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Decreased expression of IL-33 in immune thrombocytopenia
International Immunopharmacology 28 (2015) 420–424
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Decreased expression of IL-33 in immune thrombocytopenia Pei-pei Li a, Xiao-mei Zhang b, Dai Yuan a, Xin Liu a, Ying Li a, Ning-ning Shan a,⁎ a b
Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jing Wu Rd, Jinan, People's Republic of China Department of Hematology, People's Hospital of Rizhao City, 126 taian Rd, Rizhao, People's Republic of China
a r t i c l e
i n f o
Article history: Received 4 March 2015 Received in revised form 22 June 2015 Accepted 23 June 2015 Available online xxxx Keywords: IL-33 sST2 Cytokine Immune thrombocytopenia Th1/Th2
a b s t r a c t Purpose: Interleukin-33 (IL-33) is an IL-1 family cytokine which signals via its ST2 receptor and is involved in several autoimmune diseases by regulating T cell immune responses. This study aims to measure the expression of soluble ST2 (sST2) and IL-33 in active immune thrombocytopenia (ITP) and during remission. Methods: Thirty-two ITP patients with active disease and 20 patients in remission were studied. IL-33 and sST2 were measured in plasma using ELISA and compared with 27 age- and sex-matched healthy controls. Real-time quantitative PCR was used to determine IL-33 and sST2 mRNA expressions. Results: The IL-33 level in plasma was significantly down-regulated in the patients with active ITP (P b 0.01). The sST2 level was up-regulated (P b 0.01) in the patients with active ITP compared with ITP in remission and the normal controls. No significant changes were detected between the patients with ITP in remission and the normal controls. We detected an obvious up-regulation of sST2 mRNA levels but no change in IL-33 mRNA expression. There was no correlation observed between IL-33 or sST2 expression and the platelet count of the patients with active ITP. The plasma and mRNA level ratios of sST2/IL-33 were up-regulated in the active disease patients (P b 0.01). However, no difference was detected between the ITP patients with remission disease and healthy controls. Conclusions: The values of sST2 and IL-33 observed in patients with ITP correlated with disease activity. Considering the role of IL-33 in regulating T cell immunity, studies on IL-33 and sST2 in ITP would further improve our understanding of the pathogenesis of ITP. © 2015 Published by Elsevier B.V.
1. Introduction Immune thrombocytopenia (ITP) is a disease characterized by a breakdown of immune tolerance and results in a decreased platelet count because of anti-platelet antibodies. T cell immunity abnormalities are involved in the pathogenesis of ITP, including abnormal T regulatory (Treg) cells, helper T cell imbalances and cytotoxic T cells involved in immune tolerance and maintaining a balance of human immunity [1–3]. Investigations into the role of cellular immunity deficiency involved in ITP have provided us with a new perspective in understanding the pathogenesis of ITP and can reveal new therapeutic strategies [4,5]. The interleukin-1 (IL-1) family is involved in many human diseases by regulating innate immunity and inflammation [6,7]. In our previous research, we determined that IL-18 is an IL-1 family member involved in the pathogenesis of ITP by regulating the Th1/Th2 balance [8,5]. As a new IL-1 family member found in 2005, IL-33 and its receptor play important roles in inflammation, cancer, allergy and autoimmune diseases
⁎ Corresponding author at: Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jing Wu Rd, Jinan, Shandong 250021, People's Republic of China. E-mail address: [email protected] (N. Shan).
http://dx.doi.org/10.1016/j.intimp.2015.06.035 1567-5769/© 2015 Published by Elsevier B.V.
