Introduction

Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder manifested by antiplatelet autoantibody-mediated thrombocytopenia. Like in many other autoimmune diseases, autoantibody production by B cells in ITP needs the help of T cells, as evidenced by its association with both T cell activation and T–B cognate interaction [1–4]. Almost all of these autoantigen-specific T cells are CD4+ T helper (Th) cells, which may play very important roles in the pathogenesis of ITP [5–9]. Recent investigations have substantiated a Th1 polarization of the immune response in ITP [10, 11].

Although the best therapeutic approach is not yet unanimous, prednisone or prednisolone has been widely recognized as the most appropriate front-line therapy for most ITP patients who need to be managed. In recent years, some reports showed that high-dose dexamethasone (HD-DXM) might be a promising alternative to prednisone as the first- or second-line treatment in ITP patients [12–14]. In this study, oral HD-DXM was used in a single 4-day course as the initial treatment schedule in previously untreated ITP patients with active disease, and the plasma Th1, Th2, and Th3 cytokines were profiled in these patients before and after HD-DXM treatment.

Materials and Methods

Patients

Fifty-two ITP patients with active disease (34 females and 18 males, age range 13–73 years, median 39 years) were enrolled in this study. Enrollment took place between April 2003 and January 2005 at the Hematology Department of Qilu Hospital and the Second Hospital affiliated to Shandong University, Jinan, China. The patients’ platelet count ranged between 1 and 49 × 109/l, with a median count of 13.5 × 109/l, all required treatment because of clinically significant bleeding. All of the cases met the diagnosis criteria of chronic ITP as previously described [15, 16]. None of them had been treated with glucocorticosteroid previously. Patients complicated with diabetes, hypertension, cardiovascular diseases, pregnancy, active infection, or connective tissue diseases, such as systemic lupus erythematosus, were excluded.

The control group consisted of 20 adult healthy volunteers (13 females and 7 males, age range 22–51 years, median 31 years). Platelet counts were ranged from 150 to 342 × 109/l, with the median count of 207 × 109/l.

The study was approved by the Medical Ethical Committee of Qilu Hospital and the Second Hospital of Shandong University. Informed consent was obtained from each patient before being included in the study.

Treatment Regimen

Dexamethasone (DXM) 40 mg/day was administered orally for four consecutive days. No maintenance or other treatment modality was used. Initial response evaluation was made at the end of the second week after treatment initiation. The response was evaluated according to the following criteria: complete response (CR) defined as platelet count ≥150 × 109/l; partial response (PR) defined as platelet count ≥50 < 150 × 109/l; minimal response (MR) defined as platelet count ≥30 < 50 × 109/l, and cessation of bleeding; no response defined as platelet count <30 × 109/l or persistence of bleeding symptoms related to thrombocytopenia; and sustained response (SR) was defined as platelet count remained ≥30 × 109/l and cessation of bleeding in 6 months of follow-up, otherwise it was defined as relapse.

Laboratory Assay

Complete blood counts were performed at recruitment, on day 15 after HD-DXM treatment, and during follow-up period. Bone marrow aspiration was performed routinely at recruitment. Blood glucose levels were measured at recruitment and after treatment with HD-DXM.

Cytokine Assay

All of the plasma samples were isolated from 10-ml ethylenediaminetetraacetic acid (Pharmacia, USA)-anticoagulated peripheral blood by centrifuging at 2,000 rpm at room temperature (RT) for 20 min twice, and then stored at −20°C for future use. Plasma samples were obtained before therapy and 2 weeks after therapy. In addition, out of all patients’ posttreatment, 33 cases including 12 SR and 21 relapsed cases, respectively, were also examined of the plasma cytokine levels at the median time of 6 months during following-up.

Plasma levels of IFN-γ, IL-2, IL-4, IL-10, and TGF-β1 were measured by enzyme-linked immunosorbent assay (ELISA, kits from R&D Systems Inc, USA) in 96-well plates for both the test group and the standard group, whereas the standard group and the blank were duplicated following instructions of the assays. All reagents, standards, and samples were prepared according to the manufacture’s instructions. Microplate strips were removed from the plate frame, and 100 μl of Assay Diluent RD1S was added to each well except blank contrast ones, and incubated for 2 h at RT. A plate layout was provided to record the standards and samples assayed. Followed by aspirating and washing with Wash Buffer (Sigma, USA) for four times. Two hundred microliters of conjugate was added to each well and incubated for 2 h at RT, then aspirated and washed as above. As followed, 200 μl of substrate solution was added to each well and incubated for 30 min at RT while protected from light. Finally, 50 μl of stop solution was added to each well and data was read at 450 nm with automatic ELISA analysis apparatus (version 5.0.1 Build 52 software system, BIO-RAD Systems Inc., USA).

Statistical Analysis

Data were expressed as mean ± SD. The differences between two groups and patients before and after treatment were evaluated using unpaired and paired Student t test, respectively. All tests were performed by SPSS 13.0 system.

