Download The use of multi-color flow cytometry for identification of

ORIGINAL ARTICLE
Łukasz Kraszula 1, Eusebio Makandjou-Ola 1, Maciej Kupczyk 2, Piotr Kuna2, Mirosława Pietruczuk1
1
Zakład Diagnostyki Laboratoryjnej, II Katedra Chorób Wewnętrznych Uniwersytetu Medycznego w Łodzi
Head: Prof. M. Pietruczuk, MD, PhD
2
Klinika Chorób Wewnętrznych, Astmy i Alergii, II Katedra Chorób Wewnętrznych Uniwersytetu Medycznego w Łodzi
Head: Prof. P. Kuna, MD, PhD
The use of multi-color flow cytometry for identification of
functional markers of nTregs in patients with severe asthma
Wykorzystanie wielokolorowej cytometrii przepływowej do identyfikacji
markerów czynnościowych limfocytów nTreg u chorych na ciężką astmę
oskrzelową
Financial disclosure: statutory activity 503/1-095-05/503-01.
Abstract
Introduction: At present, severe asthma is a particular clinical problem. An important role is attributed to dysfunction of
nTreg subpopulations of lymphocytes in the pathogenesis of asthma. Therefore, the purpose of this study was to identify
markers of nTreg cell function in patients with severe and mild to moderate asthma.
Material and methods: The study included sixty patients with asthma (30 with severe and 30 with mild to moderate
asthma). The control group comprised 30 healthy volunteers. The diagnosis of asthma was confirmed accordance with
generally accepted recommendations (GINA 2008). nTreg immunophenotype CD4/CD25/CD127/FoxP3/GITR/CD152/CCR5/
/CCR7 was evaluated by multicolor flow cytometry.
Results: We showed a significant reduction in the percentage of nTreg (76%) cells and the expression of CD152 (46.2%) in
patients with severe asthma compared with mild-moderate asthma (85.5% and 86.7%; p < 0.05). It was observed that the
transcription factor FoxP3 expression in nTreg cells positively correlated with FEV1 in patients with severe asthma (r = 0.53;
p < 0.05). It was also found that the ratio nTregCCR5+/TeffCCR5+ was significantly reduced in patients with severe asthma
(0.91) compared with mild-moderate (1.58) asthma and control groups (1.55; p < 0.001).
Conclusions: There are phenotypic differences in nTreg lymphocytes between patients with severe and mild-moderate
asthma. This fact may confirm nTreg cell dysfunction and indicate that the potential markers (FoxP3, CD152, CCR5), can be
used to monitor the effectiveness of treatment of bronchial asthma, especially severe disease.
Key words: asthma, nTreg, FoxP3, CD152, GITR, CCR5, CCR7
Pneumonol. Alergol. Pol. 2012; 80, 5: 389–401
Introduction
Bronchial asthma is characterised by chronic
inflammation of the respiratory tract. The classic
description of the disease’s pathomechanism involves increased cytokine production by Th2 lymphocytes, excessive production of allergen-specific IgE antibodies, and the presence of activated
mast cells and granulocytes [1]. However, asthma
is a heterogeneous disease, with varying clinical
courses and different pathomechanisms. Severe
asthma poses a particular clinical challenge, with
resistance to glycocorticosteroids occurring in
many patients [2]. New treatment options are therefore being investigated for severe disease, and
there are high hopes for pharmacological or immu-
A ddress for correspondence: Łukasz Kraszula, MD, Zakład Diagnostyki Laboratoryjnej, II Katedra Chorób Wewnętrznych, ul. Kopcińskiego 22, 90–153 Łódź,
tel.: + 48 (42) 677 69 81, faks: + 48 (42) 678 28 33, e-mail: [email protected]
Manuscript received on: 12.08.2011 r.
Copyright © 2012 Via Medica
ISSN 0867–7077
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Pneumonologia i Alergologia Polska 2012, vol. 80, no 5, pages 389–401
nological modification of regulatory CD4+ T-cell
function.
