Content uploaded by Francis Beauvais
Author content
All content in this area was uploaded by Francis Beauvais on Sep 07, 2015
Content may be subject to copyright.
Journal ofLeukocyte Biology Volume 57,June 1995 851
Human eosinophils in culture undergo a striking and rapid
shrinkage during apoptosis. Role of K channels
Francis Beauvais, Laurence Michel, and Louis Dubertret
INSERM U312, Hopital Saint-Lou Paris, France
Abstract: In the absence of appropriate stimulus, eosi-
nophils in vitro rapidly exhibit the features of apoptotic
cells (nuclear pycnosis, cell shrinkage, DNA fragmenta-
tion). By using electronic cell sizing, we precisely meas-
ured the volume distribution of human eosinophils
during apoptosis. We observed that apoptosis of eosino-
phils was accompanied by a marked cell volume decrease
(approximately 60%). Moreover, analysis of the volume
distribution in different experimental conditions (kinet-
ics of apoptosis, inhibition of apoptosis by cytoidnes)
revealed that the cell shrinkage, once triggered, was a fast
process in which the intermediate states between normal
and shrunken volume had a short half4ife. As a model of
apoptosis, the eosinophil model allowed us to test the
hypothesis that apoptotic cell shrinkage was linked to
osmotic changes due to leakage of internal ions. Indeed,
in the presence of K channel blockers, the shrinkage was
inhibited in a dose-dependent manner. In conclusion,
our results suggest that eosinophil shrinkage during
apoptosis is a strildng and rapid phenomenon and os-
motic changes due to K efflux could be responsible, at
least in part, of the volume decrease.J. Leukoc. Biol. 57:
851-855; 1995.
Key Words: cell viability #{149}K channel blockers #{149}cell volume
regulation
INTRODUCTION
Blood eosinophils are end-stage cells that derive from
bone marrow. Their life in the Mood stream is thought to
be 6-12 h and then they migrate into tissues-preferen-
tially in submucosal sites-where they survive several days
by unknown mechanisms [1]. During allergic and inflam-
matory processes, eosinophils accumulate in tissues,
where they release mediators responsible for inflamma-
tion and tissue damages [2].
In vitro models for eosinophil survival have been devel-
oped and some factors have been demonstrated to in-
crease survival of eosinophils in culture. Indeed,
conditioned medium from endothelial cells [3], inter-
leukin-3 (IL-3) [4], IL-5 [5], granulocyte-macrophage col-
ony-stimulating factor (GM-CSF) [6], and coated
fibronectin [7] promote the survival of human eosino-
phils in vitro. Furthermore, eosinophils can produce IL-3,
IL-5, and GM-CSF, thus supporting their own survival [1].
It has been demonstrated that IL-3, IL-5, and GM-CSF
prolong eosinophil survival in vitro by inhibiting apop-
tosis, which, in cytokine-deprived eosinophils, occurs
spontaneously [8-10]. Apoptosis in eosinophils exhibits
typical morphological and biochemical modifications
such as nuclear and cytoplasmic condensations, decreased
cell size, and DNA fragmentation [10]. In this study we
precisely measured the volume decrease of eosinophils
and observed that the eosinophil was a useful model with
which to study apoptotic shrinkage, since volume de-
crease was particularly marked during eosinophil apop-
tosis. Indeed, little is known about the mechanisms
leading to decreased cell size due mostly to cytoplasmic
condensation and about its possible role in the apoptosis
process.
In this study, we observed that the cell shrinkage of
eosinophils during apoptosis, once triggered, was a rapid
phenomenon. Moreover, K channel blockers inhibited
the cell volume decrease, suggesting that K efflux played
an important role in apoptotic shrinkage.
MATERIALS AND METHODS
Percoll gradient preparation
Normodense human peripheral blood eosinophils from healthy donors
were isolated using Percoll gradients as described with only minor modi-
fications [1 1]. Briefly, solutions of Percoll (Pharmacia, Uppsala, Sweden)
with densities of 1.070, 1.080, 1.085, 1.090, 1.095, and 1.100 were
obtained by mixing light and heavy Percoll solutions at defined ratios.
