Alterations in immune cell subsets and their cytokine secretion profile in childhood idiopathic thrombocytopenic purpura (ITP)

Authors

  • R. M. Talaat,

    Corresponding author
    1. Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat City, Egypt
    • Correspondence: R. M. Talaat, Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat City 11211, Egypt.

      E-mail: Robamtalaat@yahoo.com

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  • A. M. Elmaghraby,

    1. Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat City, Egypt
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  • S. S. Barakat,

    1. Pediatrics Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt
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  • M. EL-Shahat

    1. Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City, Sadat City, Egypt
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Summary

Immune thrombocytopenic purpura (ITP) is acquired autoimmune disease in children characterized by the breakdown of immune tolerance. This work is designed to explore the contribution of different lymphocyte subsets in acute and chronic ITP children. Imbalance in the T helper type 1 (Th1)/Th2 cytokine secretion profile was investigated. The frequency of T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes, natural killer (NK) (CD16+56+) and regulatory T (Treg) [CD4+CD25+highforkhead box protein 3 (FoxP3)+] cells was investigated by flow cytometry in 35 ITP children (15 acute and 20 chronic) and 10 healthy controls. Plasma levels of Th1 cytokines [interferon (IFN-γ) and tumour necrosis factor (TNF-α)] and Th2 [interleukin (IL)-4, IL-6 and IL-10)] cytokines were measured using enzyme-linked immunosorbent assay (ELISA). The percentage of Treg (P < 0·001) and natural killer (NK) (P < 0·001) cells were significantly decreased in ITP patients compared to healthy controls. A negative correlation was reported between the percentage of Treg cells and development of acute (r = −0·737; P < 0·01) and chronic (r = −0·515; P < 0·01) disease. All evaluated cytokines (IFN-γ, TNF-α, IL-4, IL-6 and IL-10) were elevated significantly in ITP patients (P < 0·001, P < 0·05, P < 0·05, P < 0·05 and P < 0·001, respectively) compared to controls. In conclusion, our data shed some light on the fundamental role of immune cells and their related cytokines in ITP patients. The loss of tolerance in ITP may contribute to the dysfunction of Tregs. Understanding the role of T cell subsets will permit a better control of autoimmunity through manipulation of their cytokine network.

Introduction

Immune (idiopathic) thrombocytopenic purpura (ITP) is an acquired organ-specific autoimmune thrombocytopenic syndrome in children characterized by immune-mediated platelet destruction due to binding of immunoglobulin (Ig)G autoantibodies against Gp IIb/IIIa or GPIb/IX platelet glycoproteins [1, 2]. It is usually a benign, self-limiting disease in children [3]. ITP incidence is ≅ 2–12/100 000 per year for adults and children, respectively, and a mortality rate of 1–3% per year in severely affected cases [4, 5]. Two forms of ITP are distinguished; acute (temporary or short-term) and chronic (long-lasting). Acute ITP occurs abruptly, often after viral or bacterial infections. It usually resolves spontaneously within 6 months of diagnosis. However, approximately 20% of children newly diagnosed with ITP progress to a chronic form, defined as persistence of thrombocytopenia (platelet counts < 100 × 109/l for more than 6 months) [3, 6].

The pathophysiology of ITP is heterogeneous and complex [7]. While the presence of antibodies against platelet glycoproteins have traditionally been considered to play a central role, several abnormalities involving the cellular mechanisms of immune modulation have been described [7-9]. The cellular immune responses of patients with ITP have the characteristic hallmarks of breakdown in their tolerance mechanism [10]. Patients with ITP possess activated platelet autoreactive B and T cells and cytokine imbalance [11, 12], suggesting loss of peripheral tolerance in ITP patients. T helper (Th) cells and the cytokine they produce play a key role in ITP [13]. Several reports on serum cytokines in ITP have pointed towards specific CD4+ T helper (Th1) cell and macrophage activation [14, 15]. Activated platelet-autoreactive T cells with increasing cytokine imbalance, especially in chronic ITP patients with some evidence of higher levels of circulating proinflammatory cytokines, has been reported previously [2, 11-13].

