamine, n-acetyl glucosamine,
and galactosamine significantly inhibited their uptake by both resting
and IL-1-stimulated SAEC. In contrast, the parent sugars glucose,
galactose, mannose, and fucose had no measurable inhibitory effect (Fig
4). Incubation of apoptotic eosinophils with the tetrapeptide RGDS significantly inhibited their uptake by both
resting and IL-1-stimulated SAEC, whereas the control tetrapeptide
RGES had no measurable effect on ingestion of apoptotic eosinophils
(Fig 5). These data provided evidence that
ingestion of apoptotic eosinophils by resting or IL-1-stimulated
SAEC is dependent on an RGD-dependent signal and that a membrane
receptor molecule of the integrin family is involved. The effects of
MoAb against v3 and CD36 were therefore assessed, because these
have been shown to be important in macrophage recognition of apoptotic eosinophils18 or neutrophils.34,35 Both MoAbs,
but not an isotype-matched control or a known adhesion blocking MoAb
against CD11b, significantly inhibited ingestion of apoptotic
eosinophils by both resting and IL-1-stimulated SAEC (Fig
6). No additive effect was observed either
when both MoAbs were used in combination or when either v3 or
CD36 MoAb was used in combination with the amino sugars glucosamine,
n-acetyl glucosamine, and galactosamine by either resting or
IL-1-treated SAEC (data not shown).
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| Fig 4.
Effect of the pretreatment of apoptotic eosinophils with
sugar solutions on their uptake by () resting or ( )
IL-1-stimulated SAEC (1010 mol/L for 20 hours).
Washed aged eosinophils were resuspended in Hank's balanced salt
solution (HBSS) containing the sugars indicated at a final
concentration of 25 mmol/L before use in the interaction assay. Each
point represents the mean ± SEM of 4 experiments
(*P < .05, **P < .001).
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| Fig 5.
The effect of the tetrapeptides RGDS and RGES on the
recognition and engulfment of apoptotic eosinophils by resting or
IL-1-stimulated SAEC (1010 mol/L for 20 hours).
Washed apoptotic eosinophils were resuspended in HBSS containing the
tetrapeptides to give a final concentration of 2 mmol/L before use in
the interaction assay. Each point represents the mean ± SEM of 5 experiments (*P < .05, **P < .001).
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| Fig 6.
The effect of preincubation of resting or
IL-1-stimulated SAEC (1010 mol/L for 20 hours) with
isotype-matched () control or MoAb to the ( ) VNR, ( ) CD36, or
() CD11b before their interaction with washed apoptotic eosinophils.
All MoAbs were used at a final concentration of 50 µg/well. Each
point represents the mean ± SEM of 4 experiments
(*P < .05, **P < .001).
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Expression of v3 and CD36 by resting
or cytokine-stimulated SAEC.
To assess whether the observed increase in the capacity of
cytokine-stimulated SAEC to ingest aged eosinophils was due to increased expression of v3 or CD36, we performed specific ELISAs on resting and IL-1-stimulated or TNF-stimulated SAEC derived from 3 different donors. We observed modest but consistent expression of v3 by resting SAEC comparable to that of ICAM-1, whereas expression of CD36 was somewhat higher. Incubation of the SAEC with
either IL-1 or TNF for 5 hours did not result in a significant increase in the expression of v3 or CD36, although expression of
ICAM-1 was enhanced to a modest degree (Table
2). Near identical results were obtained
when the incubation time with either IL-1 or TNF was extended to
24 hours (data not shown).
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Table 2.