Regulatory CD4+ T-cells play a role in immunological homeostasis by inhibiting immune
response directed against an organism’s own antigens and excessive response to extrinsic factors,
including allergens. The subpopulation of CD4+
Treg lymphocytes is heterogeneous as to their
phenotype and function, and includes natural regulatory T-cells (nTregs), characterised by their
CD4+CD25highCD127lowFoxp3+ phenotype as
well as inducible regulatory T-cells (iTregs),
expressing CD4+CD25highFoxp3+, CD4+CD25IL-10+IL-4- (Tr1), or CD4+TGF-b+ (Th3) [3]. Regulatory T-cells may play a suppressor role by
directly or indirectly regulating the function of
effector cells taking part in inflammatory reactions. Prerequisite for Treg function is surface
expression of various molecules that characterise the phenotype of this cell population. These
are: cytotoxic T lymphocyte-associated antigen
4 (CTLA-4, CD152), glucocorticoid-induced TNF
receptor (GITR), and tumour growth factor-b
(TGF-b) as well as cytokines, including TGF-b
and IL-10 [3, 4].
Natural Treg cells may exert their suppressive function both in the lymphatic tissue and in
other sites where the immune response takes place [5]. Adequate distribution of Treg cells is necessary for an effective and normal immune response, whereas disturbed migration of these cells
may lead to decreased immune suppression in vivo.
Migration of Tregs to the site of inflammatory reaction is mediated by chemokines and their receptors. The nTreg subpopulation was initially characterized by expression of CCR4 and CCR8, but
later studies demonstrated the presence of other
chemokine receptors on the surface of these cells,
including CCR5 and CCR7 [6, 7]. Regulatory T-cells
devoid of CCR5 receptor have a weaker suppressive effect, whereas a lack of CCR7 receptor expression results in decreased Treg migration to the inflammation site, which results in an excessive
immune response [6, 7].
Unequivocal identification of nTreg lymphocytes is the major challenge in this field of study.
No single marker specific to the entire population
of these cells could be identified. Most markers
found on nTreg lymphocytes can also be found on
activated effector T-cells [8]. Multicolour (eightcolour) flow cytometry permits evaluation of nTreg
lymphocytes sorted from peripheral blood and at
the same time enables investigation of functional
markers and chemokine receptors present on these cells.
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Studies on the role of CD4+ regulatory T-cells
in pathogenesis of asthma give contradictory results; however, most researchers suggest that dysfunction of natural Treg cells may be of importance in this setting [9, 10]. There are no published
experimental studies reporting evaluation of eight
different markers on nTreg cells in patients with
asthma. The presented study was based on the
available data, and aimed to identify functional
markers of nTreg cells using multicolour flow cytometry in samples from patients with severe or
mild-to-moderate asthma.
Material and methods
The study group included 60 patients with
diagnosed bronchial asthma, including 30 persons
with severe and 30 subjects with mild or moderate disease. The control group consisted of 30 healthy volunteers. Tobacco smokers and patients with
unstable disease were excluded from the study. Demographical and clinical data was retrieved from
patients’ medical journals.
Diagnosis of bronchial asthma was made basing on anamnestic data and results of bronchial
reversibility tests according to the currently accepted recommendations (GINA 2008) [1]. Degree of
disease severity was assessed according to the definitions used in the ENFUMOSA study [11].
Patients with severe disease required constant
medication with high doses of inhaled corticosteroids (≥1,600 μg/day budesonide or beclomethasone, 800 μg/day fluticasone or equivalent). Patients requiring long-term peroral steroid therapy
received 800 μg/day budesonide or beclomethasone, alternatively 400 μg/day fluticasone or equivalent. In cases of severe disease, patients received
long-term therapy with inhaled long-acting betareceptor agonist (LABA), including formoterol 9 μg/
dose twice daily or salmeterol 50 μg/dose twice
daily, alternatively oral theophylline (200–300 mg,
1–2 × daily). Despite intensive anti-inflammatory
treatment, asthma was not optimally controlled in
this group of patients, and during the preceding
year each patient had at least one exacerbation.
Before definitive diagnosis of severe asthma was
made, other concomitant diseases had to be
excluded. Patient’s lack of compliance was also an
exclusion criterion, as these are the two main factors impeding optimal asthma control. Patients
diagnosed with mild-to-moderate disease received
maximally 800 μg budesonide or beclamethasone
daily, alternatively 500 μg/daily fluticasone or
equivalent. None of the included patients received any
immunosuppressive drugs other than corticosteroids.
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Łukasz Kraszula et al., The use of multi-color flow cytometry for identification of functional markers of nTregs...