Additional mechanisms of peripheral tolerance may be returned to a functionally distinct subpopulation of CD4+ T cells known as regulatory T cells (Tregs) [16], defined by the expression of CD4+CD25+ surface markers and the transcription factor forkhead box P3 (FoxP3). Tregs represent 5–10% of the normal CD4+ T cell population [17]. They play a leading role in protecting individuals from autoimmunity, thus maintaining immune tolerance and homeostasis [18]. Different populations of Tregs have been described, including naturally occurring and inducible Tregs [19]. The former are thymically derived and suppress general autoreactive responses under non-inflammatory conditions, although they can also become activated and expanded in an antigen-specific manner [20]. Inducible Tregs are generated in the periphery through exposure to antigen, but once activated they are thought to mediate suppressive activity against other antigens by the local release of specific cytokines [21]. Several reports have demonstrated Treg alterations in a number of autoimmune diseases [22].

In this regard, several independent studies have found significantly decreased Treg percentages in adults and children with ITP [23, 24], although the mechanism of suppression induced by Tregs and the factors that regulate all subsets of CD4+ cells (Th and Treg), CD8+ or other immune cells in ITP have not been elucidated. However, the mechanisms of both acute and chronic ITP are immune-mediated; different immunological mechanisms may be in charge and establishing such differences may permit identification of those children with acute ITP likely to develop the chronic form of this disorder. Thus, the objective of the present study was to examine the level of lymphocyte subsets [CD3+, CD4+, CD8+, T cell receptor (TCR)-αβ/γδ, CD19+], Treg cells (CD4+, CD25+, FoxP3) and natural killer (NK) (CD16+CD56+) cells in acute and chronic ITP children. The dysregulation in the cytokine network [interleukin (IL)-4, IL-6, IL-10, interferon (IFN)-γ and tumour necrosis factor (TNF)-α] with the disease course was also investigated.

Materials and methods

Patients and controls

Thirty-five children and adolescents with ITP (19 males and 16 females), with a mean age of 5·5 ± 3·2 years (range 1–12), were enrolled into this study. They were recruited from the Pediatric Hematology Clinic, Children's Hospital, Alexandria University, Alexandria, Egypt. In parallel, 10 healthy children were taken as controls. Diagnosis of ITP was based on the presence of thrombocytopenia (platelet count < 100 × 109/l), with no evidence of red or white blood cell abnormalities, normal or increased number of megakaryocytes with normal myeloid and erythroid element in bone marrow smears, absence of splenomegaly and exclusion of other known causes of thrombocytopenia, such as connective tissue disease, malignancy and/or drug-induced thrombocytopenia [25]. Informed consent was obtained from the parents of each participant prior to the study. All investigations were performed in accordance with the Alexandria University, Health and Human Ethical Clearance Committee guidelines for Clinical Research. The local ethics committee approved the study protocol.

At diagnosis, all patients and controls were subjected to a full history record, laying stress upon disease duration, drug intake, preceding viral infection and bleeding manifestations. A thorough clinical examination was conducted, stressing organomegaly and lymphadenopathy. A full blood picture was conducted. ITP patients were divided into two groups: 15 patients with acute ITP (disease duration less than 6 months) and 20 patients with chronic ITP (disease duration more than 6 months). At the time of sampling, all patients were at least 1 month out of steroid or immunosuppressive therapy to avoid their effect on membrane markers of lymphocytes, as documented previously [26].

