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Original article
Fundam Clin Pharmacol
1998
;
12
:
446-50
0
Elsevier, Paris
In vitro effects of Uriage spring water on the apoptosis
of
human eosinophils
F
Beauvais
l,
JL
Garcia-Mace
2,
F
Joly
I*
'
SEPhRA (Socidtd d'Etudes en Pharmacologie
:
Recherche -Applications),
41.
Avenue du Giniral Sarrail,
75016
Paris;
Laboratoires du Docteur E. Bouchara,
68,
Rue Marjolin.
92300
Levallois-Perret. France
(Received 24 December
1997;
revised
2
February
1998;
accepted 26 March
1998)
Summary
-The influx
of
eosinophils in tissues plays a central role in the pathophysiology of allergic diseases such as allergic rhinitis, allergic
asthma or atopic dermatitis. The death
of
eosinophils by apoptosis is an important factor for the resolution
of
hypereosinophilia. In the present
study, we have shown that Uriage spring water induced in vitro the apoptosis of IL-5-primed eosinophils. This effect was dose-dependent and
was statistically significant at Uriage water concentrations above
20%.
The induction of apoptosis was related to the Ca2+ content
of
Uriage
water. Indeed, Ca2+ at the same concentration as in Uriage water mimicked the apoptotic effect of the spring water. Furthermore, EGTA
reversed the apoptotic effect
of
Uriage water. These results suggest that topically applied, Uriage water could contribute to the resolution of
eosinophilic inflammation.
0
1998
Elsevier, Paris.
Uriage spring water
I
eosinophils
I
apoptosis
I
allergy
INTRODUCTION
The role of eosinophils in the pathophysiology of
allergic diseases (allergic rhinitis, asthma, allergic
conjonctivitis
or
atopic dermatitis) is now clearly
established
[6].
These cells are recruited from
peripheral blood and migrate into the tissues by
chemotaxis after adhesion to endothelial cells. In tis-
sues, they release mediators of inflammation, cyto-
kines and enzymes thus amplifying the inflammatory
reaction.
The homeostasis of blood and tissue eosinophilia
is
the result of an equilibrium between the production
of new mature eosinophils from hematopo'ietic stem
cells and the loss of eosinophils by apoptosis. Apop-
tosis is a physiological process of cell death (pro-
grammed cell death) which
-
by contrast with necro-
sis
-
does not involve the release of pro-inflammatory
enzymes and granule proteins. Indeed, apoptotic cells
are rapidly phagocytized, without further inflamma-
tion and without tissue sequelae. Recently, it has
been shown that apoptosis inhibition by local
IL-5
secretion was responsible for tissue hypereosinophilia
[71.
Any substance able to promote eosinophil apoptosis
is potentially beneficial in the treatment of diseases
with hypereosinophilia. Thus, glucocorticoids
are
able
in vitro to induce the apoptosis
of
eosinophils
[2].
Recently, it was shown that the beneficial effect of
inhaled glucocorticoids in allergic patients was corre-
lated to the increase of apoptotic eosinophils
[8].
Other
compounds, such as ketotifen
or
theophylline previ-
ously known for their anti-allergic properties, are also
able to induce the apoptosis of eosinophils in vitro,
thus suggesting that their therapeutical effects are
linked
-
at least in part
-
to eosinophil apoptosis
[3,5].
The water from the Uriage thermal station
is
iso-
tonic to plasma, rich in sulphide, calcium, chloride,
sodium ions and trace elements. Uriage water is pre-
scribed in inflammatory cutaneous diseases, in infec-
tious and inflammatory diseases in otorhinolaryngol-
ogy and rheumatology. Recently, it has been shown
that Uriage water inhibited the mast cell degranulation
induced by substance
P
[4].
Given the important role of eosinophil apoptosis in
the resolution of chronic allergic processes. we have
studied the in vitro effect of Uriage
viva1 of purified human eosinophils. water
on
the sur-
*Correspondence and reprints.
Uriage water and eosinophil survival
447
MATERIALS AND METHODS
Purification of human eosinophils
Venous blood from healthy subjects was sedimented
in
the
presence of dextran. The cell-rich supernatant was layered onto
a cushion of Ficoll (density
1.077).