and are potential therapeutic targets [9–12]. However, the role of IL-33 in ITP remains unresolved. Because of the role IL-33 plays in regulating T cell immunity, we investigated the involvement of IL-33 in the pathogenesis of ITP patients. Herein, we report decreased levels of IL-33 in the plasma of ITP patients. Interestingly and rather unexpectedly, IL-33 mRNA was not detectable in ITP. 2. Material and methods 2.1. Patients and controls Thirty-two adult primary ITP patients with active disease (19 females and 13 males, age range 18–74 years, median 54 years) were enrolled in this study between July 2013 and February 2014 at the Department of Hematology, Shandong Provincial Hospital Affiliated with Shandong University, Jinan, China. The patients were diagnosed according to recently published criteria including a patient history, physical examination, complete blood count and peripheral blood smear examination consistent with ITP [13]. The patients' platelet counts ranged between 2 and 27 × 109/l, with a median count of 9.5 × 109/l. All the patients required treatment because of clinically significant bleeding. None had been treated with glucocorticosteroids
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prior to sampling. Twenty ITP patients (10 females and 10 males, age range 17–67 years, median 49 years) were in remission with platelet counts ranging between 151 and 330 × 109/l with a median count of 231 × 109/l. All twenty ITP patients with remission disease including in our study were accepted glucocorticosteroid therapy. Only one patient was used in the combination therapy of glucocorticosteroid and rituximab (100 mg, qw, for 4 weeks) for the poor response to glucocorticosteroid therapy alone. The normal control group consisted of twenty-seven adult healthy volunteers (17 women and 10 men; range: 18–65 years; median age 42 years). Informed consent was obtained from each participating patient. Ethical approval for the study was obtained from the Medical Ethical Committee of Shandong Provincial Hospital Affiliated to Shandong University. 2.2. Plasma and peripheral blood mononuclear cell (PBMC) preparation Peripheral blood was collected into heparin-anticoagulant vacutainer tubes. Plasma was obtained from all the subjects, centrifuged and stored at − 80 °C to measure cytokine levels. Mononuclear cells were isolated from heparinized blood by a gradient centrifugation on a Ficoll-Paque (Pharmacia Diagnostics, Uppsala, Sweden). PBMCs were processed in an RNeasy mini-column (Qiagen GmbH, Hilden, Germany) according to the manufacturer's recommendations. Total RNA was eluted with 15 μl of RNase-free water and stored at −80 °C. 2.3. Enzyme-linked immunosorbent assay (ELISA) IL-33 and sST2 levels in the plasma were determined using an ELISA assay. Plasma IL-33 and sST2 were measured by commercial enzymelinked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (eBioscience and R &D, respectively). The lower detection limits of the IL-33 and sST2 kits were 7.8 and 31.3 pg/ml, respectively. All the measurements were repeated three times. 2.4. Reverse-transcription polymerase chain reaction (RT-PCR) assays Total RNA was isolated from PBMCs using a TRIzol reagent and cDNA generated using reverse-transcription reagents following the manufacturer's instructions. Amplification was performed using a SYBR Premix Ex Taq on a Roche 480 system. Each reaction system comprised 10 μl of SYBR Premix Ex Taq™, 7.2 μl of distilled water, 0.4 μl of each primer (10 μM) and 2 μl of cDNA (diluted 1:10). Light Cycler 480 Gene Scanning Software version 1.5 was used to analyze PCR results. All PCR products were visualized using a 2% agarose gel electrophoresis stained by ethidium bromide. The sequences of the amplification primers are listed in Table 1. We used a comparative Ct method (using arithmetic formulae) for relative quantification of cytokine mRNA according to relative expression software tool (REST©). The amplification efficiency between the target (i.e., IL-33) and reference control (i.e., β-actin) was compared using a delta delta Ct (ΔΔCt) calculation.
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The numerical RT-PCR results were analyzed using the REST software. A correlation analysis was performed using a Pearson correlation analysis test. A probability value of P b 0.05 was considered statistically significant. 3. Results 3.1. Plasma concentration of IL-33 and sST2 in the ITP patients and controls As shown in Fig. 1A, the plasma concentrations of IL-33 in the ITP patients with active disease were significantly lower than those of the healthy controls (24.2 ± 15.3 vs. 69.6 ± 26.1 pg/ml, P b 0.01). The plasma sST2 levels in the ITP patients with active disease were significantly increased compared with the healthy controls (1375.7 ± 733.4 vs. 934.5 ± 359.7 pg/ml, P b 0.01) (Fig. 1B). Significantly lower IL-33 and higher sST2 plasma levels were found in the active ITP patients compared with the ITP patients in remission (24.2 ± 15.3 vs. 41.5 ± 31.8 pg/ml, P b 0.01 and 1375.7 ± 733.4 pg/ml vs. 1130.3 ± 480.7 pg/ml, P b 0.01, respectively). However, no significant differences in the IL-33 and sST2 plasma levels were found between the ITP patients in remission and healthy controls. 3.2. mRNA expression levels of IL-33 and sST2 in the ITP patients and controls Because of the decreased plasma concentrations of IL-33 in active ITP, we measured the mRNA expression of IL-33 and its receptor sST2. Using the REST software, the data were presented as a fold change in gene expression normalized to an endogenous reference gene and relative to healthy controls. Consistent with the plasma sST2 level, the relative mRNA gene expression of sST2 was increased 5.1-fold in the active patients compared with the healthy controls (P b 0.01) and 2.9-fold compared to the patients in remission (P b 0.01). Different from the level of sST2 in plasma, the sST2 mRNA expression of the patients in remission was still higher than the controls (P b 0.01). Interestingly, the expression of IL-33 was unchanged in the patients compared with the controls (Fig. 2). There was no significant difference between the patients in remission and the control group. 