The use of multi-color flow cytometry for identification of functional markers of nTregs in patients with severe asthma

(1)

A A A A

Address for correspondence:ddress for correspondence:ddress for correspondence:ddress for correspondence: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: lukasz.kraszula@umed.lodz.pl

Manuscript received on: 12.08.2011 r.

Łukasz Kraszula¹, Eusebio Makandjou-Ola¹, Maciej Kupczyk², Piotr Kuna², Mirosława Pietruczuk¹

1Zakład Diagnostyki Laboratoryjnej, II Katedra Chorób Wewnętrznych Uniwersytetu Medycznego w Łodzi Head: Prof. M. Pietruczuk, MD, PhD

2Klinika 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 invo- lves 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-

(2)

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+CD25- IL-10+IL-4- (Tr1), or CD4+TGF-b+ (Th3) [3]. Re- gulatory T-cells may play a suppressor role by directly or indirectly regulating the function of effector cells taking part in inflammatory reac- tions. Prerequisite for Treg function is surface expression of various molecules that characteri- se 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 suppressi- ve function both in the lymphatic tissue and in other sites where the immune response takes pla- ce [5]. Adequate distribution of Treg cells is ne- cessary 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 cha- racterized 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 suppressi- ve 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 (eight- colour) 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.

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 importan- ce 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. De- mographical and clinical data was retrieved from patients’ medical journals.

Diagnosis of bronchial asthma was made ba- sing 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 de- finitions 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 beclometha- sone, 800 μg/day fluticasone or equivalent). Pa- tients requiring long-term peroral steroid therapy received 800 μg/day budesonide or beclomethaso- ne, alternatively 400 μg/day fluticasone or equivalent. In cases of severe disease, patients received long-term therapy with inhaled long-acting beta- receptor 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.

(3)

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 con- sent 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 (K2ED- TA). Eight millilitres of venous blood were taken from each subject and added to 12 ml Histopaque- 1077 medium (Sigma-Aldrich). Peripheral blood mononuclear cells (PBMCs) were isolated by cen- trifugation 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 Bio- tec, USA). Mononuclear cells were sorted out by density gradient, then the mixture of various bio- tin-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 bio- tin, 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. Anti- bodies against FoxP3-antigen were then added to the cell suspension, with concentration according to the manufacturer’s recommendations (BD Bio- science). The well-mixed cell suspension was fur- ther 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 Table 1. Characteristics of the study group

Severe Mild-to-moderate

asthma asthma

n 30 30

Age (years ± SD) 49 ± 14.5 42 ± 13.5

Sex (F:M) 16:14 15:15

Duration of the disease 16 ± 8.7 10 ± 6.3 (years ± SD)

Atopy (% ± SD) 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

(4)

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+CD25hi- ghCD127lowFoxp3+. The percentage of cells expressing GITR, CD152, CCR5, and CCR7 as well as their mean fluorescence intensity (mean fluore-

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+ T- cells, 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.

Table 2. Monoclonal antibodies used for immunophenotyping of natural regulatory T cells

Antibody Fluorochrome Clone Manufacturer

Anti-human CD4 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™

Anti-human GITR PE 621 BioLegend

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+

(5)

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 cor- relation to FEV1 expected values in patients with severe asthma (r = 0.53; p < 0.05). No such corre- lation 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. Percen- tage 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 fluore- scence 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)

(6)

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 Control group

asthma

% CD4+CD25highCD127lowFoxP3+ Median 76.0 85.5 93.6

Upper and lower quartile 57.5–81.7 78.9–93.1 84.4–95.7

Min–max 4.7–96.2 63.2–98.1 59.0–96.6

% CD4+CD25highCD127lowFoxP3+GITR+ Median 60.1 83.9 89.0

Min–max 21.1–95.5 43.6–95.6 70.3–93.2

% CD4+CD25highCD127lowFoxP3+CD152+ Median 46.2 86.7 91.4

Min–max 23.8–96.6 67.7–95.1 75.9–92.1

MFC nTreg GITR+ Median 830 1470 1508

Upper and lower quartile 570–990 612–2664 1445–1535

Min–max 519–2734 590–3720 760–1754

MFC nTreg CD152+ Median 1274 1166 1040

Upper and lower quartile 700–1400 990–1230 987–1060

Min–max 500–1470 847–1290 880–1093

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 mil- der 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

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+CD25hi- g h C D 1 2 7 l o w F o x P 3 + G I T R + C D 1 5 2 + 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)

(7)