The cell pellet containing
eosinophils and neutrophils was harvested after hypotonic lysis
of red cells. An immunomagnetic cell separation system
(MACS system; Miltenyi Biotec) was used to purify the eosin-
ophils by negative separation with anti-CD16 magnetic micro-
beads. After the passage through the column for magnetic sep-
aration, the purity
of
the cell suspension (above
95%)
was
assessed after specific staining
of
the eosinophils. Freshly puri-
fied
eosinophils were suspended
(
1.6
x
106
celldml) in RPMI
1640
(Gibco BRL) supplemented with
0.1
mM non-essential
amino acids,
100
U/mL penicillin,
100
pg/mL streptomycin,
10
mM HEPES,
2
mM L-glutamine and
10%
(v/v) FCS.
Treatment of eosinophils by the survival factor
(IL-5)
Aliquots
(1
20
pL) of the cell suspension were placed into
96-well flat-bottomed microtiter plates (Costar) containing
20
pL of recombinant human
IL-5
(Genzyme) at defined
concentrations
(0.1,
1
and
10
pM; final concentrations)
or
20
pL
of
diluting medium.
Treatment of eosinophils with Uriage water
Uriage water was purchased by ‘Laboratoires du Docteur E.
Bouchara’ (Levallois, France). Its Ca2+ concentration was
444
mg/mL. Stock solution of Uriage water was supplemented
with glucose
(1
mg/mL) and HEPES
(10
mM)
and then diluted
at defined concentrations
(25,
50,
75
and
100%)
in the RPMI
1640
culture medium. 80
pL
of Uriage water at
25,50,75
and
100%
(final concentrations:
10,
20,
30
and 40%)
or
control
medium were added to eosinophils. The plates were main-
tained at
37
“C
in
a
5%
CO, atmosphere for 48
h.
In
some
experiments, a saline physiological solution (Hanks’ solution:
NaCl8 g, KCI 0.4 g, glucose
1
g/L,
KH,P04
60
mg,
N%HP04
47.5
mg, HEPES
10
mM)
and a Ca*+-rich saline physiological
solution (Hanks’ solution suplemented with Ca2+
10
mM)
were tested
in
the same conditions as Uriage water.
Treatment
of
eosinophils by dexamethasone
The effect of dexamethasone
(1
pM, final concentration) as
a positive control was studied
in
the same conditions as Uri-
age water.
Measure
of
eosinophil viability
The viability of the eosinophils was assessed by trypan blue
exclusion. The percentages of viable cells were calculated
Uriage water
0
0%
I
20%
E
m
60
I
30%
E.
80
10%
.-
0
-
.-
-
loo
I
0
0
0.1
0
A
AT
*
1
10
IL-5
(pM)
B
0.1
1
10
IL-5
(pM)
Fig
1.
Effect of Uriage water on the survival and the apoptosis
of
human eosinophils in culture. Purified human eosinophils survived
in
culture in the presence of
IL-5
at defined concentrations. Uriage water
(supplemented with HEPES
10
mh4;
pH
7.4)
was added at defined
concentrations. After
48
h, the percentages of
survival
(A) and of
apoptosis
(B)
were measured. The results are expressed
as
mean
f
S.E.M.
(n
=
4
independent experiments).
*P
<
0.05;
**
P
<
0.01.
over the number
of
viable cells present at the beginning of
the culture (i.e. around
100%
of viable cells).
Measure of eosinophil apoptosis
Cell suspensions were cytocentrifuged and the slides were
stained after fixation in methanol
(kit
RAL
555,
Prolabo,
France). The percentages of eosinophils exhibiting morpho-
logic features of apoptosis was assessed as previously
described
[I].
Apoptotic eosinophils exhibited characteristic
morphology: pycnotic nucleus with condensed chromatin
or
absence
of
nucleus, decrease of cell diameter, cytoplasmic
condensation). At least
200
cells were examined on each slide.
448
F
Beauvais et a1
100
80
c
I0
c
C
-
.-
8
60
8
40
.*
-
.-
p
20
0
A
'e
0
'.
\
I
I
I
I
I
0
10
20 30 40
100
-
80
s
5
60
8
._
40
-
u)
-
._
P
c
a
ll
20
0
B
0
10
20
30
40
Uriage
water
(
0
)
or saline solution
(
o
)
(%)
Fig
2.