3.3. Correlations between plasma IL-33 and platelet count in the ITP patients with active disease Correlations between the plasma IL-33 and sST2 concentrations and platelet counts were analyzed in the active ITP patients. The data demonstrated no correlation between the IL-33 levels and platelet counts (r = 0.03305, P = 0.3194, Pearson correlation analysis; Fig. 3A). No correlation between sST2 concentrations and platelet counts was found in the ITP patients with active disease (r = 0.005, P = 0.6836, Pearson correlation analysis; Fig. 3B). 3.4. The ratios of sST2/IL-33 in the normal controls, patients with active disease and patients in remission
2.5. Statistics analysis The data were analyzed by Spss18.0 for Windows. The results were from three independent experiments and recorded as the mean ± SD. Table 1 Primers and conditions for the RT-PCR experiments performed in this study. Gene
Sequence (5′ → 3′)
T (°C)
Product (bp)
sST2
F: 5-GGATTGAGGCCACTCTGCTC-3 R: 5-CCGCCTGCTCTTTCGTATGT-3 F: 5-ATCCCAACAGAAGGCCAAAG-3 R: 5-CCAAAGGCAAAGCACTCCAC-3 F: 5-TTGCCGACAGGATGCAGAA-3 R: 5-GCCGATCCACACGGAGTACT-3
60
269
60
198
60
101
IL-33 β-Actin
The plasma levels of IL-33 in the ITP patients with active disease were significantly decreased, and the plasma levels of sST2 were significantly increased compared with the normal controls. The ratio of sST2/IL-33 in the patients with active disease was significantly increased compared with the normal and remission groups (P b 0.01). However, there was no significant difference between the patients in remission and normal controls (Fig. 4A). The ratio of sST2 mRNA/IL-33 mRNA in the ITP remission group had a trend of elevation, but did not reach statistical significance compared with the control group. The ratio of sST2 mRNA/IL-33 mRNA was significantly different between the active and control groups (P b 0.01). Statistically significant differences were found in the active patients compared with the patients in remission (P b 0.01) (Fig. 4B). No correlation between the
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Fig. 1. Expression of IL-33 and sST2 in plasma of ITP patients and normal controls. (A) Plasma concentrations of IL-33 in 32 ITP patients with active disease, 20 patients in remission and 27 normal controls were measured using ELISA method. The results of the three groups are 24.2 ± 15.3, 41.5 ± 31.8 vs. 69.6 ± 26.1 pg/ml, respectively. #P b 0.01, active ITP compared with normal controls; *P b 0.01, active ITP compared with ITP in remission; &P N 0.05, ITP in remission compared with normal controls. (B) Plasma sST2 levels were detected in the aforementioned groups. sST2 levels in ITP patients in the three groups were 1375.7 ± 733.4, 1130.3 ± 480.7 and 934.5 ± 359.7 pg/ml, respectively. #P b 0.01, active ITP compared with normal controls; *P b 0.01, active ITP compared with ITP in remission; &P N 0.05, ITP in remission compared with normal controls. The results are shown as the mean ± SD. P b 0.05 is considered significant.
ratio of sST2/IL-33 and platelet count was found in the ITP patients with active disease (r = 0.012, P = 0.5502, Pearson correlation analysis; Fig. 5). 4. Discussion T cells play a critical role in maintaining immunity homeostasis [14]. T cells are grouped into the following subsets based on their function and surface markers: cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, etc. T cell abnormalities play a central role in the pathogenesis of many autoimmunity diseases and are a potential therapeutic target in some diseases [15]. As an acquired autoimmune disease, dysregulation of T cell immunity is highly correlated with the pathogenesis of ITP [1,16]. In ITP, the percentage of Treg cells is significantly down-regulated. A lower percentage of Treg cells has been proven to be associated with increased activity of the disease [17,18]. Decreased Treg cell numbers are associated with deficient immune tolerance to self-antigens, which is demonstrated in ITP patients [19,2,18]. This deficiency can be recovered after treatment with rituximab, indicating a complex regulation pathway of immune cells in ITP patients [20]. Plasma levels of cytokines reflect an imbalance of Th1/Th2 cells in ITP. In patients with active ITP, there is generally a Th1 polarization represented by aberrant Th1 cytokine expression [21,22]. An increased Th1/Th2 ratio that is detected by measuring mRNA expression of cytokines from PBMCs in the peripheral blood has been proposed to correlate with ITP disease activity [21,23,24]. IL-33, which functions as a nuclear factor and cytokine, is a newly described member of the IL-1 family. It has structural similarities to
Fig. 2. mRNA levels of IL-33 or sST2 in PBMCs of ITP patients. mRNA expressions of IL-33 or sST2 in PBMCs from the three groups were measured by RT-PCR. There is no obvious difference in IL-33 expression between the two groups. sST2 in the active ITP patient group was increased 5.1-fold (#P b 0.01) and 2.9-fold (*P b 0.01) compared with the healthy controls and patients in remission, respectively. sST2 mRNA expression of the patients in remission was higher than the controls (&P b 0.01). The results were normalized to βactin. The results of the experiments were representative of three independent replicates.