The heavy solution was made by mixing 100 ml of Percoll (density
1.130) with 9 ml of lOx PIPES buffer and 2.4 ml of distilled water. The
pH was adjusted to 7.4 with HC1. The light Percoll solution was obtained
by mixing 10 ml of heavy Percoll solution with 90 ml of minimum
essential medium with Earle’s salts (Gibco BRL, Gaithersburg, MD). The
respective densities of the heavy and light Percoll solutions were calcu-
lated by precisely weighing 50 ml of the solution. The solutions with
defined densities were layered in 16-mI tubes: 1.5 ml for the 1.100
solutions and S ml for the 1.095, 1.090, 1.085, and 1.080 solutions.
Cell preparation
Venous blood was obtained from the French National Center for Blood
Transfusion. The granulocyte population was recovered by dextran sedi-
mentation and centrifugation through diatrizoate-Ficoll (d 1.077; Euro-
bio, Les Ulis, France). After washing, the residual erythrocytes were
eliminated by hypotonic lysis. The cell suspension was resuspended in
the 1.070 Percoll solution at a concentration of 50 x 106 cells/ml. Two
milliliters of this cell suspension was layered on Percoll gradients and
centrifuged at 600g for 20 mm. The cells were harvested from the
bottom through a pinhole and collected in 500-tl fractions. Cytocentri-
fuge smears of the different fractions were prepared, fixed in methanol,
and stained successively with eosin and methylene blue in order to
assess eosinophil percentage (Kit RAL 555, Prolabo, Paris, France). Only
the two first 500-id fractions that contained more than 95% eosinophils
were usually kept for experiments. The only contaminating cells were
neutrophils. The-ability of freshly isolated eosinophils to exdude trypan
Abbreviations: IL-3, interleukin-3; GM-CSF, granulocyte-macrophage
colony-stimulating factor.
Reprint requests: F. Beauvais, INSERM U312, Pavilion de Make, H#{244}pi-
tal Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France.
Received September 27, 1994; acceptedJanuary 1 1, 1995.
blue was 98%. The nucleus morphology of apoptotic and normal cells
was evidenced by acridine orange. For this, an aliquot of cell suspension
was added to an equal volume of acridine orange (Sigma Chemical Co.,
St. Louis, MO) at 10 tg/ml in phosphate-buffered saline and observed
by fluorescence microscopy.
Cell culture
Freshly isolated eosinophils (1.5-3.0 x iO cells/mi) were suspended in
RPM! 1640 (Gibco BRL) supplemented with 0.1 mM nonessential
amino acids, 100 U/mI penicillin, 100 ig/ml streptomycin, 10 mM
HEPES, 2 mM L-glutamine, and 10% (v/v) fetal calf serum. Aliquots of
180 Ml were placed into 96-well flat-bottomed microtiter plates (Costar,
Cambridge, MA) containing 20 uI of IL-3, IL-5, or GM-CSF (Genzyme,
Boston, MA), 4-aminopyridine, sparteine, or quinidine (Sigma) at de-
fined concentrations or diluting medium alone. The plates were main-
tamed at 37*C in a 5% CO2 atmosphere for 48 h. For long-term cultures
(up to 10 days), 2 ml of eosinophil suspension (3-5 x 105/ml) was
layered onto 35-mm culture dishes containing or not cytokines at de-
fined concentrations. For these long-term cultures the medium was
changed at 48-h intervals by centrifugation and resuspension of the cells
in fresh medium.
Electronic volume analysis
An aliquot of the cells was diluted in phosphate-buffered saline and the
volume distribution and the number of eosinophils were determined by
electronic analysis using a Coulter counter (model ZM) coupled to a
Channelyzer (Coulter Electronics, Hialeah, FL) [12]. The following set-
tings were used: gain 1, attenuation 2, aperture current 1000 j.tA, aper-
ture 140 tm, lower threshold 3.5, upper threshold 100. The instrument
was calibrated with latex microspheres (Coulter) to allow calculation of
absolute volumes.
DNA fragmentation
The DNA fragmentation ofeosinophils was evidenced as described [10].
Briefly, purified eosinophils (2 x iO cells) were washed in phosphate-
buffered saline at 4’C and resuspended in lysis buffer (100 mM NaCl,
10 mM Tris, 1 mM EDTA, 1% sodium N-lauroylsarcosine, pH 8) contain-
ing proteinase K (0.2 mg/mi) (Sigma). The suspensions were incubated
overnight at 37C and the DNA was extracted twice with phenol and
24 : 1 chloroform-isoamylalcohol (1 : 1 ,v/v). Sodium acetate was then
added (0.3 M final) and the DNA was precipitated in 80% final ethanol.