Comparison of Uriage water and isotonic physiological solution on eosinophil survival and apoptosis. Human eosinophils were incuba-
ted
in
the presence of
IL-5
(1
pM). Uriage water (closed circles)
or
isotonic physiological solution (open circles) at defined concentrations
were added. After
48
h, the percentages of survival
(A)
and of apoptosis (B) were measured. One experiment representative of
two
indepen-
dent experiments.
In situ
DNA
fragmentation
(TUNEL
method)
The presence of fragmented DNA was evidenced
in
situ
using
the TUNEL method. This method was used according to the
manufacturer (Apoptosis detection system, fluorescein;
Pro-
mega). Briefly, the eosinophils of each well were cytocentri-
fuged
and
fixed into a formaldehyde solution at
4%
(25
min;
4
"C).
After washing, the preparations were permeabilized
in
a 2% Triton
X-100
solution
(5
min;
20
"C).
The cells were
then incubated
(I
h;
37
"C)
in
the presence of
a
solution con-
taining fluorescein- 12-dUTP and terminal deoxynucleotidyl
transferase. The samples were examined under
a
fluorescence
microscope using
a
fluorescein filter set.
Statistical analysis
The results were analysed
using
the
ANOVA
test. The
means
were
then compared
using
the Student's ?-test.
For
multiple comparisons, Dunnet's method was used. A
P
value
under
5%
was considered
to
be
statistically significant.
RESULTS
In the absence of survival factor
(IL-5),
eosinophils in
culture spontaneously underwent apoptosis. After
48
h, the percentage of viable eosinophils decreased to
8
*
3%
(n
=
4;
mean
f
1
S.E.M.;figure
I).
This
decrease of viability was correlated with the appari-
tion
of
eosinophils with the typical morphology
of
apoptotic eosinophils
(84
f
6%).
In the presence of
IL-5
(from
0.1
to
10
pM), the viability of eosinophils
increased and the percentages of apoptotic eosinophils
decreased in a dose-dependent manner. Thus,
IL-5
inhibited spontaneous apoptosis of eosinophils and
prolonged their survival in culture.
Uriage water at defined concentrations
(
10
to 40%
in
the culture medium) was tested in this model. A dose-
dependent decrease of eosinophil survival, with an
increase of apoptotic eosinophils was observed in the
presence
of
Uriage water figure
I).
This
effect
was
maximal for the intermediate
IL-5
concentration
(1 pM). The percentage
of
apoptotic eosinophils
increased from
27
f
8% (in the absence of Uriage
water)
to
60
f
8% (Uriage water 40%;
n
=
4;
P
c
0.01).
At the higher
IL-5
concentration
(10
pM), Uriage water
still induced eosinophil apoptosis, from
6
f
3% to
25
f
6%
(P
<
0.01). Dexamethasone used as positive control
increased eosinophil apoptosis from
27
f
12%
to
74
*
23%
(n
=
3;
P
c
0.05),
in the presence of
IL-5,
1
pM.
We checked that the effect observed was indeed due
to Uriage water itself and not simply to deprivation of
factors from the medium culture necessary for cell
survival. We thus compared the effect
of
Uriage
water, with a saline physiological solution used
at
40%
concentration on eosinophils primed with
IL-5
at
1
pM. Neither
loss
of viability, nor increase of apop-
totic eosinophils in the presence of the control saline
solution were observed. Uriage water, tested
in
par-
allel in the same experiments, induced, as expected,
the apoptosis of eosinophils
(figure
2).
Apoptosis of eosinophils induced by Uriage water
was typical. Some eosinophils exhibited pycnotic nuclei
(round, dense nuclei) due to chromatin condensation
and a decreased cell diameter
cfigure
3A
and
B).
How-
Uriage water and eosinophil survival
449
Fig
3.
Cytology and
in
situ DNA fragmentation of eosinophils incubated in the presence
of
Uriage water or isotonic physiological solution.
IL-5
(I
pM)-primed eosinophils were incubated in the presence
of
Uriage water (40%)
or
isotonic physiological solution at the same concen-
tration for
48
h. In this experiment,
44%
of
the eosinophils were apoptotic in the presence
of
Uriage water
(B).