other IL-1 family members [25,26]. IL-33 is involved in a variety of autoimmune diseases [27–30]. In multiple sclerosis, IL-33 has been proven to be overexpressed in peripheral leukocytes and the central nervous system, but can be inhibited by an HDAC inhibitor [31,27]. Plasma and mRNA levels of IL-33 are up-regulated in Behcet's disease (BD). In BD with retinal vasculitis and skin lesions, IL-33 is further up-regulated [30]. In intermittent allergic rhinitis and asthma, elevated IL-33 levels are associated with disease severity [32,33]. Thus, it is of interest to investigate the involvement of IL-33 in the pathogenesis of ITP. We first detected IL-33 expression in the plasma of ITP patients. The results showed that IL-33 is decreased in the patients with active ITP compared with the patients in remission and normal controls. However, no significant differences were detected between the patients in remission and normal controls.
Fig. 3. Correlation between concentration of IL-33 or sST2 with platelet count. (A) There was no significant correlation between the IL-33 expression in plasma and platelet count in active ITP (P = 0.3194). (B) The correlation between the sST2 expression in plasma and platelet count platelet was not significant in ITP (P = 0.6826).
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Fig. 4. The ratio of sST2/IL-33 in ITP patients and normal controls. (A) The ratio of sST2/IL-33 in plasma in the patients with active ITP, ITP in remission, and normal controls was compared. In the patients with active ITP, this ratio is significantly up-regulated compared with the patient with ITP in remission (*P b 0.01) or the normal controls (#P b 0.01). (B) The mRNA ratio of sST2/IL-33 in the active ITP group was significantly higher than in the other two groups (**P b 0.05 and #P b 0.01, respectively).
IL-33 mediates its biological effects by combining with IL-1 receptorrelated protein ST2 to activate MAK kinases and NF-κB [10]. ST2 can be detected in Th2 cells, mast cells, dendritic cells, NK cells, basophils, macrophages, CD8 T cells, etc. [10,34,35]. IL-33 binding to the ST2 receptor can induce secretion of Th2-associated cytokines such as IL-4, IL-5 and IL-13 [10,36]. IL-33 can induce a Th1-associated cytokine cooperation with IL-12 [36]. However, in a study of human visceral leishmaniasis, IL-33 was associated with and suppressed Th1 responses [37]. IL-33 also expands suppressive Treg cells thereby enhancing immunesuppression [38,39]. Deletion of the IL-33 receptor also influences the immune activity of NK cells, CD8+T cells, etc., by affecting their cytokine secretion [40]. Our results indicate that IL-33 is involved in the pathogenesis of ITP. We deduced that IL-33 regulates T cell immunity. However, the exact role of IL-33 in the pathogenesis of ITP needs to be further investigated. In addition to the membrane form of ST2, there is also soluble ST2 in plasma which has no trans-membrane sequence and can be a decoy receptor for IL-33 [41]. IL-33 binding to sST2 can inhibit the IL-33/ST2 pathway [41]. In systemic lupus erythematosus (SLE), sST2 was proven to be associated with an active disease state in patients. This was assessed by the level of anti-dsDNA antibodies and SLEDAI [28]. In ITP patients, our results showed that sST2 in plasma was significantly upregulated in the patients with active disease compared with the patients in remission and normal controls. Additionally, no significant difference was detected between the patients in remission and normal controls. mRNA expression levels in the ITP patients and healthy controls were measured to evaluate the pathogenesis of IL-33 down-regulation. No statistical significance was detected in IL-33 mRNA expression between the two groups. The sST2 mRNA was up-regulated in the ITP patients with active disease. These results suggest that there exists no genetic variants in the IL-33 gene. The overexpression of sST2 could be correlated with the up-regulation of sST2 in plasma, which further inhibits the function of IL-33. The precise functions of IL-33 and its producing cells
and receptor expression in ITP patients need to be elucidated to better understand the pathogenesis of ITP. T cells are involved in the production and secretion of antibodies by B cells. To evaluate whether IL-33 or sST2 expression is associated with severity of platelet destruction, we performed a correlation analysis between IL-33 or sST2 expression and platelet count in the active ITP patients. No obvious correlation was detected in our study. Previous reports noted that IL-33 and sST2 cooperate in the pathogenesis of diseases [42–44]. Understanding differences in the IL-33/sST2 ratio in ITP patients and normal controls would help us better understand the function of the IL-33/ST2 pathway. Therefore, we compared ratios of the plasma and mRNA levels of sST2/IL-33 in active ITP, ITP in remission and normal control groups. Consistent with the trend of IL-33 and sST2 expressions in plasma, the ratio of sST2/IL-33 in plasma was up-regulated in the patients with active ITP compared with the other two groups. However, it was not associated with platelet count. At the mRNA level, the ratio of sST2/IL-33 was up-regulated in the patients with active ITP compared with the normal controls. There was a no statistically significant difference detected between the patients with ITP in remission and normal controls, though the ratio was up-regulated in the patients with ITP in remission. In the future, we can verify this trend by increasing the number of samples. There were significant differences between the patients with active ITP and ITP in remission, but there were no significant differences between the patients with ITP in remission and normal controls. 