ELISA Demonstrating Expression of v3, CD36, or
ICAM-1 by Resting and IL-1-Stimulated or TNF-Stimulated SAEC
(1010 mol/L Final Concentration for 5 Hours)
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DISCUSSION |
In the present study, we demonstrate for the first time that human SAEC
recognize and ingest apoptotic eosinophils via specific recognition
mechanisms and that this process is upregulated by the proinflammatory
cytokine IL-1 and, to a more modest degree, TNF. We have used
several different approaches to confirm that apoptotic eosinophils were
engulfed by SAEC and that internalized eosinophils are within
phagosomes (Fig 1D). This might represent an additional pathway by
which intact apoptotic tissue eosinophils are safely removed from the
tissues surrounding the airways, thus preventing in situ leakage of
their ubiquitous, granule-derived, highly cytotoxic proteins. It can be
envisaged that failure to remove apoptotic eosinophils might result in
secondary necrosis and subsequent leakage of their potent
proinflammatory mediators, thereby making a major contribution to
asthma pathogenesis. Thus, induction of apoptosis in eosinophils and
their subsequent removal by macrophages and resident cells such as
epithelial cells represents a potentially important therapeutic
strategy in asthma treatment. Moreover, our observation that
stimulation of SAEC with IL-1 or TNF enhances their phagocytic
capacity for apoptotic eosinophils is interesting in the light of the
fact that these same cytokines are thought to promote eosinophil
accumulation through upregulation of endothelial adhesion receptors,
most notably vascular cell adhesion molecule-1 (VCAM-1),
which interacts with eosinophil VLA-4.36 However, because
inflammation is normally a beneficial and self-limiting response, it
would make sense that the cytokines involved in eosinophil accumulation
would also prepare resident cells to remove apoptotic eosinophils, a
process analogous to that described for clearance of apoptotic
neutrophils by cytokine-stimulated monocyte-derived
macrophages.37
The mechanisms by which apoptotic cells are recognized appears to vary
according to the cell type responsible for their engulfment. To date,
at least 4 recognition mechanisms for apoptotic cells have been
described: (1) an uncharacterized lectin-dependent
interaction38; (2) a complicated charge-sensitive process
involving the CD36/vitronectin receptor complex (v3) on the
macrophage surface interacting with unknown moieties on the apoptotic
neutrophil surface via a thrombospondin bridge34,35; (3) a
stereo-specific recognition of phosphatidylserine that is expressed on
the surface of the apoptotic cell after the loss of membrane
asymmetry39,40; and (4) macrophage scavenger
receptors.41,42 In the present study, we observed that, in
some respects, SAEC recognition of apoptotic eosinophils is similar to
that of the human monocyte-derived macrophage reported by Stern et
al.18 We found that the amino sugars glucosamine, n-acetyl
glucosamine, and galactosamine significantly inhibited uptake of aged
human eosinophils by both resting and IL-1-stimulated SAEC, as did
RGD-containing tetrapeptides. MoAbs to both v3 and CD36 inhibited
the phagocytic process. However, data from a sensitive ELISA indicated
that the increased capacity of SAEC to ingest aged eosinophils after
stimulation with either IL-1 or TNF did not appear to be a
consequence of increased expression of either v3 or CD36, both of
which had low but consistent constitutive expression by SAEC. These
data suggest that factors other than a simple increase in the
expression of v3 or CD36 are involved, such as a conformational
change in these receptors leading to increased affinity for their
ligand. Expression of v3 or CD36 has not been reported in studies
that have examined the expression of integrins by the bronchial
epithelium.43 The possibility that expression of v3
or CD36 by SAEC is an artefact of their culture in vitro cannot
therefore be excluded, although uniform positive immunostaining with
MoAb to cytokeratin peptide 19, CD9, CD44, and ICAM-1 confirmed the
epithelial identity of the SAEC. Moreover, bronchial airway epithelial
cell expression of integrins is complex and, in particular, appears
dependent on a number of diverse factors, including exposure to
pollutants such as ozone, malignancy, and their stage of development or
repair.44-46 Thus, other receptors, including v3 or
CD36, might also be expressed by the bronchial epithelium under certain
circumstances. Our data suggest that both resting and
cytokine-activated SAEC recognize apoptotic eosinophils via
VnR-dependent and sugar-lectin-dependent mechanisms. However, it
remains to be determined whether other receptors shown to be involved
in both professional and nonprofessional phagocyte recognition and
engulfment of apoptotic cells, including CD14,47,48
CD68,49 or the ABC1 transporter system,50 might also be involved in the recognition of apoptotic eosinophils by SAEC.
Macrophages are thought to be one of the most important and efficient
cells in the recognition and engulfment of apoptotic cells. These
professional phagocytes have the ability to ingest multiples of
apoptotic cells that, combined with rapid digestion of their apoptotic
meal, probably explains much of their efficiency.51 A
number of studies have demonstrated that several nonprofessional phagocytic cell types are also capable of recognizing and ingesting apoptotic cells.19-21 Moreover, a recent report has
confirmed that dendritic cells recognize and ingest apoptotic cells via
the scavenger receptor CD36 and also use a receptor not expressed by
macrophages, namely v5,52 whereas human liver Kupffer
cells phagocytose apoptotic lymphocytes via lectin-dependent
recognition of increased expression of n-acetylgalactosamine,
D-galactose, and mannose residues.53 Hall et
al19 have shown that human fibroblasts, including those
derived from the lung, recognize and ingest apoptotic neutrophils and
that this involves the participation of the vitronectin receptor and a
mannose-/fucose-specific lectin. However, although SAEC also use the
vitronectin receptor in recognition of apoptotic eosinophils, we did
not observe a role for the mannose-/fucose-specific lectin, because
neither sugar had any inhibitory effect on recognition or engulfment.