Results

Clinical Therapeutic Effect of HD-DXM

Responses were reached in all patients: CR in 29 (55.77%), PR in 19 (36.54%), and MR in 4 (7.69%). Median platelet count was 152.5 × 109/l (min–max 41–387), and only four patients had a platelet count of less than 50 × 109/l after HD-DXM treatment (Table I). No bleeding or other obvious complications was observed throughout the treatment.

Table I Clinical Characteristics and Initial Responses of ITP Patients
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Side effects

No serious side effects of glucocorticosteroids occurred, such as metabolized abnormality of multiple systems and withdrawal symptoms; blood glucose level increased temporarily in two patients and insomnia occurred in two patients during short-term DXM therapy, therefore HD-DXM therapy was well tolerated.

Follow-up

The median follow-up period was 6 months (range 5.5–12 months). Among all of the 52 patients, 24 (46.15%) had SR with platelet count of more than 30 × 109/l after a single course of HD-DXM without any further therapy during follow-up. The remaining 28 patients (53.85%) had a relapse within 6 months that required additional treatment.

Cytokine Changes in ITP Patients

Compared with the normal controls, in ITP patients with active disease, the plasma levels of both IFN-γ and IL-2 were increased significantly (P < 0.01). On the contrary, IL-4, IL-10, and TGF-β1 levels were considerably decreased (P < 0.01) (Table II).

Table II Plasma Cytokine Levels in ITP Patients’ Pre- and Posttreatment, SR, and Relapse
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Cytokine Profile Correction After Treatment

Both of IFN-γ and IL-2 levels were decreased significantly (P < 0.01) and normalized after HD-DXM treatment. The levels of both IL-4 and IL-10 were increased significantly (P < 0.01) after treatment. There was no significant difference between the treated patients and the normal controls (P > 0.05). TGF-β1 was increased significantly (P < 0.01) after treatment, but was still lower than that of the normal controls (P < 0.05) (Table II).

The cytokine profiles in the SRs remained stable compared to the posttreatment level (P > 0.05). On the contrary, IFN-γ and IL-2 levels raised up, and IL-4, IL-10, and TGF-β1 levels reduced again in the relapsed patients (P < 0.01) (Table II).

Discussion

In our study, all of the patients exhibited initial responses to 4 days of HD-DXM treatment. Remarkably, more than half of the patients (55.77%) reached CR, while only four patients (7.69%) had posttreatment platelet counts of less than 50 × 109/l. Even more remarkable, 46.15% of the ITP patients obtained SR that did not need additional treatment during the 6-month follow-up. The patients’ compliance, efficacy, and safety profile of the 4-day HD-DXM treatment in the present study was satisfactory compared with usual prednisone or prednisolone scheme, supporting the proposal of HD-DXM as an alternative initial therapy for ITP [12–14, 17, 18].

It has been described that depletion of CD8+ T cells with anti-CD8 monoclonal antibodies and complement did not reduce the proliferative capacity of the responding peripheral blood mononuclear cells from patients with ITP, indicating that CD4+ T helper cells may be responsible for the response [5]. A recent study assessed the intracellular IL-4 and IFN-γ production in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin in patients with ITP, and found that the Th1/Th2 ratio in the untreated group was significantly higher than that in the control group [10]. In accordance with previous reports [10, 19, 20], the plasma levels of IFN-γ, IL-2, IL-4, and IL-10 in the patients included in the present study further implied a Th1-dominated cytokine profile in ITP with active disease. In addition, our data show that TGF-β1 concentration varied inversely with the disease activity; the significantly lower level of TGF-β1 observed in this cohort should be associated with a downregulated Th3 response in active ITP patients, but it significantly elevated after HD-DXM management, so this suggest that Th3 may play an important role in bystander immune suppression in ITP [21–23].

More interestingly, we found that HD-DXM therapy for ITP could cause a shift in the Th1/Th2 cytokine balance to the same levels as normal controls, leading to a more balanced Th1/Th2/Th3 cytokine profile response in vivo [10, 24, 25]. Glucocorticoids (GC) were known to affect cytokine synthesis in T cells by binding to and activating cytoplasmic GC receptors. The receptor–corticosteroid complex then translocates to the nucleus, where it regulates the transcription of target genes through several mechanisms [4, 26, 27]. GC may directly inhibit Th1 cytokine production in T cells and potentially enhance Th2 cytokine synthesis by inhibiting IL-12 production in antigen-presenting cells [28]. The HD-DXM-mediated Th1/Th2 cytokine profile alterations observed in this study could be the results of a downregulation of Th1 cytokines while permitting the production of Th2 cytokines. The precise mechanisms of HD-DXM await further elucidation. Furthermore, our data displayed that in the patients who maintained the response could also sustain their cytokine profiles very much resembling their posttreatment pattern; however, the cytokine profiles tended to get back to the baseline values in the relapsed patients. These data indicated that relapsed ITP rooted in recurring of the Th cytokine imbalance, and may need repeated or additional treatment.

In summary, our results support that ITP is a Th1-predominant autoimmune disease and a 4-day course of HD-DXM is an effective and safe initial therapy for ITP. The Th1 polarization could be corrected by HD-DXM therapy, which might be a chief mechanism of HD-DXM immunotherapy for ITP.