5. Conclusion As a new family member of the IL-1 family, IL-33 has various functions depending on its target cells and microenvironment. Recently, IL-33 has been treated as a potential therapeutic target for immunemediated diseases such as atopic disease, hepatitis and tumors, in which it has been shown to have a protective role [35,41,45,46]. Further investigating the role of IL-33 in the pathogenesis of ITP may provide insight into the disease and provide a new therapeutic target for ITP therapy. Competing interests The authors declare that they have no competing interests. References
Fig. 5. Correlation between the ratio of sST2/IL-33 and platelet count of the active ITP patients. There was no significant correlation between the ratio of sST2/IL-33 in plasma and platelet count of the active ITP patients (P = 0.5502).
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Decreased expression of IL-33 in immune thrombocytopenia Pei-pei Li a, Xiao-mei Zhang b, Dai Yuan a, Xin Liu a, Ying Li a, Ning-ning Shan a,⁎ a b
Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jing Wu Rd, Jinan, People's Republic of China Department of Hematology, People's Hospital of Rizhao City, 126 taian Rd, Rizhao, People's Republic of China
a r t i c l e
i n f o
Article history: Received 4 March 2015 Received in revised form 22 June 2015 Accepted 23 June 2015 Available online xxxx Keywords: IL-33 sST2 Cytokine Immune thrombocytopenia Th1/Th2
a b s t r a c t Purpose: Interleukin-33 (IL-33) is an IL-1 family cytokine which signals via its ST2 receptor and is involved in several autoimmune diseases by regulating T cell immune responses. This study aims to measure the expression of soluble ST2 (sST2) and IL-33 in active immune thrombocytopenia (ITP) and during remission. Methods: Thirty-two ITP patients with active disease and 20 patients in remission were studied. IL-33 and sST2 were measured in plasma using ELISA and compared with 27 age- and sex-matched healthy controls. Real-time quantitative PCR was used to determine IL-33 and sST2 mRNA expressions. Results: The IL-33 level in plasma was significantly down-regulated in the patients with active ITP (P b 0.01). The sST2 level was up-regulated (P b 0.01) in the patients with active ITP compared with ITP in remission and the normal controls. No significant changes were detected between the patients with ITP in remission and the normal controls. We detected an obvious up-regulation of sST2 mRNA levels but no change in IL-33 mRNA expression. There was no correlation observed between IL-33 or sST2 expression and the platelet count of the patients with active ITP. The plasma and mRNA level ratios of sST2/IL-33 were up-regulated in the active disease patients (P b 0.01). However, no difference was detected between the ITP patients with remission disease and healthy controls. Conclusions: The values of sST2 and IL-33 observed in patients with ITP correlated with disease activity. Considering the role of IL-33 in regulating T cell immunity, studies on IL-33 and sST2 in ITP would further improve our understanding of the pathogenesis of ITP. © 2015 Published by Elsevier B.V.
1. Introduction Immune thrombocytopenia (ITP) is a disease characterized by a breakdown of immune tolerance and results in a decreased platelet count because of anti-platelet antibodies. T cell immunity abnormalities are involved in the pathogenesis of ITP, including abnormal T regulatory (Treg) cells, helper T cell imbalances and cytotoxic T cells involved in immune tolerance and maintaining a balance of human immunity [1–3]. Investigations into the role of cellular immunity deficiency involved in ITP have provided us with a new perspective in understanding the pathogenesis of ITP and can reveal new therapeutic strategies [4,5]. The interleukin-1 (IL-1) family is involved in many human diseases by regulating innate immunity and inflammation [6,7]. In our previous research, we determined that IL-18 is an IL-1 family member involved in the pathogenesis of ITP by regulating the Th1/Th2 balance [8,5]. As a new IL-1 family member found in 2005, IL-33 and its receptor play important roles in inflammation, cancer, allergy and autoimmune diseases
⁎ Corresponding author at: Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jing Wu Rd, Jinan, Shandong 250021, People's Republic of China. E-mail address: [email protected] (N. Shan).
