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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 2  |  Page : 37-41

Study of T-regulatory cells in patients with acute, idiopathic thrombocytopenic purpura


1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Date of Submission14-Dec-2012
Date of Acceptance23-Jan-2014
Date of Web Publication30-Aug-2014

Correspondence Address:
Maher Borai
Department of Clinical Pathology, Faculty of Medicine, Zagazig University, 44517 Zagazig
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.139751

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  Abstract 

Introduction Idiopathic thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder that occurs because of enhanced peripheral platelet destruction. Antibodies and T cells are involved in the pathogenesis of the disease and, like other autoimmune diseases, patients with ITP have a peripheral deficiency in regulatory T cells (Treg) numbers and function that may be responsible for loss of tolerance. Our aim was to measure Tregs (CD4 + CD25 +high FoxP + 3) and levels of interleukins (IL-10 and IL-12) in peripheral blood mononuclear cell (PBMC) cultures from patients with ITP and analyze their relationship with the clinical features and outcome of treatment of ITP.
Participants and methods Forty-five participants were included in this study, divided into two groups. Group I included 15 healthy children as a control group. Group II included 30 pediatric patients with ITP. According to treatment, group II was divided into three subgroups: group IIa (no treatment) included two (6.7%) patients, group IIb (steroid treatment) included 10 (33.3%) patients, and group IIc (steroid+intravenous immunoglobulin treatment) included 18 (60%) patients. ITP is diagnosed by platelet count less than 100 Χ 103/μl. Tregs were analyzed by flow cytometry. IL-10 and IL-12 in the supernatants of basal and lipopolysaccharide-stimulated PBMC cultures were estimated using an enzyme-linked immunosorbent assay.
Results A significantly lower percentage of Tregs was found in patients than in controls (1.46 ± 0.97 vs. 7.09 ± 1.5%) and the lowest percentage of Tregs was recorded in group IIc. A positive correlation was observed between Tregs% and platelet count in the patient group. PBMCs from patients had significantly higher basal levels of IL-10 and IL-12, with a marked reduction in responsiveness to lipopolysaccharide in vitro compared with the controls.
Conclusion Children with ITP had reduced Tregs% and IL-10/IL-12 imbalance. Thus, Tregs may play a role in modifying immune responses in these patients, resulting in new strategies of treatment and monitoring of disease activity.

Keywords: flow cytometry, idiopathic thrombocytopenic purpura, IL-10, IL-12, peripheral blood mononuclear cells, Tregs


How to cite this article:
Baraka A, Borai M, Hesham MA, Almalky MA. Study of T-regulatory cells in patients with acute, idiopathic thrombocytopenic purpura. Egypt J Haematol 2014;39:37-41

How to cite this URL:
Baraka A, Borai M, Hesham MA, Almalky MA. Study of T-regulatory cells in patients with acute, idiopathic thrombocytopenic purpura. Egypt J Haematol [serial online] 2014 [cited 2017 Dec 12];39:37-41. Available from: http://www.ehj.eg.net/text.asp?2014/39/2/37/139751


  Introduction Top


Idiopathic thrombocytopenic purpura (ITP) is a bleeding disorder considered an autoimmune disease characterized by the production of autoreactive antibodies to platelet antigens, resulting in both accelerated destruction of platelets and reduced platelet production [1] . Regulatory T cells (Treg) are a group of immune suppressive cells that have been studied intensively in recent years. Regulatory T cells (Tregs) play a central role in protecting the host from autoimmune diseases. A decreased number of CD4 + CD25 +high cells were found in patients with autoimmunity [2] . Most previous studies have labeled CD4 and CD25 on Treg, but CD25 is also expressed on the surface of functional T cells. The accuracy and robustness of Treg detection by CD4 and CD25 labeling studies suggested that CD127 expression on the cell surface was related negatively to the forkhead/winged helix transcription factor p3 (FoxP3); meanwhile, FoxP3 has been proven to be the most reliable and specific marker of Treg [3] .

The presence of autoreactive T cells to platelet glycoprotein (GP) IIb-IIIa can be found in the peripheral blood of normal individuals and are involved in the production of GPIIb-IIIa autoantibodies in ITP [4] .

Naturally arising CD4 + CD25 +high T cells regulate autoreactive T cells. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion [5] . Precise understanding of the immune-suppressive mechanism of Treg cells remains elusive, although there is increasing evidence that Tregs manifest their function through a myriad of mechanisms that include the secretion of immunosuppressive soluble factors such as IL-9, IL-10, and TGFβ, cell contact-mediated regulation through the high-affinity TCR, and other costimulatory molecules such as CTLA-4, GITR, and cytolytic activity [6] . An increased number of CD4 + CD25 +high cells were found in patients with autoimmunity, cancer, or chronic infection [7] .