Table 1. Characteristics of the study group
Severe
asthma
n
Age (years ± SD)
Sex (F:M)
Duration of the disease
(years ± SD)
Atopy (% ± SD)
Mild-to-moderate
asthma
30
30
49 ± 14.5
42 ± 13.5
16:14
15:15
16 ± 8.7
10 ± 6.3
82%
88%
FEV1 (l ± SD)
2.6 ± 0.5
3.5 ± 0.8
FEV1 (% ± SD)
55% ± 15.9
89% ± 16.1
cIgE (kUA/L ± SD)
272 ± 284
251 ± 92
ACT (score ± SD)
10.4 ± 2.9
22 ± 3.5
Spirometry, skin prick tests, and asthma control
test (ACT) were performed in every subject. Atopy
was defined as at least one positive result (> 3 mm)
in skin prick tests using an allergen panel [12].
Patient characteristics in respective groups are
presented in Table 1.
All patients were informed of the study goal
and methodology. Each person gave written consent for participation in the study. Design of the
study was approved by the local ethics committee
of the Medical University of Lodz (decision no.
RNN/17/09/KE).
Studies were performed in peripheral blood
samples with added potassium versenate (K2EDTA). Eight millilitres of venous blood were taken
from each subject and added to 12 ml Histopaque1077 medium (Sigma-Aldrich). Peripheral blood
mononuclear cells (PBMCs) were isolated by centrifugation at 2500/min for 30 minutes by density
gradient. Absolute number and percentage of mononuclear cells were obtained using the pentra DX
120 analyser in order to verify the amount of isolated viable cells. Cell purity of more than 80%
lymphocytes was a prerequisite for the sample to
be proceeded to sorting.
Sorting procedure
A single-step procedure was used for negative selection of CD4+ T-lymphocytes (Miltenyi Biotec, USA). Mononuclear cells were sorted out by
density gradient, then the mixture of various biotin-conjugated antibodies directed against cell surface antigens (CD8, CD14, CD16, CD19, CD36,
CD56, CD123, TCR g//d, CD235a (glycophorin A)
was added, and the sample was incubated at 4°C
for 10 minutes. Monoclonal antibodies against biotin, conjugated with magnetically charged microbeads, were then added and incubated for 15 mi-
nutes at 4°C. Cell suspension was then processed
through the LD column for magnetic-field separation. The CD4+ lymphocytes passed through the
column and were collected in a tube, whereas all
CD4-negative cells remained inside the column.
The purity of the sorted CD4+ cells was verified
using antibodies against CD4 (95% ± 2.7) according to the manufacturer’s recommendations (BD
Bioscience). A BD FACS CANTO II cytometer was
used for cell studies.
The CD4+CD25+ lymphocytes were sorted
out by positive separation using a two-step procedure (Miltenyi Biotec, USA). Magnetic microbeads
with antibodies against CD25 were added to the
previously isolated CD4+ lymphocytes and incubated for 15 minutes. Thereafter, the cell suspension was passed through the MS column, leaving
the CD4+CD25+ lymphocytes bound with microbeads inside; these were then washed out using
buffer solution and a piston. The CD4+CD25- lymphocytes passed through the column and were then
collected to a separate tube. The purity of the obtained cell population was then verified using antibodies against CD4 and CD25 (93.8% ± 2.45)
according to the manufacturer’s recommendations
(BD Bioscience). A BD FACS CANTO II cytometer
was used for cell studies.
Cell marking procedure
Monoclonal antibodies directed against cell
surface antigens CD4, CD25, CD127, GITR, CD152,
CCR5, and CCR7 were added to 100 μl sorted lymphocytes; concentrations of respective antibodies
were taken from the manufacturer’s recommendations (BD Bioscience). The cell suspension was
then mixed and incubated in darkness at room temperature (20–25°C) for 20 minutes. The cells were
then fixed for 10 minutes using Human FoxP3
Buffer A (BD Pharmingen), followed by cell membrane permeabilisation step using the C buffer (BD
Pharmingen), vigorous mixing, and incubation at
room temperature (20–25°C) for 30 minutes. Antibodies against FoxP3-antigen were then added to
the cell suspension, with concentration according
to the manufacturer’s recommendations (BD Bioscience). The well-mixed cell suspension was further incubated in darkness for 30 minutes at room
temperature (20–25°C). Between 10,000 and 15,000
CD4+ and CD4+CD25+ lymphocytes were collected for analysis.