Blood sampling

Five ml of venous blood was withdrawn in ethylenediamine tetraacetic acid (EDTA) sterile tubes and centrifuged at 2000 g for 3 min. Plasma samples were collected and stored at −80°C for subsequent cytokine analysis. Human peripheral blood mononuclear cells (PBMCs) were obtained as described previously by Talaat et al. [27] from heparinized blood by centrifugation through a Ficoll-Hypaque separating media (Biowest SAS, Nuaillé, France) for 30 min in 24°C at 400 g (Sorvall centrifuge; Thermo Scientific, Pittsburgh, PA, USA). The PBMCs were collected from the interface and washed twice with RPMI-1640 supplemented with L-glutamine (200 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) and 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) buffer (1 M) (all from Gibco Life Technology Co., Carlsbad, CA, USA).

Flow cytometric detection of Tregs, B and T lymphocytes, TCR and NK cells

For detection of CD4+CD25+FoxP3+ Treg cells in the isolated PBMCs, samples were enumerated using fluorescein isothiocyanate (FITC)-labelled monoclonal anti-CD4, allophycocyanin (APC)-labelled anti-CD25 and phycoerythrin (PE)-labelled monoclonal anti-FoxP3 (BD Bioscience, San Jose, CA, USA). PBMCs (0·1–0·2 × 106) suspended in 100 μl of complete RPMI-1640 culture media [RPMI-1640 supplemented with 10% fetal bovine serum (FBS)] were incubated with 10 μl of anti-CD4+ and anti-CD25+ antibodies for 20 min at room temperature in the dark. At the end of the incubation, cells were washed with 2 ml of fluorescence activated cell sorter (FACS) buffer [1% FBS, 0·01% NaN3 in phosphate-buffered saline (PBS); Sigma, St Louis, MO, USA)]. To fix the cells, 2 ml of buffer (A) were added then incubated in the dark for 10 min at room temperature. The cells were washed with 2 ml of FACS buffer then fixed using 500 μl buffer (C) for 30 min at room temperature in the dark. After washing with 2 ml of FACS buffer, 20 μl of anti-FoxP3+ antibody was added to the cells. After 30 min incubation in the dark, the cells were washed with 2 ml of FACS buffer and fixed with 500 μl of 1% paraformaldehyde (Sigma-Aldrich Chemie GmbH, Munich, Germany).

For detection of the remaining cell receptors (CD19, CD3, CD4, CD8 and CD16/56), 100 μl of PBMCs were incubated with 10 μl of FITC-labelled monoclonal anti-CD4+, anti-CD3+ and anti-TCR-γδ, PE-labelled monoclonal anti-CD8+, anti-CD19+ or anti-CD16+56+ (all from BD Bioscience, San Jose, CA, USA). After incubation in the dark for 20 min at room temperature, the cells were washed with 2 ml of FACS buffer then fixed with 500 μl of 1% paraformaldehyde.

In acquisition of 25 000 events in a lymphocyte gate, the percentages of CD19+ (B cells), CD3+ (T lymphocytes), CD4+ (Th cells), CD8+ (T cytotoxic), TCR-γδ (T cell receptor) and CD16+56+ (NK cells) were assessed. For the Treg phenotype, lymphogating followed by CD4+CD25+ gating by fluorescence was performed. A gating strategy by morphology, then side-scatter versus forward-scatter (FSC/SSC) was used to identify FoxP3+ Treg cells. In each sample, data (<250 000 events) were acquired with four-colour FACSCaliber flow cytometry (BD Biosciences) and were compensated off-line and analysed with FlowJo software (Tree Star, Ashland, OR, USA).

Measurement of plasma levels of IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF-α

Concentration of IL-6 was measured in the plasma of 35 ITP Egyptian patients and 10 healthy controls using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions. IL-4, IL-10, IFN-γ and TNF-α were estimated by in-house ELISA, as described previously by Talaat (2007) [27], with slight modifications. The intensity of the developed colour was measured by reading optical absorbance at 450 nm using a microplate reader (Sunrise; Tecan Group Ltd, Männedorf, Switzerland). The ELISA reader-controlling software (Softmax; Molecular Devices, Sunnyvale, CA, USA) processed the digital data of raw absorbance values into a standard curve from which the cytokine concentrations were derived. Results were expressed as picogram of cytokine per millilitre plasma (pg/ml).

