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Increased apoptosis of mononuclear cells in atopic patients...
209
Increased apoptosis of mononuclear cells
in atopic patients – the effect of pollen season
and stimulation with specific antigen
Zwiększona apoptoza komórek jednojądrzastych u atopowych pacjentów –
wpływ sezonu pylenia i pobudzenia swoistym antygenem
ANNA PIŁAT 1, JANINA GRZEGORCZYK 2, MAREK L. KOWALSKI 3
Central University Clinical Hospital, Lodz, Poland
Department of Microbiology and Laboratory Medical Immunology, Medical University of Lodz,
Poland,
3
Department of Clinical Immunology and Allergy, Medical University of Lodz, Poland
1
2
Study supported by Medical University of Lodz research grant No 502-11-389 (22).
Streszczenie
Summary
Wprowadzenie. Zwiększona aktywność proliferacyjna komórek mononuklearnych (MN), stwierdzana we krwi obwodowej u chorych
z alergią atopową, może być związana ze zmienionym czasem przeżycia
komórek.
Introduction. Elevated proliferative activity of mononuclear cells (MNCs)
in peripheral blood of atopic patients may be associated with shorter cell
survival.
Cel pracy. Ocena apoptozy komórek jednojądrowych krwi obwodowej
od chorych z alergią atopową, przed i w czasie sezonu pylenia, w porównaniu do osób nieatopowych.
Materiały i metody. Komórki MN, wyizolowano z krwi 15 pacjentów
z sezonowym nieżytem nosa i astmą oskrzelową, przed i w czasie sezonu
pylenia oraz od 16 osób nieatopowych. Apoptozę oceniano w mikroskopie fluorescencyjnym, po wybarwieniu oranżem akrydyny i bromkiem
etydyny oraz potwierdzono poprzez analizę fragmentacji DNA. Badano
zarówno komórki adherentne, jak i nieadherentne.
Wyniki. Zarówno przed jak i w czasie sezonu pylenia, komórki MN od pacjentów z alergią, wykazywały istotnie nasiloną spontaniczną apoptozę
po 24, 48 i 72 godzinach hodowli. Komórki MN od chorych atopowych,
wykazywały również zwiększoną apoptozę po inkubacji z konkanawaliną
A. W czasie sezonu pylenia u osób uczulonych, apoptoza limfocytów znamiennie wzrastała (p<0,05). Inkubacja nieadherentnych komórek MN
z alergenem tymotki, nasilała apoptozę komórek przed sezonem pylenia
(średnio 33% w 48 godzinie) i istotnie hamowała apoptozę limfocytów
w czasie sezonu pylenia (średnio 27% w 48 godzinie).
Wnioski. Komórki MN od osób uczulonych na pyłki, wykazują nasiloną
apoptozę, a proces ten jest modulowany zarówno przez naturalną ekspozycję, jak i dodawany in vitro alergen.
Słowa kluczowe: apoptoza, alergia na pyłki, astma oskrzelowa, nieżyt nosa, komórki mononuklearne, zapalenie alergiczne
© Alergia Astma Immunologia 2012, 17 (4): 209-215
www.alergia-astma-immunologia.eu
Przyjęto do druku: 04.01.2013
Aim. Assessment of the apoptosis of peripheral blood mononuclear cells
(MNCs) from atopic patients before and during symptomatic pollen season as compared to non-atopic controls.
Materials and methods. MNCs were obtained from 15 pollen-sensitive
patients with seasonal rhinitis/asthma (SR/A) before and during the pollen season, and from 16 non-atopic controls. Apoptosis was assessed by
fluorescent microscopy after staining with acridine orange and ethidium
bromide, and confirmed by DNA fragmentation analysis. Both adherent
and non-adherent MNCs were analysed.
Results. Both before and during the pollen season, MNCs from pollensensitive patients, as compared to controls, demonstrated significantly
enhanced spontaneous apoptosis at 24h, 48h and 72h of culture. MNCs
isolated from atopic patients demonstrated also significantly enhanced
ConA induced apoptosis as compared to non-atopic patients. During the
pollen season in allergic patients, spontaneous apoptosis of MNCs and
lymphocytes was significantly higher as compared with preseasonal values (p<0.05). Incubation of non-adherent MNCs with timothy allergen
increased the cell apoptosis before the grass pollen season (on average
by 33% at 48h), but significantly inhibited apoptosis of lymphocytes studied during the pollen season (on average by 27% at 48 h).