BD Cytometer Setup and Tracking Beads (BD
Bioscience) were used for setting up the cytometer detector voltage. Compensation for eight respective colours was performed separately for each
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Pneumonologia i Alergologia Polska 2012, vol. 80, no 5, pages 389–401
Table 2. Monoclonal antibodies used for immunophenotyping of natural regulatory T cells
Antibody
Fluorochrome
Clone
Manufacturer
AmCyan
SK3
BD
Anti-human CD25
FITC
M-A251
BD Pharmingen
Anti-human CD127
PerCP-Cy5.5
A019D5
BioLegend
Anti-human FoxP3
V-450
236A/E7 BD
Horizon™
PE
621
BioLegend
Anti-human CD4
Anti-human GITR
Anti-human CD152
APC
BNI3
BD Pharmingen
Anti-human CCR5
APC Cy7
2D7/CCR5
BD Pharmingen
Anti-human CCR7
PE-Cy7
3D12 BD
Pharmingen
Figure 1. Gating strategy for natural regulatory T cells expressing CD4+CD25highCD127lowFoxP3+GITR+CD152+CCR5+CCR7+, sorted
from peripheral blood, by eight-colour flow cytometry. A. Gating for CD4+ CD25high cell subset. B. Gating for CD4+CD25highCD127low
cells. C. Gating for nTreg subset expressing FoxP3+. D. Gating for CD152+ nTreg subset. E. Gating for GITR+ cells. F. Gating for CCR5+
cells. G. Gating for nTreg subset with CCR7+
fluorochrome in the applied panel, using a sample marked with a single antibody (Tab. 2). Eight
channels of the cytometer were used at the same
time, which posed problems with reciprocal compensation for respective channels; coexpression
of GITR, CD152, CCR5, and CCR7 could not be
analysed and such an analysis was abandoned
after an initial trial. Control samples for the analysed antigens (fluorescence minus one, FMO)
were prepared, with the analysis gates set in the
plots so that all the cells in the control samples
were negative for respective markers. Analysis of
respective marker expression was performed
using FACS DIVA 6.2 software. The nTreg gate
was set on cells expressing CD4+CD25highCD127lowFoxp3+. The percentage of cells
expressing GITR, CD152, CCR5, and CCR7 as well
as their mean fluorescence intensity (mean fluore-
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scence channel, MFC) was then assessed in this cell
population (Fig. 1).
The obtained results were submitted to statistical analysis using STATISTICA 8.0 PL software. Quantitative variables were expressed as median values, upper and lower quartiles, and minimal
and maximal values. Independent groups were compared using U Mann-Whitney test. Spearman rank
coefficient was calculated for analysis of correlations.
Statistical significance was assigned for p < 0.05.
Results
The percentage of natural regulatory CD4+ Tcells, as well as expression of functional markers,
was reduced in patients with severe bronchial asthma as compared to subjects with mild-to-moderate disease and healthy controls.
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Łukasz Kraszula et al., The use of multi-color flow cytometry for identification of functional markers of nTregs...
A statistically significant reduction of
CD4+CD25highCD127lowFoxP3+ nTreg population was found in patients with severe disease
when compared to the mild or moderate disease
groups (p <0.05) and compared to control subjects
(p < 0.01) (Fig. 2A, Tab. 3). The percentage of
FoxP3+ nTreg lymphocytes showed a positive correlation to FEV1 expected values in patients with
severe asthma (r = 0.53; p < 0.05). No such correlation was found in patients with mild-to-moderate asthma. There was no difference in the amounts
of GITR-positive nTreg cells between the severe
and the mild-to-moderate asthma subgroups. A
statistically significant difference in expression of
this marker was observed when comparing severely ill patients to healthy controls (p < 0.05) (Fig. 2B,
Tab. 3). A significant difference was also found between the percentage of CD152-positive nTreg cells
in patients with severe asthma and subjects with
mild or moderate disease (p < 0.05). No such difference was found between the severely ill patient
group and healthy volunteers (Fig. 2C, Tab. 3).
Patients with severe asthma had significantly
lower mean fluorescence intensity (MFC) for GITR
receptor when compared to less gravely ill subjects
and healthy persons (Fig. 2D, Tab. 3). No differences were found between the groups regarding
CD152 MFC (Fig. 2D, Tab. 3).
Analysis of expression of CCR5 and CCR7 chemokine receptors on nTreg cells isolated from peripheral blood showed no significant differences between the respective patient groups (Fig. 3A, Tab.4).