Statistical analysis

All statistical analyses were performed using spss version 13 (SPSS, Inc., Chicago, IL, USA). Descriptive statistics including the mean and standard deviation (s.d.) for quantitative variables and number and percentage for qualitative variables were performed. For categorical variables, the statistical significances of case–control differences were tested by the χ2 test. The Kruskal–Wallis test (for subgroup analysis), the Mann-Whitney U-test (for unpaired analysis) and Spearman's correlation test were performed. In all tests the level of significance was set at P < 0·05.

Results

Clinical and laboratory characteristics of ITP patients versus healthy controls

The demographic and clinical data of all children studied are summarized in Table 1. Of the 35 ITP patients who were enrolled at the onset of the disease, 15 had acute ITP and 20 (10 female and 10 male) had chronic ITP, with an age range of 1·11–12 years (mean age 6·1 ± 3·2). Healthy control children were run in parallel, with an age range of 1·3–12·6 years (mean age 8·6 ± 3·9). None of our patients presented with enlarged liver, spleen or significant lymphadenopathy. ITP patients had a significant reduction in mean platelet count (P < 0·001) compared to control subjects, with maximum reduction in acute ITP (30 ± 31·4) rather than chronic (77·2 ± 44·6) subjects (P < 0·001).

Table 1. Baseline characteristics of immune thrombocytopenia purpura (ITP) and normal controls.
ParameterControl (n = 10)ITP (n = 35)Correlation with disease
  1. *P < 0·05; ***P < 0·001. Hb = haemoglobin; MPV = mean platelet volume; WBC = white blood cells; F/M = female/male; n.s. = not significant.
Age (years)6·8 ± 3·95·5 ± 3·2*n.s.
Sex (F/M)4/616/19n.s.
Petichaeal rash35/35
Brushing35/35
Epistaxis (mild)32/35
Preceding febrile illness 30/35
Splenomegaly0/35
Hepatomegaly 0/35
Duration of thrombocytopenia 
Less than 6 months 15/35 
More than 6 months 20/35 
Platelet count (×109/l)280·3 ± 46·456·9 ± 45·6***r = −0·901, P < 0·001
WBC count (×109/l)6·2 ± 1·69 ± 3·9*r = 0·32, P < 0·05
Hb (g/dl)11·1 ± 0·911·6 ± 1·2n.s.
MPV, fl11·4 ± 1·67·2 ± 5*r = −0·362, P < 0·05

Flow cytometric detection of Tregs, B and T lymphocytes and NK cells

Representative dot-plots of flow cytometry analysis of tested cells are shown in Fig. 1 and results are presented in Table 2. Regarding the lymphocyte subsets, the percentage of T lymphocytes (CD3+), Th cells (CD4+) and B lymphocytes (CD19+) was increased in ITP patients compared to controls. However, this elevation is statistically insignificant. The percentage of NK cells (CD16+56+) (P < 0·001), TCR-γδ, CD4+CD25+ (P < 0·05) and Treg (CD4+CD25+FoxP3) (P < 0·001) was decreased significantly in ITP patients in relation to healthy children. T cytotoxic (CD8+) cells exhibited almost the same percentage in ITP patients compared with healthy subjects.

Figure 1.

Flow cytometric detection of T lymphocytes, B lymphocytes, natural killer (NK) cells and CD4+CD25+highforkhead box protein 3 (FoxP3)+ regulatory T cells. Forward- and side-scatter histograms were used define the lymphocyte population. A1, A2 and A3: the expression of CD4+and CD8+ was assessed in the total lymphocyte population. B1, B2 and B3: the expression of CD3+ and CD19+ was assessed in total lymphocytes population. C1, C2 and C3: the expression of CD3+ and CD16+56+ was assessed in the total lymphocyte population. D1, D2 and D3: the expression of TCR-γδ was assessed in the total lymphocyte population. E1, E2 and E3: the expression of CD4+ and CD25+ in total lymphocytes was detected and different gates were performed to define CD4+CD25+high cells; F1, F2 and F3 show the percentage of CD4+CD25+highFoxP3.