http://dx.doi.org/10.1016/j.intimp.2015.06.035 1567-5769/© 2015 Published by Elsevier B.V.
and are potential therapeutic targets [9–12]. However, the role of IL-33 in ITP remains unresolved. Because of the role IL-33 plays in regulating T cell immunity, we investigated the involvement of IL-33 in the pathogenesis of ITP patients. Herein, we report decreased levels of IL-33 in the plasma of ITP patients. Interestingly and rather unexpectedly, IL-33 mRNA was not detectable in ITP. 2. Material and methods 2.1. Patients and controls Thirty-two adult primary ITP patients with active disease (19 females and 13 males, age range 18–74 years, median 54 years) were enrolled in this study between July 2013 and February 2014 at the Department of Hematology, Shandong Provincial Hospital Affiliated with Shandong University, Jinan, China. The patients were diagnosed according to recently published criteria including a patient history, physical examination, complete blood count and peripheral blood smear examination consistent with ITP [13]. The patients' platelet counts ranged between 2 and 27 × 109/l, with a median count of 9.5 × 109/l. All the patients required treatment because of clinically significant bleeding. None had been treated with glucocorticosteroids
P. Li et al. / International Immunopharmacology 28 (2015) 420–424
prior to sampling. Twenty ITP patients (10 females and 10 males, age range 17–67 years, median 49 years) were in remission with platelet counts ranging between 151 and 330 × 109/l with a median count of 231 × 109/l. All twenty ITP patients with remission disease including in our study were accepted glucocorticosteroid therapy. Only one patient was used in the combination therapy of glucocorticosteroid and rituximab (100 mg, qw, for 4 weeks) for the poor response to glucocorticosteroid therapy alone. The normal control group consisted of twenty-seven adult healthy volunteers (17 women and 10 men; range: 18–65 years; median age 42 years). Informed consent was obtained from each participating patient. Ethical approval for the study was obtained from the Medical Ethical Committee of Shandong Provincial Hospital Affiliated to Shandong University. 2.2. Plasma and peripheral blood mononuclear cell (PBMC) preparation Peripheral blood was collected into heparin-anticoagulant vacutainer tubes. Plasma was obtained from all the subjects, centrifuged and stored at − 80 °C to measure cytokine levels. Mononuclear cells were isolated from heparinized blood by a gradient centrifugation on a Ficoll-Paque (Pharmacia Diagnostics, Uppsala, Sweden). PBMCs were processed in an RNeasy mini-column (Qiagen GmbH, Hilden, Germany) according to the manufacturer's recommendations. Total RNA was eluted with 15 μl of RNase-free water and stored at −80 °C. 2.3. Enzyme-linked immunosorbent assay (ELISA) IL-33 and sST2 levels in the plasma were determined using an ELISA assay. Plasma IL-33 and sST2 were measured by commercial enzymelinked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (eBioscience and R &D, respectively). The lower detection limits of the IL-33 and sST2 kits were 7.8 and 31.3 pg/ml, respectively. All the measurements were repeated three times. 2.4. Reverse-transcription polymerase chain reaction (RT-PCR) assays Total RNA was isolated from PBMCs using a TRIzol reagent and cDNA generated using reverse-transcription reagents following the manufacturer's instructions. Amplification was performed using a SYBR Premix Ex Taq on a Roche 480 system. Each reaction system comprised 10 μl of SYBR Premix Ex Taq™, 7.2 μl of distilled water, 0.4 μl of each primer (10 μM) and 2 μl of cDNA (diluted 1:10). Light Cycler 480 Gene Scanning Software version 1.5 was used to analyze PCR results. All PCR products were visualized using a 2% agarose gel electrophoresis stained by ethidium bromide. The sequences of the amplification primers are listed in Table 1. We used a comparative Ct method (using arithmetic formulae) for relative quantification of cytokine mRNA according to relative expression software tool (REST©). The amplification efficiency between the target (i.e., IL-33) and reference control (i.e., β-actin) was compared using a delta delta Ct (ΔΔCt) calculation.