Moreover, these workers did not observe any uptake of apoptotic
neutrophils by human epidermal keratinocytes or mammary epithelial
cells. These and the observations presented here suggest that
epithelial cells at different organ sites vary in their ability to
recognize and engulf apoptotic cells. The reasons for such differences
are unclear. We also observed variation in the ability of SAEC to
ingest more than 1 apoptotic eosinophil. This was particularly marked
after IL-1 stimulation, with the majority of SAEC ingesting 2 or 3 aged eosinophils, whereas most unstimulated SAEC had only 1 or 2 ingested cells. The most likely explanation for this observation is the
nature of the conditions required for the culture of SAEC. All of the
experiments presented in this study were performed with SAEC that were
80% to 90% confluent. Culturing SAEC to 100% confluence is not
possible, because this results in contact-dependent cell morbidity and
loss. Thus, a minority of the SAEC in the monolayers used in these
studies were at different stages of cell division and/or maturity that
might explain in part the variability in their ability to ingest
apoptotic eosinophils.
The present study has focused on clearance of apoptotic eosinophils by
human small airway epithelial cells given the potential relevance of
this process to asthma pathology. We therefore did not examine the
ability of SAEC to ingest apoptotic neutrophils and neither did we
examine the engulfment ability of large airway epithelial cells.
Apoptosis is associated with the swift recognition of intact cells by
macrophages or resident cells followed by their engulfment and
degradation with the result that detection in vivo can be difficult.
This fact is the most likely explanation for the reason that, to date,
there have been no reports of the presence of apoptotic eosinophils
inside epithelial cells in, for example, bronchial biopsies from
patients with mild asthma.
Apoptotic cells are recognized and cleared by macrophages or
nonprofessional phagocytes by a specific recognition process. Crucially, not only does the rapid phagocytosis of apoptotic cells prevent local tissue injury or inflammation, but it also suppresses activation of the macrophages' usual proinflammatory secretory response.54 Similarly, engulfment of apoptotic eosinophils
exerts profound effects on the functional ability of the ingesting
macrophage, inducing an anti-inflammatory cytokine and mediator
secretory profile, ie, TGF and prostaglandin E2. In contrast,
ingestion of necrotic eosinophils induces a proinflammatory cytokine
and mediator profile, ie, release of thromboxane B2 and
granulocyte-macrophage colony-stimulating factor
(GM-CSF).18 A number of studies have demonstrated that
airway epithelial cells are also potent sources of proinflammatory
substances, including cytokines and chemokines.55 The
question as to whether ingestion of apoptotic eosinophils by SAEC
modulates their ability to release chemokines and/or cytokines will be
the subject of a future report.
In summary, we have demonstrated that human SAEC are capable of
recognizing and ingesting apoptotic eosinophils and that this process
can be enhanced by stimulation of SAEC with the proinflammatory cytokines IL-1 and, to a lesser extent, TNF. The membrane
receptors involved in SAEC recognition of apoptotic eosinophils appear
similar to those reported for human monocyte-derived macrophages. These observations demonstrate that, together with macrophages and other resident cells, human SAEC might be active participants in the removal
of apoptotic eosinophils and therefore play an important role in the
resolution of eosinophilic inflammation in the asthmatic lung.
| |
ACKNOWLEDGMENT |
The authors thank Prof Andy Rees for his valuable comments on the
manuscript and Sharon Gordon for providing some of the eosinophil preparations.
| |
FOOTNOTES |
Submitted October 15, 1998; accepted June 16, 1999.
Supported by the Wellcome Trust (044988/2/95/2).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Garry M. Walsh, PhD, Department of Medicine
& Therapeutics, IMS Building, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, UK; e-mail: g.m.walsh{at}abdn.ac.uk.
| |
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ABSTRACT
INTRODUCTION