Conclusion. Peripheral blood mononuclear cells from asymptomatic
pollen-sensitive patients exhibit increased apoptosis, and this process is
modulated by in vitro stimulation with specific allergen and by the exposure during natural pollen season.
Keywords: apoptosis, pollen allergy, asthma, rhinitis, mononuclear
cells, allergic inflammation
Adres do korespondencji / Address for correspondence
Marek L. Kowalski M.D.
Department of Immunology, Rheumatology and Allergy,
Faculty of Medicine, Medical University of Lodz
251 Pomorska St., 92-213 Lodz, Poland
Tel: +48 42 678 15 36, Fax: +48 42 678 22 92
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Alergia Astma Immunologia 2012, 17 (4): 209-215
INTRODUCTION
Mononuclear cells isolated from peripheral blood of
atopic patients (specifically T lymphocytes) are activated
and release cytokine profile, which is different from nonatopic, cells [1-5]. This profile of cytokines is corresponding
to TH2 type response with enhanced generation of IL-4, IL-5
and little production of interferon gamma. More recently
a central role for T regulatory cells (including Th17 and Th9
cells) for control and maintenance of allergic inflammation
has been implicated [6,7]. During allergen specific immunotherapy the profile of cytokines can be shifted from Th2 to
a Th1-dominated immune response in parallel with activation of regulatory T cell subsets and reduction of the
antigen-induced activation of peripheral blood lymphocytes (PBL) [8]. Peripheral blood leukocyte activation and
increased cytokine generation may parallel allergen induced upper and or lower airway symptoms, and is alleviated
during clinical improvement suggesting, that the activity of
PBLs may be related to the activity of allergic inflammation
in the airway mucosa [9-11].
Antigen stimulated lymphocytes, depending on the state of preactivation and the microenvironment may either
proliferate or undergo apoptosis (programmed cell death).
Mechanisms involved in lymphocyte death and elimination
have critical role in the immune response providing a homeostatic mechanism for controlling the magnitude of an
immune system activation [12-14]. Elimination of pathogenic, allergen-specific T(H)2 cells is an essential step in
induction of tolerance during natural exposure or following
allergen specific immunotherapy [15,16]. Although defective apoptosis of lymphocytes has been implicated in the
pathomechanisms of autoimmune diseases, little is known
on the apoptotic activity of PBL in atopic diseases. Increased
apoptosis of T lymphocytes was reported in patients with
bronchial asthma and, decreased serum levels of soluble
Fas (sFAS) was found in patients with allergic rhinitis suggesting, an impairment of apoptotic pathways in allergy [1719]. In our previous study an increased spontaneous and
mitogen-induced apoptosis of MNC from peripheral blood
of atopic patients was found and serum levels of sFas and
ICE/caspase-1 were correlated with apoptosis, suggesting
abnormal regulation of apoptotic process in peripheral blood mononuclear cells [20].
The goal of our study was to assess the apoptosis of
peripheral blood mononuclear cells from atopic patients
before and during symptomatic pollen season as compared to control non-atopic subjects. In addition the effect of
stimulation with specific, grass pollen antigen of apoptosis
of MNC was studied before and that during the pollen season.
PATIENTS AND METHODS
Patients
The study included 15 patients with seasonal rhinitis or
seasonal rhinitis and atopic bronchial asthma, diagnosed
for at last two years [8 male and 7 female; mean age 38
years (range 20-56)]. All patients had positive prick tests to
a battery of grass pollens including the timothy allergen.
None of the patients had taken oral corticosteroids. Antihistamine drugs were stopped at least 72 hours before obtaining their blood samples (Table I). Patients were studied
twice: in January-March, before grass pollen season and in
May-July during the pollen season. The control group comprised 16 healthy subjects without any history concerning
respiratory tract and with negative skin prick tests to a battery of inhalant allergen.
