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 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 38  |  Issue : 4  |  Page : 136-140

Clinical value of B-cell-activating factor receptor in childhood idiopathic thrombocytopenic purpura


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

Date of Submission06-Apr-2013
Date of Acceptance16-Jul-2013
Date of Web Publication19-Jun-2014

Correspondence Address:
Wafaa A. El-Neanaey
MD, Department of Clinical Pathology, Alexandria University, 22314 Alexandria
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000434283.56505.a9

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  Abstract 

Background

B cells play a key role in the pathogenesis of autoimmune diseases by secreting autoantibodies, presenting antigens, and secreting cytokines. Activation of autoreactive B cells is largely mediated by a cytokine called B-cell-activating factor (BAFF), which mediates its signal mainly through BAFF-R. The present study aimed to clarify the impact of BAFF/BAFF-R dysregulation on disease activity and response to therapy in childhood thrombocytopenic purpura (ITP).

Materials and methods

Forty-three patients (23 acute and 20 chronic ITP) and 20 age-matched and sex-matched healthy controls were enrolled in this study. Serum BAFF was determined using the enzyme-linked immunosorbent assay technique and the expression of BAFF-R on CD19+ B cells was detected by flow cytometry.

Results

In serum, BAFF level and BAFF-R expression were significantly higher in both acute and chronic ITP patients than in the controls (P⩽0.001). No significant difference was found in the BAFF level and BAFF-R expression between acute and chronic ITP (P=0.456 and 0.124, respectively). After treatment with immunosuppressive therapy, BAFF level and BAFF-R expression were significantly decreased in acute ITP compared with levels before treatment (P⩽0.001). No significant difference was found in the serum BAFF level, whereas BAFF-R expression was significantly higher in active ITP than inactive ITP (P=0.544 and 0.015, respectively).

Conclusion

Our data suggest that BAFF-R is more relevant than serum BAFF as an activity biomarker in ITP patients. It will be interesting to assess the possible implications of this upregulation of BAFF-R on BAFF-targeted therapy in ITP patients, especially those with active disease.

Keywords: B-cell-activating factor, flow cytometry, thrombocytopenic purpura


How to cite this article:
El-Neanaey WA, Swelem RS, Nazir HF. Clinical value of B-cell-activating factor receptor in childhood idiopathic thrombocytopenic purpura. Egypt J Haematol 2013;38:136-40

How to cite this URL:
El-Neanaey WA, Swelem RS, Nazir HF. Clinical value of B-cell-activating factor receptor in childhood idiopathic thrombocytopenic purpura. Egypt J Haematol [serial online] 2013 [cited 2019 Dec 15];38:136-40. Available from: http://www.ehj.eg.net/text.asp?2013/38/4/136/134780


  Introduction Top


Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which the patient’s immune system is activated by platelet autoantigens, resulting in immune-mediated platelet destruction and/or suppression of platelet production 1. The autoantibodies produced by autoreactive B cells against self-antigens, specifically IgG antibodies against GPIIb/IIIa and/or GPIb/IX, are considered to play a crucial role 2. In addition, several abnormalities involving the cellular mechanisms of immune modulation, such as the decreased number or defective suppressive function of regulatory T cells and the platelet destruction by cytotoxic T cells, have been described 3,4.

B-cell-activating factor (BAFF) is part of the tumor necrosis factor family. It is essential for B-cell development, survival, immunoglobulin production, and T-cell costimulation 5–7.

BAFF binds to three receptors: B-cell maturation antigen, transmembrane activator and calcium-modulating cyclophilin ligand interactor, and BAFF receptor (BR3/BAFF-R) 8,9.

BAFF-R is identified as the crucial receptor for B-cell survival and is expressed on a wide range of B-cell subsets, including immature, transitional, mature, memory and germinal center B cells, as well as on plasma cells 10. Excess amounts of BAFF result in the rescue of self-reactive B cells from anergy. Therefore, BAFF overexpression alters immune response in the periphery and predisposes to the development of autoimmune disease 11. In accordance with such data, some studies have reported elevated serum levels of BAFF in various autoimmune diseases 12–15. However, it is not clear whether B-cell activation in ITP is only because of an increase in BAFF or could also be modulated by the expression of BAFF-R. To our knowledge, the data on BAFF-R in ITP are very limited. The present study aimed to clarify the impact of BAFF/BAFF-R dysregulation on disease activity and response to therapy in children with ITP.


  Materials and methods Top


The present study was carried out on 43 pediatric patients with ITP (23 acute and 20 chronic) admitted to the Hematology unit of Alexandria University Children’s hospital. In addition, 20 age-matched and sex-matched healthy controls were included. At the time of sampling, all acute ITP and eight of chronic ITP had active ITP (31 patients) with platelet count less than 50×109/l and the remaining 12 chronic ITP patients were inactive with platelet count of at least 50×109/l. All acute ITP patients were indicated for immunosuppressive therapy according to the evidence-based practice guidelines of the American Society of Hematology 16.