No significant difference in fluorescence intensity (MFC) for CCR5 and CCR7 receptors on nTreg
cells was observed between patients with severe and
subjects with mild-to-moderate asthma (Fig. 3B,
Tab. 4). However, significantly lower MFC for CCR5
(p < 0.001) and for CCR7 receptors (p < 0.05) was
found on nTreg cells between the severe asthma
subgroup and the control group (Fig. 3B, Tab. 4).
Analysis of chemokine receptor analysis on
effector lymphocytes was also performed. Percentage of CD4+CD25-FoxP3- CCR5+ cells was significantly higher in patients with severe asthma as
compared to other analysed groups (p < 0.01) (Fig.
4A, Tab. 5). There was no such difference as to
percentages of CD4+CD25-FoxP3-CCR7+ cells
between these groups, however (Fig. 4A, Tab. 5).
Figure 2. Percentages of CD4+CD25+CD127lowFoxP3+ natural regulatory T lymphocytes expressing GITR and CD152, and mean fluorescence intensity (mean fluorescence channel, MFC) for GITR and CD152 on nTregs in patients with severe asthma (SA), mild-moderate
asthma (MA), and in the control group (NC)
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Pneumonologia i Alergologia Polska 2012, vol. 80, no 5, pages 389–401
Table 3. Variables (median, lower, and upper quartile, minimum and maximum) concerning percentages of CD4+ natural T
regulatory cells, expression of their surface markers GITR and CD152 as well as mean fluorescence intensity
(MFC) for GITR and CD152 on nTregs
nTreg
Quantitative parameters Severe asthma
Mild-to-moderate
asthma
Control group
% CD4+CD25highCD127lowFoxP3+
Median
Upper and lower quartile
76.0
57.5–81.7
85.5
78.9–93.1
93.6
84.4–95.7
Min–max
4.7–96.2
63.2–98.1
59.0–96.6
Median
Upper and lower quartile
Min–max
60.1
51.4–83.1
21.1–95.5
83.9
74.3–90.5
43.6–95.6
89.0
77.1–90.1
70.3–93.2
% CD4+CD25highCD127lowFoxP3+CD152+
Median
Upper and lower quartile
Min–max
46.2
36.5–92.2
23.8–96.6
86.7
79.5–93.6
67.7–95.1
91.4
88.4–91.5
75.9–92.1
MFC nTreg GITR+
Median
Upper and lower quartile
Min–max
830
570–990
519–2734
1470
612–2664
590–3720
1508
1445–1535
760–1754
MFC nTreg CD152+
Median
Upper and lower quartile
Min–max
1274
700–1400
500–1470
1166
990–1230
847–1290
1040
987–1060
880–1093
% CD4+CD25highCD127lowFoxP3+GITR+
Figure 3. Percentage nTreg cells expressing CCR5 and CCR7 chemokine receptors and mean fluorescence intensity (mean fluorescence
channel, MFC) for CCR5 and CCR7 in patients with severe asthma (SA), mild-moderate asthma (MA) and in the control group (NC)
No significant differences were found in mean fluorescence intensity (MFC) for CCR5 and CCR7
receptors on Teff lymphocytes between the two patient subgroups with asthma of different intensity
grades (Fig. 4B, Tab. 5). On the other hand, MFC for
CCR5 (p < 0.001) and CCR7 (p < 0.05) receptors on
Teff cells was found between patients with severe
asthma and the control group (Fig. 4B, Tab. 5).
The calculated nTregCCR5+/TeffCCR5+ coefficient was significantly lower in patients with
severe asthma as compared to patients with milder asthma and healthy controls (p < 0.001) (Fig.
5A, Tab. 6). No such difference was found for
nTreg/Teff or nTreg CCR7+/Teff CCR7+ coefficient (Fig. 5B, Tab. 6) and respective coefficients
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for MFC when analogical comparisons were made
(Fig. 5A and 5B, Tab. 6).
Discussion
Many studies concerning the role of nTreg
lymphocytes in pathogenesis of various diseases
have been published to date; however, there are
no reports on lymphocytes expressing
C D 4 + C D 2 5 h i g h C D 1 2 7 l o w FoxP3+GITR+CD152+CCR5+CCR7+ in patients
with severe bronchial asthma. The presented study showed that multicolour flow cytometry is a
useful method of characterising CD4+CD25highCD127lowFoxP3+GITR+CD152+
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Łukasz Kraszula et al., The use of multi-color flow cytometry for identification of functional markers of nTregs...