Table 2. Lymphocyte subsets in patients with acute and chronic immune thrombocytopenia purpura (ITP) and normal controls.
ParameterControl (n = 10)ITP (n = 35)Acute (n = 15)Chronic (n = 20)
  1. aSignificance compared to controls. bSignificance compared to acute patients (
  2. *P < 0·05; **P < 0·01; ***P < 0·001). Treg = regulatory T cells.
CD3+%56·2 ± 2·662·3 ± 3·160·6 ± 5·163·9 ± 3·8
CD4+%34·4 ± 2·937·9 ± 2·437·3 ± 4·838·5 ± 2·4
CD8+%20·5 ± 2·320·9 ± 1·119·2 ± 1·622·2 ± 1·0
CD16+56+%4·3 ± 0·81·9 ± 0·3a***0·7 ± 0·2a***2·9 ± 0·5b***
CD19+%0·04 ± 0·010·07 ± 0·020·09 ± 0·060·06 ± 0·02b*
TCR-γδ5·9 ± 1·14·1 ± 0·44·1 ± 0·54·2 ± 0·6
CD4+CD25+%0·3 ± 0·030·2 ± 0·05a*0·4 ± 0·10·2 ± 0·03b*
Treg%77·9 ± 2·152·9 ± 3·1a***51 ± 4a***54·4 ± 4·5a**

As shown in Table 2, the highest percentage of B lymphocytes (CD19+) was reported in acute ITP followed by little reduction (P < 0·05) in chronic patients. A maximum reduction (P < 0·001) in NK cells (CD16+56+) was estimated in acute ITP patients compared to controls. A significant elevation in chronic patients (P < 0·001) was recorded in relation to acute patients. A statistically significant negative correlation (r = −838, P < 0·001) between change in NK cells and acute form of the disease was recorded. Although there was a reduction in the percentage of TCR (TCR-γδ) in acute and chronic ITP patients in relation to controls, it was not statistically significant. The percentage of total CD4+CD25+ was slightly increased in acute ITP in comparison with controls. In contrast, a statistically significant reduction in the percentage of CD4+CD25+ in chronic patients (P < 0·05) was demonstrated compared to their acute counterparts. The significant reduction of the percentage of Tregs was statistically significantly in acute (P < 0·001) and chronic (P < 0·001) ITP patients compared to controls. A negative correlation was reported between the percentage of Treg cells and the development of acute (r = −0·737; P < 0·01) and chronic ITP (r = −0·515; P < 0·01).

Plasma cytokine secretion levels

In Figure 2, the bar histogram shows that the levels of Th1 cytokines (IFN-γ and TNF-α) (P < 0·05 and P < 0·001) and Th2 (IL-4, IL-6 and IL-10) (P < 0·05, P < 0·05 and P < 0·001) were increased significantly in ITP patients compared to healthy controls. After classification of ITP patients into acute and chronic stages, most cytokines were increased in acute patients. TNF-α (P < 0·01), IL-4 (P < 0·05) and IL-10 (P < 0·001) were elevated statistically in relation to controls. A positive correlation was found between the acute form of ITP and IL-4 (r = 0·487, P < 0·05), IL-10 (r = 0·849, P < 0·001) and TNF-α (r = 0·611, P < 0·001). Chronic ITP patients produced more IL-6 than controls or acute patients (P < 0·05). The highest amounts of TNF-α were demonstrated in children with chronic ITP (P < 0·001). A positive correlation between the levels of IL-6 (r = 0·547, P < 0·01) and TNF-α (r = 0·727, P < 0·001) and progress to chronicity was demonstrated.