The DNA precipitates were recovered by centrifugation, air dried, and
resuspended in 20 p1 of Tris-EDTA buffer. Two microliters of loading
buffer was added to each sample. Electrophoresis was performed at 6
V/cm of gel in Tris-EDTA-acetate buffer. After electrophoresis, DNA
was visualized by soaking the gel in distilled water containing 1 pig/ml
ethidium bromide.
#{149}O.
B
C24h 48h
‘S
‘.
( ...
: :
:,
852 Journal of Leukocyte Biology Volume 57, June 1995
RESULTS
Eosinophils cultured in the absence of cytokine undergo
a striking volume decrease
Purified human eosinophils from peripheral blood were
cultured for 48 h in enriched medium without addition
of cytokine (Fig. 1A). At the end of the culture, the fea-
tures of apoptosis were observed in almost all the eosino-
phils. Indeed, in almost all the cells, after a step of nuclear
pycnosis mostly observed at 24 h (not shown), the nuclear
structures became indiscernible in the cytoplasm of the
eosinophils, which then appeared as “granule bags” (Fig.
1B). Moreover, the hallmark of apoptosis, the DNA frag-
mentation, was at the limit of detection at 24 h and was
clearly evidenced at 48 h (Fig. 1C). The viability of the
eosinophils was 80.7 ± 4.2% (n 6) at 24 h and 24.0 ± 2.2
(n 18) at 48 h.
Another feature of apoptosis, the decrease of the cell
size, was particularly demonstrative in aged eosinophils
on cytocentrifuge preparations (Fig. 1B). This strong de-
crease was precisely measured on eosinophil suspensions
by using electronic cell sizing. As seen in Figure 2, the cell
volume of freshly isolated human eosinophils was distrib-
uted according to a Gaussian shape with a mean of 408 ±
29 tm3 (n =13). This corresponds to a diameter of 9.2 ±
0.2 tim, if we assume that eosinophils are spherical (Fig.
2). During the 48-h culture, a clearly distinct cell popula-
tion progressively appeared with a decreased volume of
150 ± 15 j.tm3 (cell diameter 6.6 ± 0.2 tm), whereas the
number of cells in the larger population decreased in
respect. At 48 h, 71.3 ± 5.9% (mean ± sr; n13) of the
eosinophils present at the beginning of the culture were
of small volume. Thus, the measure of eosinophil volume
by electronic cell sizing confirmed the observations with
cytocentrifuge preparations: eosinophils underwent a
strong volume decrease (63.2 ± 3.6%) when cultured in
the absence of cytokine.
We studied whether a volume decrease comparable to
that of eosinophils was also observed in neutrophils, be-
cause these polymorphonuclear cells also spontaneously
Fig. 1. Apoptosis ofeosinophils in culture in the absence ofadded cytokine. Human eosinophils from peripheral blood were purified and suspended
in enriched medium (A). After culture for 48 h, without cytokine addition, the nuclear structures became undiscernible among the numerous granules
in almost all the eosinophils and the cell diameter was strongly reduced (B). DNA was extracted at 24 and 48 h from duplicate wells and electrophoresis
on agarose was performed. The DNA fragmentation with a ladder pattern was at the limit ofdetection at 24 h and was clearly evidenced at 48-h (C).
U)
a,
0
‘4-
0
a,
-E
z
0 200 400 600 800 0 200 400 600
Cell volume (,am3)
Fig. 3. Volume decrease of IL-5-treated eosinophils in 48-h culture.
Eosinophils were cultured in enriched medium containing control buffer
alone (1) or IL-5 at 10” M (2), iO’ M (3), 10” M (4), 10 M (5), 10” M
(6). The cell volumes were measured at the beginning of the culture and
after 48 h.
t=i,/{\\s,L
I __
0 200 400 600 800 0 200 400 600 800
3
Cell volume (m )
Fig. 4. Volume decrease ofIL-5-treated eosinophils in long-term culture.