Interleukin (IL) 10 is known to promote antibody production by B cells, and it has been suggested that it might play a role in the development of autoantibody-mediated diseases such as systemic lupus erythematosis (SLE) [8] and ITP. However, it has been shown that, in human autoimmune diseases, IL-12 promotes cell-mediated immunity through its ability to induce interferon g, but suppresses humoral immune responses and autoantibody production [9] . Therefore, studies of peripheral blood lymphocyte-monocyte activation and IL production are valuable tools for investigating the relationship between the immunoregulatory cytokine network and ITP [10] .

Aim of this work

This study aimed to measure Tregs (CD4 + CD25 +high FoxP + 3) and levels of ILs (IL-10) and (IL-12) in peripheral blood mononuclear cell (PBMCs) culture from patients with ITP and analyze their relationship with the clinical features and outcome of treatment of ITP.


  Participants and methods Top


Participants

The study was carried out at the Clinical Pathology and Pediatric Departments (Hematology Unit) of Zagazig University Hospitals. The study protocol was approved by the ethical committee of the Faculty of Medicine, Zagazig University.

This study included 45 participants divided into two groups. Group I included 15 healthy children as a control group (nine males and six females). Group II included 30 pediatric patients with ITP (15 males and 15 females). According to treatment, group II was divided into three subgroups as follows: group IIa (no treatment) included two (6.7%) patients, group IIb (steroid treatment) included 10 (33.3%) patients, and group IIc (steroid+intravenous immunoglobulin treatment) included 18 (60%) patients.

Methods

Informed consents were obtained from the parents of the participants, who were subjected to the following:

  1. Full assessment of history together with careful clinical examination.
  2. Laboratory investigations that included the following: liver function, kidney function by ADVIA - 1650, Chemistry auto analyser (Bayer Diagnostics Laboratories, Berkeley, CA), complete blood count by sysmex S.F. 3000, prothrombin time, and partial thromboplastin time by Sysmex CA (Sysmex Corp., KOBE, Japan) 1500.
  3. Leishman-stained peripheral blood smears and bone marrow aspirates were examined. The diagnosis of ITP was made on the basis of the presence of thrombocytopenia (platelets < 100 × 10 3 /μl), with exclusion of patients with other causes of thrombocytopenia, for example, aplastic anemia, hypersplenism, leukemia, and drug-induced thrombocytopenia.


Treg detection by flow cytometry

Lymphocyte separation

0Heparinized blood sample was taken from all participants for Treg detection. PBMCs were purified by Ficoll-Hypaque gradients (Seromed-Biochrom, Berlin, Germany). Separated PBMCs for the flow cytometric assay were washed twice with FACs washing solution; the cell pellet was suspended in FACs buffer at a concentration of 1.0 × 10 6 /ml.

Treg detection by flow cytometry

Treg markers were detected using a specific fluorochrome, mouse anti-human monoclonal antibodies (mAbs), anti-CD25 fluorescein isothiocyanate, anti-CD4 peridinin-chlorophyll protein conjugate, and anti-CD3 phycoerythrin conjugate (eBioscience, San Diego, California, USA).

Sample preparation and staining

Surface staining was performed for the detection of CD3, CD4, and CD25 by adding 20 μl of each mAb to 100 ml of separated PBMCs, followed by incubation for 30 min in the dark at 4°C, and then the tubes were washed twice with FACs washing buffer. Intracellular staining was performed for the detection of FoxP3 according to the manufacturer's protocol (eBioscience). Cells were first stained with the surface mAb of interest (anti-CD4/anti-CD25) and washed twice with PBS. After permeabilization with a Fixation/Permeabilization Buffer, 20 μl anti-human Foxp3 mAb were added and the cells were incubated for 30 min in the dark at 4°C. Finally, 0.5 ml of PBS was added to the washed cells and the samples were ready for the measurement of the CD4 + CD25 + and FoxP3 using FACScalibur flowcytometry (Becton Dickinson, San Jose, California, USA).

To avoid nonspecific Fc receptor staining, appropriate isotype controls of mouse anti-human mAbs were used. FACs acquisition and analysis were carried out using FACs Cell Quest software (BD Biosciences, San Jose, CA). Samples were first examined for the frequency of CD3 + CD4 + T cells. The percentage of CD4 + CD25 +high FoxP + 3 T cells in the total CD4 + T-cells population was then determined, and the intensity of CD25 surface expression was measured using the mean fluorescence intensity (MFI) as described by Sakaguchi et al. [11] . T cells with CD25 expression of at least 120 were considered CD25 +high (average MFI, 160 ± 25).