Serum BAFF and BAFF-R expressions were determined before and after treatment in acute ITP. Chronic ITP patients did not receive immunosuppressive therapy at least 6 months before starting the study and they were assessed only once on admission.

Informed consent was obtained from all patients and the study was approved by the Ethics Committee of Faculty of Medicine, Alexandria University.

All patients enrolled in the study were subjected to the following:

  1. Full assessment of history and thorough clinical examination.
  2. Two-laboratory investigations including:
    1. Complete blood picture using a Sysmex XT-1800 automated blood cell counter (Siemens Health Care Diagnostics Inc., Newark, Delaware, USA).
    2. Bone marrow examination (for patients only).
    3. Measurement of serum BAFF by the enzyme-linked immunosorbent assay technique using human soluble BAFF/BLYS/TNFSF13B immunoassay (R&D System, Minneapolis, Minnesota, USA) 17.
    4. Detection of BAFF-R (PE-CD268), clone: 11C1 (Biolegend, San Diego, California, USA) and FITC-CD19, clone: HD37 (Dako, Glostrup, Denmark) using Miltenyi Biotec (Miltenyi Biotec Inc., California, USA), MACS Quant flow cytometer Analyzer equipped with MACS Quantify software version 2.3, USA 18,19.


Sampling

Venous blood was collected into two vacutainer tubes: the EDTA tube for complete blood picture and flow cytometry and the plain tube for serum BAFF level estimation. Clotted blood samples were centrifuged at room temperature for 30 min and separated serum was stored in small aliquots at −70°C until tested for BAFF.

Flow cytometric analysis of BAFF-R (CD268)

A volume of 100 μl of whole EDTA blood was mixed with 10 μl of CD268 MoAb and incubated for 10 min at room temperature. Two milliliters of lysing solution was added, mixed, and left for 10 min in the dark, then centrifuged for 5 min at 2000 rpm, and the supernatant was discarded. The cells were washed twice with PBS for 2 min each at 2000 rpm.

After the last wash, the cells were analyzed on a flow cytometer. During analysis, a gate was set around the required cell population that was positive for CD19.

Statistical analysis

The data were tabulated and statistically analyzed using Microsoft Excel 2007 and the statistical package for social sciences (SPSS Inc., Chicago, Illinois, USA).


  Results Top


The demographic data of all ITP patients are presented in [Table 1].
Table 1: Demographic data of ITP patients under study

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The mean values of serum BAFF levels were significantly higher in ITP patients than in controls (1415.57±269.35 vs. 1168.70±213.94, P<0.001 for acute ITP and 1510.30±213.68 vs. 1168.70±213.94, P<0.001 for chronic ITP). Similarly, BAFF-R levels were significantly higher in ITP patients than in controls (13.99±4.22 vs. 7.79±1.96, P<0.001 for acute ITP and 15.61±7.60 vs. 7.79±1.96, P<0.001 for chronic ITP). However, no significant difference was observed in the mean values of BAFF and BAFF-R between acute ITP and chronic ITP (1415.57±269.35 vs. 1510.30±213.68, P=0.456 for BAFF and 13.99±4.22 vs. 15.61±7.60, P=0.124 for BAFF-R). The BAFF level did not correlate with platelet count either for acute or for chronic ITP (r=−0.037, P=0.867 and r=0.311, P=0.094, respectively). However, BAFF-R expression was inversely correlated with platelet count in chronic ITP (r=−0.636, P=0.00). After immunosuppressive therapy, the mean values of BAFF and BAFF-R were significantly decreased in acute ITP compared with those before treatment (1118.80±283.83 vs. 1415.57±269.35, P=<0.001 for BAFF and 11.28±4.992 vs. 13.99±4.22, P=<0.001 for BAFF-R). In terms of disease activity, serum BAFF level was not significant between active and inactive ITP (P=0.544), whereas BAFF-R expression was significantly higher in active than in inactive ITP (P=0.015) [Table 2] and [Figure 1] and [Figure 2].
Figure 1: BAFF-R expression in ITP patients. BAFF, B-cell-activating factor; ITP, idiopathic thrombocytopenic purpura.

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Figure 2: BAFF-R expression in healthy controls. BAFF, B-cell-activating factor.