Cells isolation and culture
Mononuclear cells were isolated with Boyüm’s method
[14,21]. Shortly: 20 ml of heparinized venous blood were
mixed with PBS in 1:3 proportion, then cautiously stratified on Histopaque 1,077 g/cm3 (Sigma) and centrifuged
at 400x g for 20 min. The ring which was formed on the
borderline of the phases was carefully collected and rinsed
in phosphate buffer (pH=7,4 without Ca++ and Mg++). Finally suspended in the medium supplemented with 0,3%
albumin, Ca++ and Mg++ and 0,036% glucose so that the
Table I. Characteristic of patients
Patient No
F/M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
F
M
F
M
M
M
F
M
M
F
M
F
F
F
M
Atopic patients
Age in
years
24
35
43
26
56
25
21
20
22
46
27
38
26
33
28
Clinically diagnosis
Rhinitis
Asthma + Rhinitis
Rhinitis
Rhinitis
Rhinitis
Rhinitis
Asthma + Rhinitis
Asthma + Rhinitis
Rhinitis
Asthma + Rhinitis
Rhinitis
Asthma + Rhinitis
Rhinitis
Rhinitis
Asthma + Rhinitis
Healthy subjects
Patient No
F/M
Age in
years
1
F
32
2
F
28
3
F
24
4
F
33
5
F
37
6
F
42
7
F
28
8
F
32
9
F
34
10
F
29
11
M
43
12
F
49
13
M
30
14
M
23
15
M
24
16
F
26
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Increased apoptosis of mononuclear cells in atopic patients...
number of cells equals 2x106/ml. One aliquot of MNC suspension was cultured and the second fractionated into
lymphocytes and monocytes according to following procedure: five millilitre aliquots of mononuclear cells were
incubated in plastic dishes at 37o C in 5% C02 for 1h. Non adherent cells were removed by vigorous washing, and the
remaining adherent cells by scraping with a rubber policeman. The adherent cells were typically >65% monocytes, as
assessed by immunofluorescence method with monoclonal
antibody CD14 [15,22].
Unfractionated peripheral blood mononuclear cells, lymphocytes and monocytes from 15 asthmatic subjects and 7
non-atopic persons were cultured according to Theuson’s
method [16,23]. After the cells were preincubated for 4h
with Con A (10 µg/ml), timothy antigen (1, 10, 100 ng/ml)
or medium and then centrifuged at 400 x g for 20 min.,
washed twice in PBS without Ca++ and Mg++ , suspended
in the primary medium volume and cultured for 24h, 48h,
72h under the same conditions.
Assessment of apoptosis
The MNC were removed from culture after 24h, 48h and
72h to study apoptosis by morphology after fluorescent
staining, as described McGahon [17,24]. In brief: 25 µl
aliquots of MNC were mixed with PBS solution containing
100 µg/ml acridine orange base (Sigma) and 100 µg/ml
ethidium bromide (Sigma). Stained MNC were transferred
to a glass slide and examined by means of an epifluorescence microscope using the filter with the length of wave
490–525 nm. A minimum of 200 cells were assessed and
the number of apoptotic, viable and necrotic cells were recorded. The percentage of apoptotic cells (apoptotic index)
was calculated as follows:
total number of cells with
apoptotic nuclei
% Apoptotic cells = ---------------------------------------x 100
total number of cells
counted (viable + apoptotic
+ necrotic cells)
DNA Gel Electrophoresis
Fragmentation of DNA was assessed by method according to Hermann [18,25]. MNCs were removed from
culture after 72 hours, washed with PBS and pelleted by
centrifugation. The cell pellets were then treated for 10 s
with lysis buffer (1% NP-40 in 20 mM EDTA, 50 mM TrisHCL, pH 7,5; 10 µl per 106 cells). After centrifugation the
supernatants were transfered to 1% SDS and treated for 2
hours with RNAse A at 56o C. Followed by digestion with
proteinase K for at lest 2 hourse at 37oC. After addition of
1⁄2 vol. 10 M ammonium acetate the DNA was precipitated
with 2,5 vol. ethanol and separated by electrophoresis in
1% agarose gell containing ethidium bromide. The samples
were visualised by UV light and analysed by gel analysing
system (Vilber Lourmat).
Statistical analysis
Data are shown as mean±SD. Statistical differences
were assessed using the Student’s d-Test and t-Test, preceded by evaluation of normality with F-Test. P-Values<0,05
were considered significant.
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RESULTS
Spontaneous apoptosis
Unfractionated mononuclear cells (lymphocytes + monocytes) and isolated from MNC lymphocytes and monocytes, demonstrated apoptosis immediately after isolation
(To) and the percentage of apoptotic cells increased with
the time of incubation in both groups. MNCs isolated from
atopic patients before and during the pollen season demonstrated significantly enhanced apoptosis, as compared
to controls, at 24h, at 48h and at 72h in patients before
and during the pollen seasonal and controls respectively of
culture (Fig. 1a). Apoptotic index for non-adherent MNCs
(lymphocytes) was significantly higher in atopic patients
studied before the pollen season or during the pollen season as compared to non-allergic subjects after 24, 48 and
72hours of culture (p<0,05) (Fig. 1b). In atopic patients the
percentages of apoptotic unfractionated MNCs analysed
during the pollen season were significantly higher as compared with the cells isolated before the pollen season at
48h and 72h (Fig 1a, 1b).
