|Year : 2016 | Volume
| Issue : 3 | Page : 116-120
Role of interleukin-27 in immune thrombocytopenic purpura and its impact on disease response
Hoda A Gad Allah, Mohammed M Moussa, Amro M.S. El-Ghammaz MS , Basma S.M. Ali
Clinical Hematology, Oncology and Bone Marrow Transplantation Unit, Internal Medicine Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||15-May-2016|
|Date of Acceptance||09-Jun-2016|
|Date of Web Publication||27-Dec-2016|
Amro M.S. El-Ghammaz
37 Mohamed Korayem Street, Nasr City, Cairo
Source of Support: None, Conflict of Interest: None
Background Aberrant cytokine profiles play important roles in immune thrombocytopenia (ITP) pathogenesis. Interleukin-27 (IL-27) has pleiotropic immunomodulatory effects. However, the role of IL-27 in ITP and its impact on disease response are still controversial.
Patients and methods This study included 60 adult ITP patients [20 de-novo patients (group 1), 20 with complete response (CR) to corticosteroids (group 2), and 20 with refractory ITP (group 3)]. Serum IL-27 level was assessed in all patients using enzyme-linked immunosorbent assay.
Results The mean IL-27 for all patients was significantly higher than that for controls (P<0.001). There were significant differences in mean IL-27 levels between group 1 and group 2 (P=0.002) and between group 2 and group 3 (P=0.030). There was a significant negative correlation between IL-27 level in all studied patients and platelet count (P=0.003). A serum level of IL-27 of 32 pg/ml had a sensitivity of 100% and specificity of 90% in differentiating de-novo ITP patients from healthy controls as detected by means of receiver operating characteristic curve. Moreover, a serum level of IL-27 of 72.5 pg/ml had a sensitivity of 65% and specificity of 75% in differentiating refractory patients from those with CR.
Conclusion Serum IL-27 is significantly elevated in ITP patients (de novo, in CR, and refractory) and it can be used as a predictor for disease occurrence and to a lower extent for its responsiveness to corticosteroid therapy.
Keywords: immune thrombocytopenic purpura, interleukin-27, prediction, response state
|How to cite this article:|
Gad Allah HA, Moussa MM, El-Ghammaz AM, Ali BS. Role of interleukin-27 in immune thrombocytopenic purpura and its impact on disease response. Egypt J Haematol 2016;41:116-20
|How to cite this URL:|
Gad Allah HA, Moussa MM, El-Ghammaz AM, Ali BS. Role of interleukin-27 in immune thrombocytopenic purpura and its impact on disease response. Egypt J Haematol [serial online] 2016 [cited 2020 Aug 13];41:116-20. Available from: http://www.ehj.eg.net/text.asp?2016/41/3/116/196177
| Introduction|| |
Primary immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by immune-mediated accelerated platelet destruction and suppressed platelet production and resulting in isolated thrombocytopenia . It has a significant incidence of about 3.3 per 100 000 adults/year . ITP is rarely fatal. Adult ITP typically has an insidious onset and rarely resolves spontaneously . Concepts surrounding the mechanisms of thrombocytopenia in ITP have shifted from the traditional view of increased platelet destruction mediated by autoantibodies to more complex mechanisms in which impaired platelet production, T-cell-mediated effects, and disturbed cytokine profiles play a role ,.
Interleukin-27 (IL-27), a heterodimeric cytokine composed of Epstein–Barr virus-induced gene 3 (Ebi3) and IL-27p28, belongs to the IL-6/IL-12 family cytokines. Its receptor is composed of gp130 and IL-27 receptor α chain (IL-27Rα, also known as WSX1 or TCCR) that activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway and the mitogen-activated protein kinase (MAPK) pathway . IL-27 is a cytokine with both proinflammatory and anti-inflammatory effects. There is accumulating evidence suggesting various roles of IL-27 in autoimmune diseases in humans (e.g. rheumatoid arthritis, Crohn’s disease, psoriasis, and multiple sclerosis) . Many studies focused on the role of IL-27 in ITP ,,. However, the role of IL-27 in ITP and its impact on disease response have not been fully elucidated and are still controversial. In this study, we assessed the serum level of IL-27 in adult ITP patients at diagnosis and in different states of response in comparison with healthy controls to evaluate its impact on treatment response.