Lymphocyte culture

Two milliliter cell suspensions were plated into 5ml plastic Petri dishes (Falcon; Becton Dickinson) and cultured at 37°C in a 5% CO 2 humidified atmosphere under basal conditions and with the addition of 100 ng/ml of lipopolysaccharides (LPS) (Sigma-Aldrich, Saint Louis, Missouri, USA) to stimulate monocyte IL-10 and IL-12 production. After 48 h, the supernatants were collected and frozen at −80°C until use.

Cytokine assay

Quantitative determination of IL-10 and IL-12 was performed using quantitative sandwich enzyme-linked immunosorbent assay (Quantikine; R&D systems, Minneapolis, Minnesota, USA). The detection limits for IL-10 and IL-12 were 4.61 and 2.0 pg/ml, respectively.

Statistical analysis

Data were analyzed using SPSS15 (SPSS, Chicago, IL, USA) software. Data were expressed as mean ± SD for quantitative variables, and as number and percentage for qualitative variables. Analysis of variance (or F-test) was carried out for comparison of means of more than two groups (P < 0.05, significant; P < 0.01, highly significant; and P < 0.001, very highly significant).


  Results Top


Thirty children with ITP and 15 healthy age-matched and sex-matched controls were enrolled in this study. PBMCs from patients and controls were first analyzed for the frequency of CD4 + CD25 + FoxP3 + T cells in the total CD3 + CD4 + T-cell population; the percentage of Tregs (CD25 +high ) was then determined using MFI [Figure 1]. The results showed a significantly lower percentage of Treg cells (CD4 + CD25 +high /FoxP3 + ) in CD4 + cells in peripheral blood in patients than in controls (1.46 ± 0.97 vs. 7.09 ± 1.5%) [Table 1]. The mean percentage of Tregs in the three patients groups (IIa, IIb, IIc) was 2.17 ± 0.31, 1.41 ± 0.79, and 0.80 ± 0.50%, respectively [Table 2].
Table 1: Percentage of Tregs in the control and patient groups

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Table 2: Percentage of Tregs in the patient group

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Figure 1: Flow cytometry analysis for the percentage of regulatory T cells (Tregs) in idiopathic thrombocytopenic purpura patients. (a) Peripheral blood mononuclear cells were stained for CD3+CD4+ T cells. (b) CD25 gate set to determine CD25+high cells, CD25intermediate, and CD25- cells in CD4+ T-cell populations. (c) FoxP3 staining was measured in populations using selected gates in which FoxP3 was brightly expressed in the CD25+high Treg population (shadowed) whereas it was negative in CD25intermediate and CD25- populations (line). FITC, fluorescein isothiocyanate; PerCP, peridinin– chlorophyll proteins.

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There were no significant differences between either groups (IIa, IIb) or group IIc in WBC, lymphocytes, and neutrophils. However, a highly significant reduction in the platelet count was recorded in group IIc [Table 3].
Table 3: Laboratory data of blood counts in the patient groups according to treatment

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A significant positive correlation was observed between Tregs% and platelet count, whereas there was no significant correlation in WBC, lymphocytes, and neutrophils in the patient groups [Table 4].
Table 4: Correlation coefficient between percentage of Tregs and different laboratory parameters among the patient group

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IL-10 and IL-12 are primarily secreted by monocytes, not T cells. We evaluated the levels of these cytokines in RPMI culture medium before and after LPS stimulation (LPS preferentially stimulates monocytes). Before stimulation, PBMCs from patients showed a constitutively increased level of IL-10 compared with the controls (132 vs. 18.4, P < 0.001). With stimulation, the levels increased seven-fold in patients (924 pg/ml) and 40-fold in the controls (728 pg/ml) [Table 5].
Table 5: Comparison of interleukins IL-10 and IL-12 in patients with ITP and healthy controls

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  Discussion Top


ITP is an autoimmune disease. Patients with ITP have activated platelet autoreactive T cells and cytokine imbalance; by far the most widely recognized is a CD4 + T-cell population that expresses high levels of CD25 (CD4 + CD25 +high ), suggesting loss of peripheral tolerance in ITP patients [12] . CD4 + Tregs play an important role in the maintenance of peripheral tolerance and are characterized by the expression of the CD25 high surface marker and the transcription factor forkhead box protein 3 (Foxp3), making up 5-10% of the normal CD4 + T-cell population [11] .

Our findings showed a highly significant decrease in the percentage of CD4 + CD25 +high /Foxp3 T cells (Tregs) in children with acute ITP compared with controls. Therefore, a defective Tregs population may contribute toward the pathogenesis of childhood ITP.