Eosinophils were maintained in enriched medium containing or not IL-5
(10” M). Fresh medium, with or without IL-5, was replaced at 48-h
intervals after washing the cell suspension by centrifugation. Because the
purpose of this figure was to show the low number of eosinophils with
intermediate volumes, the results presented here are relative cell numbers
and we did not take into account the loss of eosinophils due to washing
process and/or to cell lysis.
Beauvais et a!. Volume decrease of apoptotic eosinophils 853
Cell volume (m3)
Fig. 2. In vitro volume decrease of eosinophils in the absence of added
cytokine. Purified eosinophils (or neutrophils; inset) were maintained in
enriched medium without addition of any cytokine. The cell volume
distribution was assessed at 0, 24, and 48 h using electronic cell sizing. In
this figure and the following, surfaces under the curves are not relative
values but are proportional to the numbers ofcells present in the culture.
undergo apoptosis in culture [13]. After an incubation
period of 24 h, which was previously shown to be optimal
for apoptosis, a decrease of the modal value (approxi-
mately 30%) of the volume distributions was observed
(Fig. 2, inset). However, in contrast with eosinophils, the
volume distributions of fresh and apoptotic cells widely
overlapped.
Effect of lL-5 on the volume decrease of eosinophil
during apoptosis
IL-S (10’ ‘-10’5M) added at the beginning of the culture
decreased, in a dose-response manner, the number of
eosinophils that became shrunken at 48 h (Fig. 3) and
inhibited the other features of apoptosis (nuclear
pycnosis, DNA fragmentation; data not shown). Notewor-
thy, IL-5 treatment resulted in a dose-dependent decrease
of the number of eosinophils that underwent apoptosis
but the bimodal aspect of the volume distribution was
maintained. This indicated that, once triggered, cell
shrinkage remained a fast event in IL-S-treated eosino-
phils. Similar results were obtained with two other cytok-
ines that also inhibit eosinophil apoptosis, GM-CSF and
IL-S (data not shown).
Long-term cultures of eosinophils were performed with
IL-S (10.1! M) and, although the IL-S-containing medium
was renewed at 48-h intervals, cell shrinkage was not in-
exorably blocked but only delayed (Fig. 4). Indeed, at day
7, approximately half of the cells present in the culture
were of small volume and at day 9 almost all the cells
present in the culture belonged to the small-volume popu-
lation. Here again, the bimodal aspect of volume distribu-
tions was preserved. Cytocentrifuge preparations at day 9
of these long-term cultured populations in the presence
of IL-S were comparable to those presented in Figure lB.
Effect of K channel blockers on eosinophil shrinkage
The marked volume decrease of eosinophils during apop-
tosis prompted us to use cytokine-deprived eosinophils as
Cell volume (m3)
0
fW .
854 Journal of Leukocyte Biology Volume 57,June 1995
a model of apoptotic shrinkage. Since K is the most
abundant intracellular ion, we tested the possibility that
loss of intracellular K was responsible for apoptotic
shrinkage.
Blockers of K channels were used to decrease the ef-
flux of K. In the presence of 4-aminopyridine (1-3 mM)
the shrinkage of eosinophils was inhibited in a dose-re-
sponse manner (Fig. 5A). At the higher concentration,
4-aminopyridine inhibited the shrinkage by approxi-
mately 60%. It should be noted that inhibition by 4-ami-
nopyridine did not preserve the bimodal aspect of volume
distributions and homogenous populations with interme-
diate volumes (between normal and shrunken) could be
observed. On cytocentrifuge preparations, apoptotic eos-
inophils treated by K channel blockers appeared as en-
larged “granule bags” as compared with nontreated
apoptotic eosinophils (Fig. 5B and C). The same results
were obtained with sparteine (3-10 mM) or quinidine
(10-100 jiM) (data not shown). In contrast, the K chan-
nel blockers were without effect on the apoptotic trans-
formation of the nucleus.
U,
a,
U
0
a,
.0
E
z
, .
-.
,.f
... /t: #{149}S
Fig. 5. Effect of K channel blockers on eosinophil volume decrease in
48-h culture. Eosinophils were cultured in enriched medium containing
control buffer alone or 4-aminopyridine at defined concentrations (1-3
mM). The cell volumes were measured at the beginning ofthe culture and
after 48 h (A). Eosinophils treated with 4-aminopyridine(3 mM) appeared
as enlarged granule bags (C) compared with nontreated apoptotic eosi-
nophils (B). The dashed line represents the population at the beginning
of the culture.