| Patients and methods|| |
This study comprised 60 adult ITP [20 de-novo patients (group 1), 20 patients with complete response (CR) to corticosteroids (group 2), and 20 patients with refractory ITP (group 3)]. Twenty age-matched and sex-matched controls were also included in the study. All patients were diagnosed according to the diagnostic criteria of ITP described in the international consensus report published in 2010 . The patients were enrolled in this study between March 2015 and January 2016. The study excluded patients with acute or chronic inflammation, associated autoimmune disease and/or malignancy, patients with Evans syndrome, and patients with secondary ITP.
CR was defined as a platelet count of at least 100×109/l measured on two occasions more 7 days apart and the absence of bleeding . Refractory ITP was defined as failure of achievement of response to initial treatment with glucocorticoids and also to splenectomy together with the presence of a risk for bleeding that in the opinion of the attending physician requires therapy .
Serum interleukin-27 level assessment
Serum level of IL-27 was assessed using IL-27 subunit α ELISA kit (E0385h, EIAab, Wuhan, China). Briefly, the microtiter plate provided had been precoated with an antibody specific to IL-27 subunit α. The samples were then added to the microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IL-27 subunit α, and avidin conjugated to horseradish peroxidase was added to each microplate well and incubated. Thereafter, a tetramethyl benzidine substrate solution was added to each well. Only those wells containing IL-27 subunit α, biotin-conjugated antibody, and enzyme-conjugated avidin exhibited a change in color. The enzyme–substrate reaction was terminated by the addition of a sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450±2 nm. The concentration of IL-27 subunit α in the samples was then determined by comparing the optical density of the samples with the standard curve. The detection range was 15.6–1000 pg/ml.
Descriptive statistical analysis of variables was carried out (mean, SD, median, range, number and percentage). Comparison of different variables was made using the analysis of variance test (for continuous variables) and the χ2-test (for categorical variables). Spearman correlation coefficients were used to assess the relation between two quantitative parameters in the same group. Receiver operating characteristic (ROC) curve was used to assess best cutoff point for serum level of IL-27 to discriminate between de-novo ITP patients and controls and between refractory patients and those in CR together with calculation of area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of serum IL-27. Statistical significance was determined at the 0.05 level. All P values were two-sided. Standard computer program SPSS for Windows, release 17.0 (SPSS Inc., Chicago, IL, USA) was used for data entry and analysis.
All procedures followed were in accordance with the ethical standards of the ethical committee of Faculty of Medicine of Ain Shams University on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all participants included in the study.
| Results|| |
Baseline patient characteristics
The mean age of patients was 31.37±5.99 years and that of controls was 30.95±7.19 years (range=20–44 years). There were 53 female patients (88.3%) and 16 female controls (80%). The patients and controls were matched as regards age and sex (P=0.798 and 0.349, respectively). The baseline patient characteristics are summarized in [Table 1].
Serum interleukon-27 in patients and controls
The mean IL-27 for all patients was 113.4 pg/ml (range=22.5–270 pg/ml) and that for controls was 13.9 pg/ml (range=2–69 pg/ml). There was a significant difference between the two levels (P<0.001) ([Figure 1]). The mean IL-27 level was 145.25 pg/ml (range=37.5–270 pg/ml) for group 1, 70.15 pg/ml (range=22.5–270 pg/ml) for group 2, and 124.8 pg/ml (range=22.5–270 pg/ml) for group 3. There were significant differences in mean IL-27 levels between group 1 and group 2 (P=0.002). Moreover, there was a significant difference in mean IL-27 between group 2 and group 3 (P=0.030). However, there was a nonsignificant difference in mean IL-27 between group 1 and group 3 (P=0.452). However, there were significant differences in mean IL-27 levels between groups 1, 2 and 3 and controls (P<0.001, P<0.001, and P<0.001, respectively).