Effect of specific allergen on apoptosis of MNC
Unfractionated MNC from allergic patients incubated
with timothy allergen before the season demonstrated
a significant increase in the apoptotic index. No influence of
allergen on apoptosis of MNC was observed during the pollen season (Fig. 2a, b). Incubation of non-adherent MNCs
(lymphocytes) with specific allergen increased the cell
apoptosis before the grass pollen season, but significantly
inhibited apoptosis of lymphocytes isolated during the
pollen season, at 48h (Fig. 3a, b). We did not observe any
effect of allergen on apoptosis of monocytes. Incubation of
mononuclear cells (unfractionated and fractionated) from
non-atopic subjects with timothy allergen did not affect
apoptotic index.
Stimulation in vitro by ConA
Incubation of cells with Con A (10 µg/ml ) resulted in
a significant increase in the proportion of apoptotic cells
in all cell population. Non-adherent MNCs (lymphocytes)
isolated from atopic patients as compared to non-atopic
subjects, demonstrated significantly enhanced of ConA
stimulated apoptosis at 24h and 72h (Fig. 4a, b). The percentages of apoptotic monocytes in allergic patients, both
before and during the season, were significantly higher as
compared to non-allergic subjects only at 72h. In allergic
patients the apoptotic index for MNCs during the pollen
season, was not different as compared with preseasonal
values.
DNA fragmentation
The presence of apoptosis of mononuclear cells was confirmed by DNA ladder. The fragmentation of DNA was observed after 72hrs of culture of MNC, only in 4 atopic patients
with percentage of apoptotic MNC above 44% as assessed
by fluorescence microscope. We did not observe any DNA
fragmentation in cells from atopic patients with apoptotic
index ≤40% or in non-atopic subjects (Table II).
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Alergia Astma Immunologia 2012, 17 (4): 209-215
1a
1b
Fig. 1. Spontaneous apoptosis of peripheral blood mononuclear cells (a) and lymphocytes (b) from healthy subjects (H) (n=16) and grasspollen-sensitive patients examined before (BS) and during the pollen season (S) (n=15). Results are expressed as the percentage of apoptotic
cells (mean+SD). The statistical differences was assessed using the Student’s t-Test and d-Test. P-value less 0,05 was considered significant
Fig. 1a. Unfractionated MNC (Lymphocytes + Monocytes)
Fig. 1b. Lymphocytes
2a
2b
Fig. 2. Effect of specific allergen on apoptosis of unfractionated mononuclear cells (lymphocytes+monocytes) from grass-pollen-sesitive patients (n=6) examined before (a) and during the pollen season (b). Results are expressed as a mean apoptotic index±SEM. The statistical differences between timothy allergen-treated and medium-treated cells was determined by Student’s d-Test; * p<0,05 as compared to control
Fig. 2a. Monocytes before the pollen season
Fig. 2b. Monocytes during the pollen season
3a
3b
Fig. 3. Effect of specific allergen on apoptosis of lymphocytes from grass-pollen-sesitive patients (n=6) examined before (a) and during the
pollen season (b). Results are expressed as a mean apoptotic index±SEM. The statistical differences between timothy allergen-treated and
medium-treated cells was determined by Student’s d-Test; * p<0,05 as compared to control
Fig. 3a. Lymphocytes before the pollen season
Fig. 2b. Lymphocytes during the pollen season
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Increased apoptosis of mononuclear cells in atopic patients...
4a
4b
Fig. 4. Comparison ConA stimulated apoptosis of lymphocytes from healthy subjects (H=7) and grass-pollen-sensitive patients (A=15) examined before (a) or during the pollen season (b). Results are expressed as the percentage of apoptotic cells (mean+SD). The statistical differences was determined by Student’s t-Test.; * p<0,05
Fig. 4a. Before the pollen season
Fig. 4b. During the pollen season
Table II. Assesment of apoptosis by morphology and electrophoresis in 1% agarose gel (fragmentation of DNA)
Patient No
Atopic patients (n=5)
Healthy subjects (n=7)
4
5
10
11
13
4
5
8
9
11
12
14
Morphological analysis of apoptosis
(apoptotic index - %)
46%
63%
46%
44%
40%
22%
32%
20%
18%
30%
27%
31%
DISCUSSION
Our study demonstrated, that isolated peripheral blood
mononuclear cells from both atopic patients and non-atopic subjects undergo apoptosis directly after isolation, and
the percentage of apoptotic cells increases with time of
culture. MNCs from atopic patients before and during the
pollen season demonstrated significantly enhanced spontaneous apoptosis as compared to non-allergic subjects.