Table 4. Variable (median, lower and upper quartile, minimum, maximum) describing percentage of nTreg cells expressing
chemokine receptors CCR5 and CCR7 mean fluorescence intensity (MFC) for CCR5 and CCR7 on nTregs
nTreg
Quantitative parameters Severe asthma
Mild-to-moderate
asthma
Control group
% CD4+CD25highCD127lowFoxP3+ CCR5+
Median
Lower and upper quartile
Min–max
43.5
33.3–50.0
13.5–85.5
30.3
27.9–47.9
10.0–88.9
36.8
26.0–44.3
20.0–76.7
% CD4+CD25highCD127lowFoxP3+CCR7+
Median
Lower and upper quartile
Min–max
78.9
63.3–84.0
7.7–87.1
72.5
54.7–81.6
40.2–92.9
74.5
66.7–79.6
50.0–98.0
MFC nTreg CCR5+
Median
Lower and upper quartile
Min–max
1120
1015–1306
864–1842
1262
1124–1788
758–2629
1812
1261–2350
962–3694
MFC nTreg CCR7+
Median
Lower and upper quartile
Min–max
1130
957–1381
708–1724
1519
1002–1797
688–2199
1576
1387–1876
1106–2898
Figure 4. Comparison of the percentage T CD4+ effector lymphocytes expressing the chemokine receptors CCR5 and CCR7 and Mean
Fluorescence Chanel (MFC) for CCR5 and CCR7 in patients with severe asthma (SA), mild-moderate (MA) and the control group (NC)
Table 5. Variables (median, lower, and upper quartile, minimum and maximum) concerning percentages of CD4+ effector T
lymphocytes expressing CCR5 and CCR7 chemokine receptors as well as mean fluorescence intensity (MFC) for CCR5 and
CCR7 on CD4+ T lymphocytes
Teff
Quantitative parameters Severe asthma Mild-to-moderate asthma
CD4+CD25-FoxP3-CCR5+
Median
Lower and upper quartile
Min–max
44.9
33.0–59.1
13.8–72.8
17.8
13.0–41.8
11.2–57.5
22.6
15.2–37.8
8.6–48.7
CD4+CD25-FoxP3-CCR7+
Median
Lower and upper quartile
Min–max
81.3
75.7–90.2
66.8–94.9
87.1
78.6–90.9
65.2–94.9
86.8
76.1–89.5
63.4–96.2
MFC Teff CCR5+
Median
Lower and upper quartile
Min–max
1293
1203–1545
1013–2301
1424
1135–1576
899–2547
1869
1456–2098
1043–3523
MFC Teff CCR7+
Median
Lower and upper quartile
Min–max
1759
1505–2198
1061–3888
2313
1273–2649
1005–3791
2454
1824–2654
1484–3293
CCR5+CCR7+ nTreg cells. The observed decrease
in the percentage of nTreg cells and reduced
expression of functional markers GITR and CD152
Control group
may suggest abnormal activity of these cells in
patients with severe asthma. The results of other
published studies are contradictory, which can be
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Pneumonologia i Alergologia Polska 2012, vol. 80, no 5, pages 389–401
Figure 5. Coefficients of CCR5 and CCR7 chemokine receptor expression on natural regulatory T-cells and effector T-cells. Coefficients of
mean fluorescence intensity (MFC) for CCR5 and CCR7 chemokine receptors on nTregs and CD4+ effector T-cells
Table 6. Variables (median, lower, and upper quartile, minimum and maximum) concerning coefficients of CCR5 and CCR7
chemokine receptor expression on natural regulatory cells and effector cells. Coefficients of mean fluorescence intensity
(MFC) for CCR5 and CCR7 on nTregs and CD4+ effector T-cells
Coefficient
Quantitative parameters Severe asthma Mild-to-moderate asthma
nTreg/Teff
Median
Lower and upper quartile
Min–max
0.12
0.09–0.14
0.05–0.27
0.11
0.09–0.12
0.06–0.2
0.11
0.07–0.13
0.03–0.18
nTreg CCR5+/Teff CCR5+
Median
Lower and upper quartile
Min–max
0.91
0.81–1.08
0.61–1.62
1.58
1.17–2.1
0.63–3.07
1.55
1.05–2.14
0.55–3.01
nTreg CCR7+/Teff CCR7+
Median
Lower and upper quartile
Min–max
0.87
0.77–1.02
0.49–1.24
0.85
0.74–0.9
0.5–1.08
0.91
0.77–0.95
0.67–1.15
MFC nTregCCR5+/MFC Teff CCR5+
Median
Lower and upper quartile
Min–max
0.84
0.69–1.04
0.58–1.16
0.99
0.75–1.29
0.48–1.90
0.98
0.75–1.15
0.55–2.42
MFC nTreg CCR7+/MFC Teff CCR7+
Median
Lower and upper quartile
Min–max
0.63
0.54–0.75
0.41–0.88
0.70
0.62–0.79
0.54–0.84
0.72
0.61–0.85
0.53–1.11
potentially attributed to different patient selection,
varying asthma phenotypes (especially when concerning severe disease), or study methodology. It
should be emphasised that only some studies contain data concerning expression of FoxP3 transcription factor. If expression of this marker, strategic
to nTreg cell population, is not assessed, definitive separation of nTregs and effector cells cannot
be performed and cells cannot be positively identified, according to current knowledge.