|Figure 1 Comparison of serum interleukin-27 (IL-27) in patients and controls [mean IL-27=113.4 vs. 13.9 pg/ml (P<0.001)].|
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Correlation of serum interleukin-27 with platelet count and other variables
There was a significant negative correlation between IL-27 level and platelet count (r=−0.375; P=0.003) ([Figure 2]). However, there was no significant correlation between IL-27 and platelet count in variable groups (for group 1, r=−0.082 and P=0.732; for group 2, r=−0.357 and P=0.122; and for group 3, r=−0.081 and P=0.733). As regards other variables, there was a nonsignificant correlation between IL-27 level and age, total leukocytic count, and hemoglobin (r=−0.037 and P=0.781; r=−0.102 and P=0.437; and r=−0.044 and P=0.741, respectively).
|Figure 2 Correlation of serum interleukin-27 (IL-27) with platelet count in immune thrombocytopenia (ITP) patients. There was a significant negative correlation (r=−0.375; P=0.003).|
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Sensitivity, specificity, positive predictive value, and negative predictive value of interleukin-27 as a screening test for immune thrombocytopenia diagnosis and response to first-line treatment
A serum level of IL-27 of 32 pg/ml had a sensitivity of 100% and specificity of 90% in differentiating de-novo ITP patients from healthy controls as detected by means of ROC curve, with a PPV of 90.9% and NPV of 100% (AUC=0.985; 95% confidence interval=0.000–1.000; P<0.001) ([Figure 3]). Moreover, a serum level of IL-27 of 72.5 pg/ml had a sensitivity of 65% and specificity of 75% in differentiating patients in CR from refractory patients as detected by means of ROC curve, with a PPV of 72.2% and NPV of 68.2% (AUC=0.683; 95% confidence interval=0.514–0.851; P=0.048) ([Figure 4]).
|Figure 3 Receiver operating characteristic (ROC) curve for serum level of interleukin-27 (IL-27) in de-novo immune thrombocytopenia (ITP) patients and healthy controls. The optimum cutoff point of IL-27 for maximum sensitivity (100%) and specificity (90%) is 32 pg/ml.|
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|Figure 4 Receiver operating characteristic (ROC) curve for serum level of interleukin-27 (IL-27) in patients in complete response (CR) and refractory patients. The optimum cutoff point of IL-27 for maximum sensitivity (65%) and specificity (75%) is 72.5 pg/ml.|
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| Discussion|| |
The pathophysiological significance of IL-27 has been investigated in Th1/Th17-mediated inflammatory diseases, such as rheumatoid arthritis , inflammatory bowel disease , and systemic lupus erythematosus . Primary ITP is an autoimmune disease with many immune dysfunctions . ITP has shown a Th1 dominant profile  and upregulated Th17 expression . IL-27 transgenic mice exhibited an increased number of megakaryocytes . However, until now the role of IL-27 in ITP has not been fully elucidated and is still controversial. A study analyzed the plasma level of IL-27 in ITP patients and demonstrated that plasma and mRNA expression levels of IL-27 in ITP patients with active disease was significantly lower than that in healthy controls or patients in remission, which was not in agreement with our results . This difference can be attributed to the inclusion of patients who have received other modalities of treatment in the other study, including vincristine, danazol, rituximab, and thrombopoietin receptor agonists, raising the suspicion of the effect of these therapies on cytokine levels.
In contrast, another study indicated that the levels of IL-27 in the plasma of untreated active ITP patients were higher than that in normal controls, which is in agreement with our results . Moreover, authors in the latter study evaluated the contribution of IL-27 to T-cell differentiation and found that IL-27 induced the differentiation of T helper-1 and T cytotoxic-1 cells . They concluded that IL-27 might play an important role in the pathogenesis of ITP by inducing the production of proinflammatory cytokines . This study points that IL-27 might be involved in the pathophysiological process of ITP and that modulation of IL-27 might provide therapeutic benefits for ITP.