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The numerical RT-PCR results were analyzed using the REST software. A correlation analysis was performed using a Pearson correlation analysis test. A probability value of P b 0.05 was considered statistically significant. 3. Results 3.1. Plasma concentration of IL-33 and sST2 in the ITP patients and controls As shown in Fig. 1A, the plasma concentrations of IL-33 in the ITP patients with active disease were significantly lower than those of the healthy controls (24.2 ± 15.3 vs. 69.6 ± 26.1 pg/ml, P b 0.01). The plasma sST2 levels in the ITP patients with active disease were significantly increased compared with the healthy controls (1375.7 ± 733.4 vs. 934.5 ± 359.7 pg/ml, P b 0.01) (Fig. 1B). Significantly lower IL-33 and higher sST2 plasma levels were found in the active ITP patients compared with the ITP patients in remission (24.2 ± 15.3 vs. 41.5 ± 31.8 pg/ml, P b 0.01 and 1375.7 ± 733.4 pg/ml vs. 1130.3 ± 480.7 pg/ml, P b 0.01, respectively). However, no significant differences in the IL-33 and sST2 plasma levels were found between the ITP patients in remission and healthy controls. 3.2. mRNA expression levels of IL-33 and sST2 in the ITP patients and controls Because of the decreased plasma concentrations of IL-33 in active ITP, we measured the mRNA expression of IL-33 and its receptor sST2. Using the REST software, the data were presented as a fold change in gene expression normalized to an endogenous reference gene and relative to healthy controls. Consistent with the plasma sST2 level, the relative mRNA gene expression of sST2 was increased 5.1-fold in the active patients compared with the healthy controls (P b 0.01) and 2.9-fold compared to the patients in remission (P b 0.01). Different from the level of sST2 in plasma, the sST2 mRNA expression of the patients in remission was still higher than the controls (P b 0.01). Interestingly, the expression of IL-33 was unchanged in the patients compared with the controls (Fig. 2). There was no significant difference between the patients in remission and the control group. 3.3. Correlations between plasma IL-33 and platelet count in the ITP patients with active disease Correlations between the plasma IL-33 and sST2 concentrations and platelet counts were analyzed in the active ITP patients. The data demonstrated no correlation between the IL-33 levels and platelet counts (r = 0.03305, P = 0.3194, Pearson correlation analysis; Fig. 3A). No correlation between sST2 concentrations and platelet counts was found in the ITP patients with active disease (r = 0.005, P = 0.6836, Pearson correlation analysis; Fig. 3B). 3.4. The ratios of sST2/IL-33 in the normal controls, patients with active disease and patients in remission
2.5. Statistics analysis The data were analyzed by Spss18.0 for Windows. The results were from three independent experiments and recorded as the mean ± SD. Table 1 Primers and conditions for the RT-PCR experiments performed in this study. Gene
Sequence (5′ → 3′)
T (°C)
Product (bp)
sST2
F: 5-GGATTGAGGCCACTCTGCTC-3 R: 5-CCGCCTGCTCTTTCGTATGT-3 F: 5-ATCCCAACAGAAGGCCAAAG-3 R: 5-CCAAAGGCAAAGCACTCCAC-3 F: 5-TTGCCGACAGGATGCAGAA-3 R: 5-GCCGATCCACACGGAGTACT-3
60
269
60
198
60
101
IL-33 β-Actin
The plasma levels of IL-33 in the ITP patients with active disease were significantly decreased, and the plasma levels of sST2 were significantly increased compared with the normal controls. The ratio of sST2/IL-33 in the patients with active disease was significantly increased compared with the normal and remission groups (P b 0.01). However, there was no significant difference between the patients in remission and normal controls (Fig. 4A). The ratio of sST2 mRNA/IL-33 mRNA in the ITP remission group had a trend of elevation, but did not reach statistical significance compared with the control group. The ratio of sST2 mRNA/IL-33 mRNA was significantly different between the active and control groups (P b 0.01). Statistically significant differences were found in the active patients compared with the patients in remission (P b 0.01) (Fig. 4B). No correlation between the
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Fig. 1. Expression of IL-33 and sST2 in plasma of ITP patients and normal controls. (A) Plasma concentrations of IL-33 in 32 ITP patients with active disease, 20 patients in remission and 27 normal controls were measured using ELISA method. The results of the three groups are 24.2 ± 15.3, 41.5 ± 31.8 vs. 69.6 ± 26.1 pg/ml, respectively. #P b 0.01, active ITP compared with normal controls; *P b 0.01, active ITP compared with ITP in remission; &P N 0.05, ITP in remission compared with normal controls. (B) Plasma sST2 levels were detected in the aforementioned groups. sST2 levels in ITP patients in the three groups were 1375.7 ± 733.4, 1130.3 ± 480.7 and 934.5 ± 359.7 pg/ml, respectively. #P b 0.01, active ITP compared with normal controls; *P b 0.01, active ITP compared with ITP in remission; &P N 0.05, ITP in remission compared with normal controls. The results are shown as the mean ± SD. P b 0.05 is considered significant.