Shi et al. sorted and studied CD4+CD25+
lymphocytes but found no differences as to the
amount of natural regulatory T-cells in peripheral
blood between patients with asthma and healthy
controls. The amount of nTreg cells increased, however, during asthma exacerbations. The population
of nTregs in stable asthma did not significantly fluctuate, and the cells were found to suppress proliferation of effector T-cells and inhibit production of
396
Control group
both Th1 and Th2 cytokines. These studies did not
include data on FoxP3 expression on nTreg cells [13].
Xue et al. demonstrated a decreased percentage of nTreg cells and decreased amount of FoxP3
mRNA in this cell subpopulation in patients with
asthma, particularly in periods of exacerbation
[14]. Similar results were reported by Mamessier
et al., who noted Treg decrease in exacerbated asthma [15].
Many authors emphasise the role of antigens
other than FoxP3 for nTreg functioning. The CD152
(CTLA-4) antigen is a negative regulator of immune response. Studies in rheumatoid arthritis confirm that abnormal CD152 expression can be linked to aberrancies of Treg lymphocyte suppressive effect [16, 17]. Transmembrane protein GITR,
which belongs to the TNF receptor family, is constitutively expressed on nTreg cells, and plays an
important role for their suppressor function [18].
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Łukasz Kraszula et al., The use of multi-color flow cytometry for identification of functional markers of nTregs...
Studies of cultured human lymphocytes showed
reduced suppressor capacity of nTreg cells when
GITR-blocking antibodies were added, which supports the role of this antigen in immune responses. In the presented study, nTreg cells showed
decreased CD152 expression in patients with severe asthma, with no change in GITR expression.
Zhang et al. showed that nTreg lymphocytes in
patients with asthma express CD152, GITR, tolllike receptor 4 (TLR4), latency-associated peptide
(LAP/TGF-b1), and FoxP3 [19]. However, these
authors did not observe significantly different percentages of FoxP3+ nTreg cells or CD152+ and
GITR+ lymphocytes in patients with asthma (severe and stable disease) and healthy controls [19].
Dysfunction of nTreg cells is believed to correlate with development of severe asthma. The currently presented results support this hypothesis, as
the percentage of FoxP3+ nTreg cells correlated
with FEV1 % expected value in subjects with severe asthma. Besides, the higher the percentage on
nTreg cells the higher the one second forced expiratory volume, which could imply that an increasing
amount of nTreg cells in patients with severe asthma could improve their clinical condition.
As mentioned previously, nTreg lymphocytes
need to recirculate to be able to fully exert their
function, which in turn is regulated by chemokines and their receptors. Activated helper cells of
the Th1 type, cytotoxic Tc1 cells, and regulatory
T-cells are known to express CCR5 chemokine receptor. This receptor is suggested to contribute to
effector lymphocyte migration to the site of inflammation. Cytokine receptor CCR5 can thus play a
pro-inflammatory role, but on the other hand, as
it is also expressed on regulatory cells, it can also
have an anti-inflammatory effect [20]. The pro-inflammatory role of CCR5 was supported by the studies showing expression of this antigen on effector T-cells found in synovium in patients with
rheumatoid arthritis, and in the central nervous
system in mice models of experimental autoimmune encephalomyelitis (EAE) [21, 22]. The CCR5
receptor was also found to take part in T-cell migration to pancreatic islands, and its expression
correlated with severity of diabetes in animal models [23]. Lack of CCR5 expression causes decreased effector cell migration to the sites of viral infection or parasitic infestation (Trypanosoma cruzei, Toxoplasma gondii) [24–26].