Taking into consideration the heterodimeric nature of IL-27  and the association of p28 promoter polymorphisms with certain diseases such as asthma , inflammatory bowel diseases , chronic obstructive pulmonary disease , and breast cancer , Zhao et al.  investigated the association of single nucleotide polymorphisms of the IL-27 genes with the risk for ITP. Unexpectedly, they found that there was no significant difference in genotype and allele distribution between ITP patient sand the healthy controls . This indicates that the IL-27 polymorphism may not be involved in susceptibility to ITP.
We observed a significantly higher mean IL-27 level in de-novo patients than in responding patients to corticosteroids and in healthy controls. Moreover, there was a significantly higher mean IL-27 level in refractory patients than in responding patients and in healthy controls. Our results are in agreement with those of Li et al . We differed from that study in that we still found a significant difference between responding patients and healthy controls. This difference can be attributed to the use of a different analysis tool in the other study (flow cytometer using a BD FACS-Aria instrument) and to the nonunified timing of sample collection for assessment of response in our study in comparison with the other study in which all blood samples were collected at 2 weeks following dexamethasone treatment . This finding raises the suspicion about the possibility of relapse of those remitted patients with persistently elevated IL-27 and warrants longer follow-up. Moreover, it is in accordance with the chronic pathology of ITP.In the present study, although an inverse correlation was detected between IL-27 level and platelet count in all patients, there was no significant correlation between IL-27 and platelet count in variable groups. This finding can be attributed to the small number of patients in each group. Our finding is in agreement with that reported by Li et al. , who stated that there was no correlation between IL-27 level and platelet count before and after treatment in patients with de-novo active ITP. However, other studied cytokines correlated significantly with platelet count, suggesting that an interaction of many cytokines play a role in the pathogenesis of ITP .
Using the ROC curve, serum IL-27 is helpful as a screening test for predicting the risk for ITP occurrence. However, serum IL-27 has been found to be less useful for the prediction of response to first-line corticosteroids. To our knowledge, no other study analyzed the sensitivity and specificity of IL-27 for prediction of ITP risk or response rate. However, studies on larger number of patients are needed to confirm these results.
| Conclusion|| |
IL-27 is significantly elevated in patients with ITP and this potentiates the previous suggestions about the role of IL-27 in the pathogenesis of ITP, but its exact role still requires to be elucidated. Moreover, we suggest using IL-27 as a predictor for disease occurrence and to lower extent for responsiveness to corticosteroid therapy but this needs to be confirmed in larger studies. We encourage performing studies for longer follow-up periods including patients with relapsed ITP to detect the impact of IL-27 on relapse risk. Finally, we recommend studying of IL-27 as a potential target of therapy in ITP patients especially in resistance state.
The manuscript has been read and approved by all authors. The requirements for authorship have been met. Each author believes that the manuscript represents honest work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Johnsen J. Pathogenesis in immune thrombocytopenia: new insights. Hematology Am Soc Hematol Educ Program
Terrell DR, Beebe LA, Vesely SK, Neas BR, Segal JB, George JN. The incidence of immune thrombocytopenic purpura in children and adults: a critical review of published reports. Am J Hematol
Mathias SD, Gao SK, Miller KL, Cella D, Snyder C, Turner R et al.
Impact of chronic immune thrombocytopenic purpura (ITP) on health-related quality of life: a conceptual model starting with the patient perspective. Health Qual Life Outcomes
Semple JW, Milev Y, Cosgrave D, Mody M, Hornstein A, Blanchette V et al.
Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity. Blood
Provan D, Stasi R, Newland AC, Bussel JB, Paula BM, Blanchette VS et al.