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 Control group

asthma

% CD4+CD25highCD127lowFoxP3+ CCR5+ Median 43.5 30.3 36.8

Lower and upper quartile 33.3–50.0 27.9–47.9 26.0–44.3

Min–max 13.5–85.5 10.0–88.9 20.0–76.7

% CD4+CD25highCD127lowFoxP3+CCR7+ Median 78.9 72.5 74.5

Min–max 7.7–87.1 40.2–92.9 50.0–98.0

MFC nTreg CCR5+ Median 1120 1262 1812

Lower and upper quartile 1015–1306 1124–1788 1261–2350

Min–max 864–1842 758–2629 962–3694

MFC nTreg CCR7+ Median 1130 1519 1576

Min–max 708–1724 688–2199 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 Control group

CD4+CD25-FoxP3-CCR5+ Median 44.9 17.8 22.6

Min–max 13.8–72.8 11.2–57.5 8.6–48.7

CD4+CD25-FoxP3-CCR7+ Median 81.3 87.1 86.8

Min–max 66.8–94.9 65.2–94.9 63.4–96.2

MFC Teff CCR5+ Median 1293 1424 1869

Min–max 1013–2301 899–2547 1043–3523

MFC Teff CCR7+ Median 1759 2313 2454

Min–max 1061–3888 1005–3791 1484–3293

CCR5+CCR7+ nTreg cells. The observed decrease in the percentage of nTreg cells and reduced expression of functional markers GITR and CD152

may suggest abnormal activity of these cells in patients with severe asthma. The results of other published studies are contradictory, which can be

(8)

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 Control group

nTreg/Teff Median 0.12 0.11 0.11

Min–max 0.05–0.27 0.06–0.2 0.03–0.18

nTreg CCR5+/Teff CCR5+ Median 0.91 1.58 1.55

Min–max 0.61–1.62 0.63–3.07 0.55–3.01

nTreg CCR7+/Teff CCR7+ Median 0.87 0.85 0.91

Min–max 0.49–1.24 0.5–1.08 0.67–1.15

MFC nTregCCR5+/MFC Teff CCR5+ Median 0.84 0.99 0.98

Min–max 0.58–1.16 0.48–1.90 0.55–2.42

MFC nTreg CCR7+/MFC Teff CCR7+ Median 0.63 0.70 0.72

Min–max 0.41–0.88 0.54–0.84 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 con- tain 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 fluc- tuate, and the cells were found to suppress prolife- ration of effector T-cells and inhibit production of

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 lin- ked to aberrancies of Treg lymphocyte suppressi- ve effect [16, 17]. Transmembrane protein GITR, which belongs to the TNF receptor family, is con- stitutively expressed on nTreg cells, and plays an important role for their suppressor function [18].

(9)

Studies of cultured human lymphocytes showed reduced suppressor capacity of nTreg cells when GITR-blocking antibodies were added, which sup- ports the role of this antigen in immune respon- ses. 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, toll- like 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 corre- late 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 expi- ratory 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 autoimmu- ne 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 in- fection or parasitic infestation (Trypanosoma cru- zei, Toxoplasma gondii) [24–26].

The anti-inflammatory effect of CCR5 receptor can be supported by the published observations of tissue infiltration by alloreactive cytotoxic T- cells 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]. Imba- lance 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 T- cells 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

(10)

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 T- lymphocytes in patients with severe or mild-to- moderate 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.

Conclusions

1. 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.

2. Patients with severe and mild-to-moderate asthma show phenotypical differences in T- cell subpopulations, with decreased expression of functional markers on nTreg cells {in severe disease}, which may suggest dysfunction of these cells.

3. 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.

Conflict of interests

The authors have no conflicts of interest to declare.

Piśmiennictwo

1. Bateman E.D., Hurd S.S., Barnes P.J. et al. Global strategy for asthma management and prevention: GINA executive summa- ry. Eur. Respir. J. 2008; 31: 143–178.

2. Holgate S.T., Holloway J., Wilson S. et al. Understanding the pathophysiology of severe asthma to generate new therapeutic opportunities. J. Allergy. Clin. Immunol. 2006; 117: 496–506.

3. Sakaguchi S., Wing K., Yamaguchi T. Dynamics of peripheral tolerance and immune regulation mediated by Treg. Eur. J.

Immunol. 2009; 39: 2331–2336.

4. Sakaguchi S., Yamaguchi T., Nomura T. et al. Regulatory T cells and immune tolerance. Cell 2008; 133: 775–787.

5. Huehn J., Hamann A. Homing to suppress: address codes for Treg migration. Trends. Immunol. 2005; 26: 632–636.

6. Schneider M.A., Meingassner J.G., Lipp M. et al. CCR7 is required for the in vivo function of CD4+ CD25+ regulatory T cells. J. Exp. Med. 2007; 204: 735–745.