In the presence
of
the control
saline solution, only
7%
of
the eosinophils were apoptotic (A). The TUNEL method was used
to
evidence in situ DNA fragmentation. In the
presence
of
the control saline solution, the nuclei were not stained (C). In the presence
of
Uriage water, apoptotic nuclei
-
clearly evidenced by
their pynoctic aspect
-
were systematically stained (D).
ever,
in
the majority of apoptotic eosinophils, nuclear
structures were lost and eosinophils then appeared as
‘granule bags’ of small diameter. The DNA fragmenta-
tion was evidenced
in
situ (TUNEL Method).
In
the
presence of the control physiological solution, the nuclei
of the 1L-5
(1
pM)-primed eosinophils were not stained
figure
3C).
The dark non-stained bilobed nuclei of non-
apoptotic eosinophils were visible on the weakly bright
background.
In
the presence of Uriage water, the pyc-
notic nuclei were systematically brightly stained
(fig-
ure
30).
No staining was observed when the nuclear
structures had disapeared.
The possible role of Ca2+ contained
in
Uriage water
on the eosinophil apoptosis was studied
in
two series
of experiments. First, the effects of a physiological
saline solution containing the same Ca’+ concentration
(10
mM) as Uriage water was compared to the spring
water. The Ca2+-rich saline solution and Uriage water
induced the apoptosis of eosinophils primed with IL-5
(1
pM) and decreased their survival
in
a very close
manner
(figure
4).
Second, the apoptosis of IL-5-
primed eosinophils induced by Uriage water at
40%
was reversed by the Ca2+ chelator EGTA in a dose-
response manner. Conversely, the cell survival
increased in the presence of EGTA
(figure
5).
DISCUSSION
The results of this study indicate that Uriage water
decreases the
in
vitro apoptosis of 11-5-primed eosino-
F Beauvais et al
u
0
10
20
30
40
60
z
a
e
'$
40
m
30
-
50
0
.-
c
20
8
I
IIIII
0
10
20
30
40
Uriagespringwater
(
0
)
or
Ca2'-rich saline
solution
(
)
(96)
Fig
4.
Comparison of Uriage water and Caz+-rich physiological
solution on eosinophil survival and apoptosis. Purified eosinophils
were incubated in the presence of IL-5
(1
pM). Uriage water
(circles) or an isotonic physiological solution containing
10
mM
Ca*+ (squares) at defined concentrations were added. After
48
h, the
percentages of cell survival (A) and eosinophil apoptosis (B) were
measured
(n
=
3
independent experiments).
0
8o
1
A
8o
1
IL.5
0
1
2
3
alone
IL-5
+
Uriage
water
4oK
+
EGTA
(mM)
IL.~
o
I
z
3
alone
IL-5
f
Urmp
watef
4W
+
EGTA
(mM)
Fig
5.
Effect of Ca2+ chelation on the induction of apoptosis and the
survival decrease induced by Uriage water. Eosinophils primed by
IL-5
(1
pM) were incubated in the presence of Uriage water
(40%).
EGTA at defined concentrations was added at the beginning of the
incubation. After
48
h, the percentages of cell survival (A) and eosi-
nophil apoptosis (B) were measured
(n
=
3
independent experiments).
phils through an apoptotic process. This effect was
statistically significant at concentrations above
20%
of Uriage water in the culture medium. The induction
of apoptosis was not simply due to deprivation of the
culture medium since a physiological saline solution
in the same experimental conditions did not influence
the survival and the apoptosis of eosinophils.
Ca2+ plays an important role in the apoptosis pro-
cess. Moreover, Uriage water has a very high Ca2+
content
(-
10
mM) and it has been shown that the
inhibitory effect of Uriage water on mast cell degran-
ulation was due to Ca2+
[4].
Indeed, the present results
show that Uriage water and a Ca2+-rich (10 mM)
physiological solution behaved similarly with very
close dose-responses on eosinophil apoptosis and
survival. Finally, the chelation of Ca2+ by EGTA
reversed the apoptotic effect of Uriage water, thus
strongly suggesting that Ca2+ was indeed responsible
for the effects observed in this in vitro model.
In conclusion, these results show that Uriage water,
even at low concentrations
(20%),
reduces the survi-
val
of
eosinophils by inducing their apoptosis. This
effect is most probably related to the high Ca2+ con-
tent of the Uriage spring water. Thus, this study sug-
gests that topical application
of
Uriage water could
contribute to reduce the tissue hypereosinophilia
of
allergic diseases.
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