Figure 2.

Plasma levels of T helper type 1 (Th1) [interferon (IFN-γ) and tumour necrosis factor (TNF-α)] and Th2 [interleukin (IL)-4, IL-6 and IL-10] cytokines. Results are expressed as mean ± standard error. (a) Groups statistically significantly different from controls. (b) Groups statistically significantly different from acute immune thrombocytopenia purpura (ITP) patients.*P < 0·05; **P < 0·01; ***P < 0·001.

Discussion

ITP is one of the common causes of thrombocytopenia in adults and children [28]. This work was undertaken to investigate the change in the immunological picture (immune cells and cytokine secretion profile) in ITP patients with both acute and chronic forms. The mean age of acute ITP was 4·7 ± 3·1 years. Lanzkowsky [25] reported that acute ITP is most common at age 2–10 years, with a peak between 4 and 8 years. The mean age of chronic patients was 6·1 ± 3·2 years. It is clear that there was a significant reduction in the age of acute patients compared with chronic patients at the time of examination. This difference is due most probably to the chronic nature of ITP, as children with the chronic form of the disease are diagnosed for a much longer period of time, while children with acute ITP are usually diagnosed at a younger age [28].

Clinical features were present in all patients (100%) in the form of petichaeal rash and 91·4% (32 of 35) with epistaxis. It was reported previously that petichiae and ecchymosis were present in 99% of patients with childhood ITP and epistaxis was the most frequent haemorrhagic manifestation [29]. In agreement with Edslev et al. [30], our patients showed a significant decrease in platelet count in acute and chronic stages, with a maximum reduction in acute patients. A significant inhibition in mean platelet volume (MPV) (≈7 fl) in ITP patients has been documented previously by Ahmed et al. [31]. They reported that acute ITP patients had a MPV of less than 8 fl.

In the present study, the percentage of B cells (CD19+) was increased in ITP patients compared with controls, with little reduction in chronic ITP compared to acute patients. This might be expected, as B cells are ultimate producers of anti-platelet antibodies, and patients with ITP have an increased generation of anti-platelet antibodies [32]. Previous studies reported an increased frequency of anti-glycoprotein IIb–IIIa antibody-producing B cells in ITP patients [33-35].

In our study, the percentage of NK cells (CD16+56+) was reduced significantly in ITP patients compared to controls, with a maximum reduction in the acute form of the disease. Few studies have examined NK cells in ITP. French and Yokoyama [36] reported that there was a reduction in NK cell numbers and function with autoimmune diseases. It is not clear whether the NK cell alterations occur as a result of the disease or are involved in its pathogenesis [37, 38]. In addition, a temporal correlation has been identified between NK cell numbers or activity and periods of disease progression or remission in autoimmune diseases, suggesting that NK cells may play an immunoregulatory role in disease pathology [39, 40]. NK cells play an important role in B cell regulation function, as they have a suppressive effect on antibody production [41]. Semple et al. [42] found an association between NK cell activity defects and an increase in B cell numbers, with an elevation in anti-platelet Th activity leading to enhanced platelet destruction. However, they reported that it is unclear if the reduction in activity of NK cells plays a role in the autoimmune processes of ITP.

In our study, we observed disequilibrium in proportion of T lymphocyte subsets with excessive Th and almost the same level of Tc cells. Since 1980, it has been recognized that the CD4+/CD8+ ratio is increased in patients with autoimmune diseases. Although some studies have not found CD8+ T cell deficiency in patients with autoimmune diseases [43], or have attributed the deficiency to hormonal factors [44], CD8+ T cell deficiency would appear to be a general feature of human chronic autoimmune diseases. This was interpreted initially as a decrease in suppressor CD8+ T cells leading to uninhibition of autoimmune responses [45, 46], but attributed later to sequestration of CD8+ T cells in the target organ [47-49], because in some autoimmune diseases CD8+ T cells are selectively enriched compared to CD4+ T cells in the target organ [48, 50].