Similar results have been reported by Liu [13] , who showed that the percentage of Treg cells was significantly decreased in ITP patients in an active and nonremission state when compared with healthy individuals [14] . Also, Zahran and Elsayh [15] reported that the percentages of CD4 + CD25 +high and CD4 + CD25 +high FoxP3 + cells were significantly decreased in patients with ITP. In contrast, Ling et al. [16] reported that the proportion of CD4 + CD25 +high T cells in the peripheral blood of patients with ITP was significantly higher than that in the normal control group. The study carried out by Workman et al. [17] showed that patients with ITP had significantly decreased percentage of n-Treg cells in peripheral blood compared with normal controls, suggesting a specific role of these cells in the pathogenesis of the disease.

It was important to document that Treg cells in our patients were not diluted with activated CD25 +high effector T cells. Although it is difficult to rule out this assumption entirely, the brightest (CD25 +high ) cells were analyzed using MFI and counted to exclude activated T cells that are usually intermediate in their CD25 expression [11] . In this respect, Fontenot and Rudensky [18] reported that FoxP3 is a unique marker of Tregs, distinguishing them from activated CD4 + CD25 + T cells and playing a pivotal role in their development and maturation.

Torgerson [19] showed that Treg cells in peripheral blood of healthy humans preferentially resided within the CD4 + CD25 high T-cell population. Moreover, our results are supported by previous studies of autoimmune diseases. Several studies in human autoimmune disorders have reported a decrease in circulating CD4 + CD25 +high Treg cells including patients with acute and chronic ITP [20] , multiple sclerosis, SLE, autoimmune liver disease, and rheumatoid arthritis [21] .

Irrespective of the mechanism, the degree of Treg-mediated suppression is dependent on the frequency of CD4 + CD25 +high T cells, access to a sufficient concentration of antigen presented by antigen-presenting cells, and localization with effector T-cell targets. The cytokine environment required to support Treg growth, maintenance, and activation of suppressor function in the periphery at the time of antigen encounter is also critical [22] .

In our study, the mean initial platelet count of patients who received no treatment was significantly higher than that of patients who received an initial treatment (steroid only and steroid + intravenous immunoglobulin). These results indicated that the decision on treatment should be made on the basis of platelet count, history of bleeding, signs and symptoms, and other factors [23] .

In a study carried out by Watts [24] , it was reported that his group continues to treat most patients with platelet counts less than 20 × 10 3 /l, whereas an Egyptian study carried out by Elalfy et al. [25] concluded that the initiation of treatment for ITP was based on low platelet count irrespective of the severity of bleeding. Both studies reported that the treatment was well tolerated, with a low incidence of recognized side effects. However, the recent guidelines suggest that treatment should be initiated according to bleeding symptoms irrespective of the platelet count and that more patients can be managed conservatively [26] .

In our study, we found a significant positive correlation between CD4 + CD25 high percentage and platelet count. This indicated a close association between Treg cells percent and the parameters known to reflect the degree of platelet destruction. These findings raise the possibility that Treg cells may regulate the disease phenotype particularly in relation to the degree of thrombocytopenia.

In agreement with our results, Lin et al. [27] found that the expression of circulating CD4 + CD25 high Treg cells derived from SLE patients correlated inversely with disease activity. Also, Zhang [20] found that the percent of circulating Tregs may be decreased during active disease and the extent of this decrease correlates with the severity of the disease.

In the present study, IL-10 and IL-12 production was significantly higher in ITP patients than in the controls before stimulation of the culture medium with LPS; this may be related to the high basal activation state in the patient group. This significant difference was not maintained following LPS stimulation, indicating a state of cell exhaustion. The absence of an IL-12 response to LPS stimulation could also be ascribed to the suppressive effect of IL-10 on IL-12 producer cells. Our results are in agreement with those of Toriani-Terenzi et al. [28] , who reported increased basal synthesis of IL-10 and levels of IL-12 in 55% of ITP PBMC cultures compared with controls. The authors concluded that the production of autoantibodies in ITP may be secondary to the imbalance between IL-10 and IL-12. Liorent et al. [22] reported that the continuous administration of anti IL-10 antibodies delays the onset of autoimmunity of SLE in experimental animals because of increase of TNF-α; administration of IL-10, in contrast, was found to accelerate disease progress as recombinant IL-10 strongly inhibits the in-vitro production of IL-12 in PBMC cultures from patients with SLE and healthy controls.


  Conclusion Top


Children with ITP had reduced Tregs% and IL-10/IL-12 imbalance. Thus, Tregs may play a role in modifying immune responses in these patients, resulting in new strategies of treatment and monitoring of disease activity.


  Acknowledgements Top


 
  References Top

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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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