International consensus report on investigation and management of primary immune thrombocytopenia. Blood
Iwasaki Y, Fujio K, Okamura T, Yamamoto K. Interleukin-27 in T cell immunity. Int J Mol Sci
Bosmann M, Ward PA. Modulation of inflammation by interleukin-27. J Leukoc Biol
Liu XG, Ren J, Yu Y, Sun L, Shi Y, Qin P et al.
Decreased expression of interleukin-27 in immune thrombocytopenia. Br J Haematol
Zhou H, Qiu JH, Wang T, Yu YY, Liu XN, Li X et al.
Interleukin 27 inhibits cytotoxic T-lymphocyte-mediated platelet destruction in primary immune thrombocytopenia. Blood
Li Q, Zhang L, Xia R, Zeng Q, Wang Y, Xia L et al.
Plasma levels of interleukin 12 family cytokines and their relevant cytokines in adult patients with chronic immune thrombocytopenia before and after high-dose dexamethasome treatment. Med Princ Pract
Neunert C, Lim W, Crowther M, Cohen A, Solberg L Jr, Crowther MA. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood
Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM et al.
Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood
Wong CK, Chen da P, Tam LS, Li EK, Yin YB, Lam CW. Effects of inflammatory cytokine IL-27 on the activation of fibroblast-like synoviocytes in rheumatoid arthritis. Arthritis Res Ther
Honda K, Nakamura K, Matsui N, Takahashi M, Kitamura Y, Mizutani T et al.
T helper 1-inducing property of IL-27/WSX-1 signaling is required for the induction of experimental colitis. Inflamm Bowel Dis
Li TT, Zhang T, Chen GM, Zhu QQ, Tao JH, Pan HF et al.
Low level of serum interleukin 27 in patients with systemic lupus erythematosus. J Investig Med
Li Q, Zhang L, Yang M, Xia R, Xia L, Liu F. Increased interleukin-27 promotes Th1 differentiation in patients with chronic immune thrombocytopenia. Scand J Immunol
Panitsas FP, Theodoropoulou M, Kouraklis A, Karakantza M, Theodorou GL, Zoumbos NC et al.
Adult chronic idiopathic thrombocytopenic purpura (ITP) is the manifestation of a type-1 polarized immune response. Blood
Zhang J, Ma D, Zhu X, Qu X, Ji C, Hou M. Elevated profile of Th17, Th1 and Tc1 cells in patients with immune thrombocytopenic purpura. Haematologica
Seita J, Asakawa M, Ooehara J, Takayanagi S, Morita Y, Watanabe N et al.
Interleukin-27 directly induces differentiation in hematopoietic stem cells. Blood
Li HY, Zhang DL, Ge J, Zhou H, Qi AP, Ma L et al.
Elevated interleukin-27 enhances the polarization of Th1/Tc1 cells and the production of proinflammatory cytokines in primary immune thrombocytopenia. Hum Immunol
Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J et al.
IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells. Immunity
Chae SC, Li CS, Kim KM, Yang JY, Zhang Q, Lee YC et al.
Identification of polymorphisms in human interleukin-27 and their association with asthma in a Korean population. J Hum Genet
Li CS, Zhang Q, Lee KJ, Cho SW, Lee KM, Hahm KB et al.
Interleukin-27 polymorphisms are associated with inflammatory bowel diseases in a Korean population. J Gastroenterol Hepatol
Huang N, Liu L, Wang XZ, Liu D, Yin SY, Yang XD. Association of interleukin (IL)-12 and IL-27 gene polymorphisms with chronic obstructive pulmonary disease in a Chinese population. DNA Cell Biol
Zhang S, Wang Y, Wang M, Ji Z. IL-27-964A>G polymorphism and the risk of breast cancer: a case-control study. Tumour Biol
Zhao H, Zhang Y, Xue F, Xu J, Fang Z. Interleukin-27 rs153109 polymorphism and the risk for immune thrombocytopenia. Autoimmunity
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