DISCUSSION
Among the features of apoptosis, DNA fragmentation
received greater attention than cytoplasmic condensation.
Yet cytoplasmic condensation is of considerable physi-
ological importance. Indeed, during necrosis the plasma
membrane loses its ability to maintain osmotic pressure
and the cell is swelling up to cell lysis. Consequently, the
content of the cell, proteins and enzymes, is released in
the surrounding tissues, inducing tissue lesions and in-
flammatory mechanisms. This is of particular importance
in the case of eosinophils, because the content of eosino-
phil granule can be highly cytotoxic if it is released [1].
On the contrary, in apoptosis, the cytoplasmic organelles
are tightly packed and the cell membrane integrity is
maintained. Moreover, it has been shown that apoptotic
eosinophils are phagocytized by macrophages [10]. Thus,
packing each eosinophil in a reduced volume could help
their ingestion by macrophages.
In the present study, by directly measuring the cell
volume, we observed prominent cell shrinkage (more
than 60% of the initial volume) undergone by eosinophils
during apoptosis. Moreover, only two cell populations
with volumes centered approximately on 150 and 400
jtm3 were observed. In the experiments described in Fig-
ures 2 and 3, it was clear that the eosinophil population
at the beginning of the culture was transformed into a
small-volume population apparently without changing
into populations centered on intermediate volume (be-
tween 150 and 400 .tm3). This is the hallmark of a fast
process. Indeed, in this case, the lifetime of the interme-
diate is brief and therefore only the values of the initial
and final states could be properly observed. Moreover,
partial inhibition of apoptosis by cytokines at suboptimal
concentrations also maintained both populations distinct.
This latter observation indicated that, at the individual
cell level, the cell shrinkage was blocked or was not
blocked. In other words, no cell was half-shrunken. Cytok-
ines only delay the onset of apoptosis but, once triggered,
cell shrinkage remains a fast process.
In neutrophils, we measured a 30% decrease between
modal values of fresh and apoptotic cell volumes and a
volume decrease of 30% was reported in thymocytes dur-
ing dexamethasone-induced apoptosis [14]. In these
cases, however, the volume distributions of the apoptotic
and normal populations largely overlapped. This does not
imply that at the individual cell level, the volume changes
C are not fast. Simply, due to large variances of volume
‘ distributions and small differences between the modal
values, studying the distribution of the volumes of the
populations did not make it possible to draw clear-cut
conclusions at the individual cell level. This strengthens
the interest in the eosinophil model for studying some
aspects of apoptosis, namely the cell shrinkage.
The mechanisms of cell shrinkage during apoptosis re-
main obscure. It is thought that this cytoplasmic conden-
sation is obtained through an isosmotic loss of water by a
mechanism that remains unknown [15]. As K is the most
abundant intracellular ion, leakage of internal K could
be a simple way to obtain a rapid loss of volume. Such a
mechanism has been suggested in hepatic cells, where the
catabolic effects of glucagon seem to involve a cell volume
decrease linked to K depletion probably through KF
channels [16]. In the present experiments, 4-aminopyrid-
me, sparteine, and quinidine, three classical K channels
blockers, down-modulated the eosinophil shrinkage dur-
Beauvais et aL Volume decrease of apoptotic eosinophils 855
ing apoptosis. However, in contrast with IL-5, which main-
tamed the bimodal aspect of volume distribution, in the
presence of K channel blockers, populations with inter-
mediate volumes between 150 and 400 J.tm3 could be
observed in a dose-response manner. Moreover, no in-
hibitory effect on the nuclear events was observed and
therefore apoptotic eosinophils treated by K channel
blockers appeared as enlarged granule bags. Taken to-
gether, this suggests that, in contrast with IL-5, which
delays the triggering of the apoptosis process itself, K
channel blockers sloied down only the shrinkage proc-
ess.
In conclusion, our results suggest that eosinophil
shrinkage during apoptosis is a fast and rapid phenome-
non in which osmotic changes due to K efflux could be
responsible, at least in part, for the volume decrease.