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Table 2: Mean values of BAFF and BAFF-R in different subgroups of ITP patients and controls

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  Discussion Top


B cells play a key role in the pathogenesis of autoimmune diseases by secreting autoantibodies, presenting antigens, and secreting cytokines. BAFF is a crucial regulator of B-cell survival and activation and mediates its signal mainly through BAFF-R, but the data on BAFF-R in ITP are very limited. In agreement with Zhou and colleagues, this study showed that serum BAFF level was significantly higher in both acute and chronic ITP patients compared with controls.

However, the mean values of serum BAFF were not significantly different between acute and chronic ITP 20. The exact mechanism underlying the relationship between excess BAFF and immune dysfunction of ITP was studied by Zhu and colleagues. They reported that the BAFF level was not associated with antiplatelet antibodies and the addition of recombinant human BAFF did not promote the production of autoantibodies in vitro. They suggested that excessive BAFF may play a pathogenic role in ITP by increasing the survival of CD19+ and CD8+ cells and increasing the apoptosis of platelets through cytotoxic T lymphocyte-mediated platelet lysis 21. Similarly, Zhou and colleagues found that BAFF levels were not correlated with platelet-associated immunoglobulins or serum immunoglobulins. They suggested that the immunologic role of BAFF in ITP may be to alter T-cell-mediated immune response other than through direct B-cell survival regulation 20. At the transcriptional level, Zhou and colleagues found that there was no statistically significant difference in BAFF-R mRNA expression between ITP patients and controls. Moreover, no statistically significant correlation was found between BAFF and BAFF-R mRNA levels 20.

In contrast, our study showed that flow cytometric expression of BAFF-R on peripheral B cells was significantly higher in both acute and chronic ITP patients than controls. Together with the correlation that we found between serum BAFF level and BAFF-R expression in acute ITP, we suggest a direct effect of BAFF on BAFF-R regulation through a post-transcriptional mechanism 20.

The present study showed that serum BAFF level and BAFF-R expression were significantly decreased in acute ITP after treatment with immunosuppressive therapy including corticosteroids and intravenous immunoglobulin. Previously, it has been shown that the BAFF promoter is controlled by nuclear factor-κB. Emmerich et al. 22 and Auphan et al. 23 suggested that immunosuppressive therapy including corticosteroids and methotrexate substances may act indirectly on BAFF expression by downregulation of these factors. However, it cannot be excluded that certain drugs bind directly to the BAFF promoter, thereby suppressing its activity 24,25.

Similar to Zhou et al. 20 we found that serum BAFF levels were not statistically different between active ITP and inactive ITP.

However, Zhu et al. 21 and Emmerich et al. 22 observed that serum BAFF levels were significantly higher in active ITP than inactive ITP and ITP in remission.

Our interesting finding is that BAFF-R expression not serum BAFF level was significantly higher in active ITP compared with inactive ITP and this finding has not been reported before.

These results suggest that BAFF-R is overexpressed during disease activity to incorporate the soluble form of BAFF. Accordingly, this will decrease the free soluble form of BAFF, which can be detected by immunoassay, and explains why we could not detect a high level of BAFF in active ITP.


  Conclusion Top


Our data suggest that BAFF-R is more relevant than serum BAFF as an activity biomarker in ITP patients. It will be interesting to assess the possible implications of this upregulation of BAFF-R on BAFF-targeted therapy in ITP patients, especially those with active disease.[25]



 
  References Top

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17.Pottler L, Bendaoud B, Youlnou P, Olivler Pers J, Darldon C.New ELISA for B cell-activating factor.Clin Chem2009;55:1843–1851.  Back to cited text no. 17
    
18.Thompson JS, Bixler SA, Qian F, Vora K, Scott ML.BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.Science2001;293:2108–2211.  Back to cited text no. 18
    
19.Pezzutto A, Dörken B, Rabinovitch PS, Ledbetter JA, Moldenhauer G, Clark EA, et al..CD19 monoclonal antibody HD37 inhibits anti-immunoglobulin-induced B cell activation and proliferation.J Immunol1987;138:2793–2799.  Back to cited text no. 19
    
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22.Emmerich F, Bal G, Barakat A, Milz J, Muhle C, Gamboa LM, Salma A.High-level serum B-cell activating factor and promoter polymorphisms in patients with idiopathic thrombocytopenic purpura.Br J Hematol2006;136:309–314.  Back to cited text no. 22
    
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24.Wang CY, Zhu XJ, Hou M, Shi Y, Peng J, Sun JZ, et al..Effect of high – dose dexamethasone on BAFF and T regs in patients with immune thrombocytopenic purpura.Zhonghua Xue Ye Xue Za Zhi2010;3:164–167.  Back to cited text no. 24
    
25.Zhu XJ, Shi Y, Sun JZ, Shan NN, Peng J, Guo CS, et al..High – dose dexamethasone inhibits BAFF expression in patients with immune thrombocytopenia.J Clin Immunol2009;29:603–610.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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