Moreover, significantly higher percentage of apoptotic mononuclear cells was present in allergic patients during the
pollen season as compared with preseasonal values. Apoptosis was assessed with morphologic fluorescent method
and validated by DNA fragmentation analysis. Although the
fragmentation of DNA was observed only in those patients
with the highest percentage of apoptotic cells ( ≥44%,) these data are consisted with previous observation [26].
Incubation of non-adherent MNCs from grass pollen
sensitive patients with timothy allergen increased the cell
apoptosis before the grass pollen season, but significantly
inhibited apoptosis of cells during the pollen season. The
apoptosis of MNCs from non-atopic patients was not chan-
Fragmentation of DNA
+
+
+
+
_
_
_
_
_
_
_
_
ged after allergen challenge. Non specific stimulation by
mitogen (ConA) significantly increased of apoptotic process
in atopic patients and non-atopic subjects as compared to
spontaneous apoptosis both before and during the pollen
season.
This study suggests, that the lifespan of mononuclear
cells in peripheral blood is different in atopic and non
atopic subjects and that in patients with clinical allergy
lymphocyte apoptosis varies depending on patient’s clinical
status. In our earlier study an increased spontaneous and
ConA induced apoptosis of peripheral blood lymphocytes
from house-dust mite sensitive patients with perennial allergic rhinitis and/or bronchial asthma was demonstrated
[20]. Increased spontaneous and Dex-induced apoptosis of
lymphocytes in atopic patients with bronchial asthma was
reported previously by Ho et al. [17] although the authors
did not analysed an atopic status of their patients.
Apoptosis or programmed cell death is one of the mechanisms for controlling proliferation and survival of cells
during allergic inflammation [27,28]. The degree of apoptosis of immunologically active cells is determined by either
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Alergia Astma Immunologia 2012, 17 (4): 209-215
intrinsic (genetic) or micro environmental factors inducing
or suppressing cell proliferation and apoptosis. This process
is not only able to regulate immune response to allergen
but also cell number and activity at the inflammatory site
affecting allergic disease activity [29]. Increased apoptosis
of lymphocytes found in atopic patients could be associated with profile of released cytokines which in atopics
corresponds to Th2 type and includes among others IL-4,
IL-5 [30,31]. However, our asymptomatic pollen-sensitive
patients, at the time of the study were in stable condition
and were not taking any oral medication suggesting, that
increased apoptosis of lymphocytes was related to patients’
atopic status rather than disease activity. On the other hand
natural exposure to allergen during the pollen season significantly increased spontaneous apoptosis of lymphocytes, suggesting that local inflammation in the airways,
probably by released to systemic circulation cytokines, may
affect apoptotic activity of peripheral blood lymphocytes.
Increased apoptosis of lymphocytes in peripheral blood of
allergic patients reported in our study and higher apoptotic susceptibility of T lymphocytes of asthmatic patients to
DEX treatment reported by Ho et al. [17] are in contrast to
decreased apoptosis observed at the site of inflammation in
the bronchial mucosa of asthmatic patients [13]. In patients
with asthma, specifically during allergy season, a different
activity of apoptotic molecules belonging to Fas-FasL system
have been reported [17,32]. A negative correlation between serum level of Fas molecule and peripheral blood MNCs
apoptotic index was observed in house-dust mite sensitive
patients [20]. In earlier study a resistance to Fas-dependent
apoptosis was demonstrated and related to altered antigen-driven, accessory cell-dependent signalling and ineffective activation of Fas signal transduction in asthma, confirming the role for soluble serum proteins in controlling of
the lymphocytes apoptosis in asthma [18,33,34].
In conclusion, our study demonstrated, that peripheral
blood non-adherent mononuclear cells (lymphocytes) from
asymptomatic pollen sensitive patients exhibit increased
apoptosis, and the process is further augmented during the
symptomatic period of allergy. Stimulation in vitro of lymphocytes with timothy allergen increased the cell apoptosis
before the pollen season, but inhibited apoptosis during
the pollen season. These data suggested that apoptotic
activity of lymphocytes may play an important role in controlling allergic inflammation.
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