The anti-inflammatory effect of CCR5 receptor can be supported by the published observations
of tissue infiltration by alloreactive cytotoxic Tcells in the setting of lacking CCR5 expression.
Studies in mice showed that CD4+CD25+ regula-
tory T-cells that lack membrane CCR5 receptor
expression are less effective in suppressing immune reaction as part of the graft versus host disease
(GvHD) [7]. Kallikourdis et al. reported that regulatory T-cells can be divided into subpopulations
of strong (CCR5+) or weak (CCR5-) immunosuppressive properties. These authors also noted that
regulatory T-cells lacking CCR5 receptor have a
weaker effect in supporting immune tolerance between mother and foetus. These data can suggest
that CCR5+ Tregs can represent an effector subpopulation of regulatory lymphocytes [27]. Imbalance between anti-inflammatory CCR5+ Treg
cells and pro-inflammatory CCR5+ Teff lymphocytes may modulate the course and intensity of the
inflammatory reaction, which is supported by the
presented results. Decreased coefficient of CCR5+
nTreg/Teff cells as well as increased percentage of
CCR5+ effector cells may suggest immunological
imbalance between these lymphocyte subpopulations in the course of severe asthma. The presented results also suggest a dominant pro-inflammatory function of this receptor, thus leading to increased inflammatory response and greater severity of asthma. Casas et al. observed similar expression of CCR5 receptor on CD4+ lymphocytes in
subjects with allergies and in healthy controls [28].
Chemokine receptor CCR7 is known to play a
role in initiating induced immune response, as it
stimulates migration of naïve and regulatory Tcells to secondary lymphatic organs. The same receptor is, however, also suggested to regulate immune response [29]. Lack of CCR7 expression on
nTreg lymphocytes is correlated with excessive
immune response, as lymphocytes demonstrate
defective migration to lymph nodes, and thus their effect at the site of inflammation cannot be achieved [6]. The presented study showed no differences in percentage of CCR7+ nTreg cells and coefficient of CCR7+ nTreg/Teff cells between the patient groups, which suggests that nTreg lymphocyte migration to secondary lymphatic organs in
severe bronchial asthma is not disturbed at the
cellular level.
Published data suggest an immunoregulatory
function of CCR5 and CCR7 receptors; therefore,
their decreased expression on regulatory and effector lymphocytes may have a negative effect on
migration of these cells, whereas increased receptor expression could contribute to a stronger cell
migration towards sites of ongoing immune response. A lower value of mean fluorescence intensity
(MFC) for chemokine receptors on nTreg and Teff
cells in severe asthma may indirectly point to decreased receptor density on the surface of these
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397
Pneumonologia i Alergologia Polska 2012, vol. 80, no 5, pages 389–401
cells. Fewer receptors on cell surface result in weaker response of these cells to chemokine receptor
agonists, thus resulting in decreased lymphocyte
migration to sites of inflammation.
Analysis of expression of functional markers
and chemokine receptors on natural regulatory Tlymphocytes in patients with severe or mild-tomoderate asthma using multicolour flow cytometry confirmed that this analytic modality is an optimal method of nTreg cell characterization and separation from effector cells. A decreased amount
of CD4+CD25highCD127lowFoxP3+GITR+ nTreg
cells and lack of immunological balance between
regulatory and effector T-cell subpopulations
expressing CCR5 chemokine receptor may point to
dysfunction of these cells. Additionally, depletion
of induced regulatory T-cells and decreased expression of functional markers on these cells in patients
with asthma cannot be excluded.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Conclusions
1.
2.
3.
Multicolour flow cytometry permits definitive identification of nTreg lymphocytes. This
can be achieved by analysis of expression of
various markers, including FoxP3 transcription, which permits separating natural regulatory and effector lymphocytes.
Patients with severe and mild-to-moderate
asthma show phenotypical differences in Tcell subpopulations, with decreased expression of functional markers on nTreg cells {in
severe disease}, which may suggest dysfunction of these cells.
FoxP3, CD152, and CCR5 may be used for evaluation of treatment efficacy, especially in severe asthma. The presented study suggests that
induction of nTreg cells with high expression
of FoxP3 and potent suppressor effect may be
a new therapeutic option, especially in severe
asthma.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Conflict of interests
The authors have no conflicts of interest to
declare.
25.
26.
Piśmiennictwo
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