REFERENCES
1. Weller, P.F. (1991) The immunobiology of eosinophils. N. EngLJ.
Med 324, 1110-1118.
2. Resnick, M.B., Weller, P.F. (1993) Mechanisms ofeosinophil recruit-
ment. Am.J. Respir. CelL MoL Biol. 8, 349-355.
3. Rothenberg, M.E., Owen, W.F.,Jr., Silberstein, D.S., Soberman, R.J.,
Austen, K.F., Stevens, R.L. (1987) Eosinophils coculturedwith endo-
thelial cells have increased survival and functional properties. Science
237, 645-647.
4. Rothenberg, M.E., Owen, W.F., Jr., Silberstein, D.S., Woods, J.,
Soberman, R.J., Austen, K.F., Stevens, R.L. (1988) Human eosino-
phils have prolonged survival, enhanced functional properties, and
become hypodense when exposed to human interleukin 3.J. Clin.
Invest. 81, 1986-1992.
5. Rothenberg, M.E., Petersen, J., Stevens, R.L., Silberstein, D.S.,
McKenzie, D.T., Austen, K.F., Owen, W.F.,Jr. (1989) IL-5-depend-
ent conversion ofnormodense human eosinophils to the hypodense
phenotype uses 3T3 fibroblasts for enhanced viability, accelerated
hypodensity, and sustained antibody-dependent cytotoxicity.J. Im-
munoL 143, 2311-2316.
6. Owen, W.F., Jr., Rothenberg, M.E., Silberstein, D.S., Gasson, J.C.,
Stevens, R.L., Austen, K.F., Soberman, R.J. (1987) Regulation of
human eosinophil viability, density, and function by granulo-
cyte/macrophage colony-stimulating factor in the presence of 3T3
fibroblasts.J. Exp. Med. 166, 129-141.
7. Anwar, A.R.F., Moqbel, R., Walsh, G.M., Kay, A.B., Wardlaw, A.J.
(1993) Adhesion to fibronectin prolongs eosinophil survival.j Exp.
Med. 177, 839-843.
8. Yamaguchi, Y.Y., Suda, T., Ohta, S., Tominaga, K., Miura, Y.,
Kashahara, T. (1991) Analysis of the survival of mature human
eosinophils: interleukin-5 prevents apoptosis in mature human eos-
inophils. Blood 78, 2542-2547.
9. Her, E., Frazer,J., Austen, K.F., Owen, W.F.,Jr. (1991) Eosinophil
hematopoietins antagonize the programmed cell death of eosino-
phils. Cytokine andglucocorticoid effects on eosinophils maintained
by endothelial cell-conditioned medium. J. Clin. Invest. 88,
1982-1987.
10. Stern, M., Meagher, L, Savill, J, Haslett, C. (1992) Apoptosis of
human eosinophils. Programmed cell death in the eosinophil leads
to phagocytosis by macrophages and is modulated by IL-5.j Iminu-
noL 148,3643-3549.
11. Fukuda, T., Dunnette, S.L, Reed, CE., Ackerman, S.J., Peters, M.S.,
Gleich, G.J. (1985) Increased numbers ofhypodense eosinophils in
the blood of patients with bronchial asthma. Am. Rev. Respir. Dis.
132, 981-985.
12. Segel, G.B., Cockelet, G., Lichtman, M.A. (1981) The measurement
oflymphocyte volume: importance ofreference particle deformabil-
ity and counting solution tonicity. Blood 57,894-899.
13. Brach, M.A., deVos, S., Gruss, H.J., Herrmann, F. (1992) Prolonga-
tion ofsurvival ofhuman polymorphonudear neutrophils by granu-
locyte-macrophage colony-stimulating factor is caused by inhibition
of programmed cell death. Bkxxi 80, 2920-2924.
14. Thomas, N., Bell, P.A. (1981) Glucocorticoid-induced cell size
changes and nuclear fragility in rat thymocytes. MoL CelL EndocvinoL
22, 71-84.
15. Cohen,J.J. (1993) Apoptosis. ImmunoL Today 14, 126-130.
16. Hallbrucker, C., vom Dahl, S., Lang, F., Gerok, W., Haussinger, D.
(1991) Modification of liver cell volume by insulin and glucagon.
I’lugrrsArch. 418, 519-521.