7. Wysocki C.A., Jiang Q., Panoskaltsis-Mortari A. et al. Critical role for CCR5 in the function of donor CD4+CD25+ regulatory T cells during acute graft-versus-host disease. Blood 2005; 106:

3300–3307.

8. Seddiki N., Santner-Nanan B., Martinson J. et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J. Exp. Med. 2006; 203:

1693–1700.

9. van Oosterhout A.J., Bloksma N. Regulatory T-lymphocytes in asthma. Eur. Respir. J. 2005; 26: 918–932.

10. Seroogy C.M., Gern J.E. The role of T regulatory cells in asthma.

J. Allergy Clin. Immunol. 2005; 116: 996–999.

11. The ENFUMOSA cross-sectional European multicentre study of the clinical phenotype of chronic severe asthma. European Network for Understanding Mechanisms of Severe Asthma. Eur.

Respir. J. 2003; 22: 470–477.

12. Kuprys-Lipinska I., Elgalal A., Kuna P. Skin prick test with inhaled allergens in the general population of Lodz province.

Pneumonol. Alergol. Pol. 2009; 77: 229–234.

13. Shi HZ., Qin XJ.: CD4CD25 regulatory T lymphocytes in allergy and asthma. Allergy 2005; 60: 986–995.

14. Xue K., Zhou Y., Xiong S. et al. Analysis of CD4+ CD25+

regulatory T cells and Foxp3 mRNA in the peripheral blood of patients with asthma. J. Huazhong. Univ. Sci. Technolog. Med.

Sci. 2007; 27: 31–33.

15. Mamessier E., Milhe F., Guillot C. et al. T-cell activation in occupational asthma and rhinitis. Allergy 2007; 62: 162–169.

16. Ehrenstein M.R., Evans J.G., Singh A. et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy. J. Exp. Med. 2004; 200: 277–285.

17. Flores-Borja F., Jury EC., Mauri C et al. Defects in CTLA-4 are associated with abnormal regulatory T cell function in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 2008; 105: 19396–19401.

18. Motta A.C., Vissers J.L., Gras R. et al. GITR signaling potenti- ates airway hyperresponsiveness by enhancing Th2 cell activity in a mouse model of asthma. Respir. Res. 2009; 10: 93.

19. Zhang Q., Qian FH., Liu H. et al. Expression of surface markers on peripheral CD4+CD25high T cells in patients with atopic asthma: role of inhaled corticosteroid. Chin. Med. J. 2008; 121:

205–212.

20. D’Ambrosio D., Panina-Bordignon P., Sinigaglia F. Chemokine receptors in inflammation: an overview. J. Immunol. Methods.

2003; 273: 3–13.

21. Bagaeva L.V., Williams L.P., Segal B.M. IL-12 dependent/IFN gamma independent expression of CCR5 by myelin-reactive T cells correlates with encephalitogenicity. J. Neuroimmunol.

2003; 137: 109–116.

22. Wang CR., Liu MF. Regulation of CCR5 expression and MIP- 1alpha production in CD4+ T cells from patients with rheumatoid arthritis. Clin. Exp. Immunol. 2003; 132: 371–378.

23. Carvalho-Pinto C., Garcia MI., Gomez L. et al. Leukocyte attrac- tion through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice. Eur. J. Immunol. 2004;

34: 548–557.

24. Glass W.G., Lane T.E. Functional expression of chemokine receptor CCR5 on CD4(+) T cells during virus-induced central nervous system disease. J. Virol. 2003; 77: 191–198.

25. Luangsay S., Kasper L.H., Rachinel N. et al. CCR5 mediates specific migration of Toxoplasma gondii-primed CD8 lymphocytes to inflammatory intestinal epithelial cells. Gastroenterol- ogy 2003; 125: 491–500.

26. Machado F.S., Koyama N.S., Carregaro V. et al. CCR5 plays a critical role in the development of myocarditis and host pro- tection in mice infected with Trypanosoma cruzi. J. Infect. Dis.

2005; 191: 627–636.

27. Kallikourdis M., Andersen K.G., Welch K.A. et al. Alloantigen- enhanced accumulation of CCR5+ «effector» regulatory T cells in the gravid uterus. Proc. Natl. Acad. Sci. USA 2007; 104: 594–599.

28. Casas R., Lindau C., Zetterstrom O. et al. Downregulation of CXCR6 and CXCR3 in lymphocytes from birch-allergic patients.

Scand. J. Immunol. 2008; 68: 351–361.

29. Worbs T., Forster R. A key role for CCR7 in establishing central and peripheral tolerance. Trends. Immunol. 2007; 28: 274–280.