Concerning TCR-γδ, we noted that there is a reduction in its percentage in ITP patients. Most mature T cells express TCR-αβ and approximately 1–15% of peripheral blood lymphocytes express TCR-γδ. The possible role of γδ T cells in autoimmune diseases may be returned to their ability to recognize self-antigens. This has been discussed in studies concerning coeliac disease, multiple sclerosis, autoimmune thyroid diseases, autoimmune liver disease and systemic sclerosis. However, in the previous diseases the accumulation of TCR-γδ cells occurred predominantly in pathologically changed tissues; their increase in peripheral blood of the same patients was less evident, or even unnoticeable [51].

In patients with ITP, the breakdown of self-tolerance may be the result of a number of non-mutually exclusive mechanisms, such as a failure of central tolerance leading to the abnormal accumulation of self-reactive T cells [52]. CD4+ Tregs play a critical role in the maintenance of peripheral tolerance by both directly and indirectly suppressing the activation and proliferation of many cell types, including T, B, dendritic, NK and NK T cells in vivo and/or in vitro [53]. Because of their ability to control homeostasis and immunopathology [54], the level of Tregs and their function are among the most informative criteria of a patient's immune status. Tregs are characterized by high expression of the CD25 molecule (the IL-2 receptor α-chain) and expression of the transcription factor FoxP3 and make up 5–10% of the normal peripheral CD4+ T cell population [55, 56]. As with a number of other autoimmune diseases, recent studies in patients with ITP have shown reduced levels of FoxP3 mRNA [57] and protein [58] in circulating mononuclear cells and abnormal Treg function in spleen biopsies [59]. The FoxP3 transcription factor can effectively distinguish Tregs from activated CD4+ T cells [60]. Although some FoxP3+ Tregs can be generated in peripheral lymphoid organs by particular modes of antigen stimulation [38], FoxP3 expression is considered to be a unique marker of Tregs [61].

In our study, there was a significant decrease in CD4+CD25+ percentage in ITP patients, with a greater reduction in chronic than acute patients who showed elevation in their percentage compared to controls. These data disagreed with Fahim and Monir [28], who found a significant decrease in CD4+CD25+ in acute than in chronic patients. Zahran and Elsayh [6] found a significant decrease in CD4+CD25+high in both types of ITP patients than in controls. Our results agreed with theirs, as the percentage of CD4+CD25+high FoxP3+ in ITP patients was decreased significantly in both types of ITP patients compared to controls. In accordance with our results, Stasi et al. [62] found that circulating Tregs were lower in ITP patients than in control subjects. Supporting our data, previous reports indicate that ITP patients with active disease have a reduced percentage of Treg cells [23, 24, 63, 64]. In contrast to our data, Mazzucco et al. [65] demonstrated that there was no significant difference in Treg counts between patients and controls at any point during the course of the study and no difference in Treg counts between the chronic and non-chronic groups.

Several cytokine abnormalities have been reported to be associated with ITP [66]. We observed that the concentration of Th1 and Th2 cytokines were significantly different within patient groups and between patients and controls. Th1/Th2 balance is known to regulate the immune system under normal conditions and it always refers to this by the balance between IL-4 and IFN-γ. In our study, we found a high ThI/Th2 ratio. These results are in agreement with the data reported previously by Wang et al. [67]. The Th1/Th2 balance has been broken in ITP patients, as Th1 cytokines including IFN-γ, TNF-α and IL-2 are involved in cell-mediated inflammatory reaction, delayed hypersensitivity and activate cytotoxic reaction, while Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13, which increase production of antibodies.

We observed an increase in the secretion of various cytokines, including IFN-γ, TNF-α, IL-4, IL-6 and IL-10. This could be related to activation of macrophages, which have been reported to be stimulated in ITP patients by platelet autoantigen and lead to activation of T cells. More IL-4, IL-10, IFN-γ and TNF-α secretion was induced by activated T cells, which may lead to the activation of B cells [8], a remarkable feature of ITP [32].

There was a significant increase in IL-4 in ITP patients with maximum elevation in the acute stage of the disease. This result is in agreement with Webber et al. [68], who reported that IL-4 plays an important role in autoantibody production. Moreover, they stressed the essential role of IL-4 for T-dependent B cell differentiation and isotype-switching to several IgG isotypes. Also, the increase in Th2 cytokine (IL-4 and IL-10) levels can affect differentiation and survival of pathogenic B cells in ITP patients. This increase is supported by the increase of the percentage of T cells reported in our patients (as the main source of IL-4 is Th2 cells).

In our study we found that there was a significant increase in IL-6 levels, with maximum elevation in chronic ITP patients. These data are in agreement with Andersson [69]. This may be expected, as IL-6 is involved in the production of platelets by activation of megakaryocytes to produce platelets. Furthermore, IL-6 has an inhibitory effect on Treg cells [70], which could explain the reduction of Tregs coinciding with the elevation of IL-6 in ITP patients. Moreover, the elevation of IL-6 coincides with the elevation of Th17 observed previously in ITP patients [71, 72].

We also found a significant increase in TNF-α in patients with acute and chronic ITP. These results are in accordance with Andersson [70] and Malinowska et al. [2], who reported that the increase of TNF-α induces the activation of macrophages. Additionally, it induces the release of IL-6, which is an important stimulator of megakaryopoiesis and thrombopoiesis; thus, this increase could be due to the activation of both macrophages and T cells [8]. We observed a significant elevation of IL-10 in both groups of ITP patients, which is in accordance with Del Vecchio et al. [73] and Zhou et al. [74], who reported that IL-10 has potent immunostimulatory effects on B cells and is an important immunoregulatory cytokine produced mainly by monocytes and T lymphocytes.

A significant increase in IFN-γ in our ITP patients is supported by the data presented by Del Vecchio et al. [73], who reported an increase in IFN-γ in ITP patients. Andersson et al. [75] suggest that patients with active disease may have a Th1-cytokine activation pattern and, when in remission, the cytokines are skewed to a Th2 pattern. An increase in T cells in our ITP patients might improve the elevation of IFN-γ (as IFN-γ is secreted by both Th and Tc cells). Moreover, the elevation of IL-17 and IFN-γ may be an important dysregulation of cellular immunity in paediatric ITP patients [72].

Conclusions

Taken together, our study demonstrates that a high frequency of CD4+ helper T cells and B cells and a low frequency of NK cells may have prognostic significance in ITP patients. Estimation of CD4+CD25+HighFoxP3+ T cells may imply a fundamental role for Treg cells in the pathogenesis of ITP. The demonstrated dysfunction of Tregs may contribute to the loss of tolerance. In general, our cytokine production data prove that, in addition to Th1 activation, activated macrophages may play a potential role in ITP patients. Hopefully, a better understanding of the role of these T cell subsets will permit better control of autoimmunity through manipulation of their cytokine network, although there are shortcomings in the current study in that it depended upon a small number of patients and controls, making the elucidation of some results a difficult and onerous task.

Acknowledgements

The authors would like to express their appreciation to Dr Iman fathy Fath-elbab, Chimes in VACSERA, Egypt, for her unlimited help throughout the work. We also would like to acknowledge Dr Maha Yosof, Assistant Professor of Haematology, for her help and support throughout the study. Deep thanks to the nursing staff of the Haematology Clinic of Alexandria University Hospital.

Author contributions

R. T. and A. E. conceived and designed the study; R. T. and A. E. provided the study materials; S. S. B. provided the study patients; A. E. collected and assembled the data; R. T. and A. E. provided the data analysis and interpretation. All authors contributed to writing the manuscript and all authors gave final approval of the manuscript.

Disclosures

All authors declare that there are no conflicts of interest with other people or organizations.

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