ratio of sST2/IL-33 and platelet count was found in the ITP patients with active disease (r = 0.012, P = 0.5502, Pearson correlation analysis; Fig. 5). 4. Discussion T cells play a critical role in maintaining immunity homeostasis [14]. T cells are grouped into the following subsets based on their function and surface markers: cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, etc. T cell abnormalities play a central role in the pathogenesis of many autoimmunity diseases and are a potential therapeutic target in some diseases [15]. As an acquired autoimmune disease, dysregulation of T cell immunity is highly correlated with the pathogenesis of ITP [1,16]. In ITP, the percentage of Treg cells is significantly down-regulated. A lower percentage of Treg cells has been proven to be associated with increased activity of the disease [17,18]. Decreased Treg cell numbers are associated with deficient immune tolerance to self-antigens, which is demonstrated in ITP patients [19,2,18]. This deficiency can be recovered after treatment with rituximab, indicating a complex regulation pathway of immune cells in ITP patients [20]. Plasma levels of cytokines reflect an imbalance of Th1/Th2 cells in ITP. In patients with active ITP, there is generally a Th1 polarization represented by aberrant Th1 cytokine expression [21,22]. An increased Th1/Th2 ratio that is detected by measuring mRNA expression of cytokines from PBMCs in the peripheral blood has been proposed to correlate with ITP disease activity [21,23,24]. IL-33, which functions as a nuclear factor and cytokine, is a newly described member of the IL-1 family. It has structural similarities to
Fig. 2. mRNA levels of IL-33 or sST2 in PBMCs of ITP patients. mRNA expressions of IL-33 or sST2 in PBMCs from the three groups were measured by RT-PCR. There is no obvious difference in IL-33 expression between the two groups. sST2 in the active ITP patient group was increased 5.1-fold (#P b 0.01) and 2.9-fold (*P b 0.01) compared with the healthy controls and patients in remission, respectively. sST2 mRNA expression of the patients in remission was higher than the controls (&P b 0.01). The results were normalized to βactin. The results of the experiments were representative of three independent replicates.
other IL-1 family members [25,26]. IL-33 is involved in a variety of autoimmune diseases [27–30]. In multiple sclerosis, IL-33 has been proven to be overexpressed in peripheral leukocytes and the central nervous system, but can be inhibited by an HDAC inhibitor [31,27]. Plasma and mRNA levels of IL-33 are up-regulated in Behcet's disease (BD). In BD with retinal vasculitis and skin lesions, IL-33 is further up-regulated [30]. In intermittent allergic rhinitis and asthma, elevated IL-33 levels are associated with disease severity [32,33]. Thus, it is of interest to investigate the involvement of IL-33 in the pathogenesis of ITP. We first detected IL-33 expression in the plasma of ITP patients. The results showed that IL-33 is decreased in the patients with active ITP compared with the patients in remission and normal controls. However, no significant differences were detected between the patients in remission and normal controls.
Fig. 3. Correlation between concentration of IL-33 or sST2 with platelet count. (A) There was no significant correlation between the IL-33 expression in plasma and platelet count in active ITP (P = 0.3194). (B) The correlation between the sST2 expression in plasma and platelet count platelet was not significant in ITP (P = 0.6826).
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Fig. 4. The ratio of sST2/IL-33 in ITP patients and normal controls. (A) The ratio of sST2/IL-33 in plasma in the patients with active ITP, ITP in remission, and normal controls was compared. In the patients with active ITP, this ratio is significantly up-regulated compared with the patient with ITP in remission (*P b 0.01) or the normal controls (#P b 0.01). (B) The mRNA ratio of sST2/IL-33 in the active ITP group was significantly higher than in the other two groups (**P b 0.05 and #P b 0.01, respectively).
IL-33 mediates its biological effects by combining with IL-1 receptorrelated protein ST2 to activate MAK kinases and NF-κB [10]. ST2 can be detected in Th2 cells, mast cells, dendritic cells, NK cells, basophils, macrophages, CD8 T cells, etc. [10,34,35]. IL-33 binding to the ST2 receptor can induce secretion of Th2-associated cytokines such as IL-4, IL-5 and IL-13 [10,36]. IL-33 can induce a Th1-associated cytokine cooperation with IL-12 [36]. However, in a study of human visceral leishmaniasis, IL-33 was associated with and suppressed Th1 responses [37]. IL-33 also expands suppressive Treg cells thereby enhancing immunesuppression [38,39]. Deletion of the IL-33 receptor also influences the immune activity of NK cells, CD8+T cells, etc., by affecting their cytokine secretion [40]. Our results indicate that IL-33 is involved in the pathogenesis of ITP. We deduced that IL-33 regulates T cell immunity. However, the exact role of IL-33 in the pathogenesis of ITP needs to be further investigated. In addition to the membrane form of ST2, there is also soluble ST2 in plasma which has no trans-membrane sequence and can be a decoy receptor for IL-33 [41]. IL-33 binding to sST2 can inhibit the IL-33/ST2 pathway [41]. In systemic lupus erythematosus (SLE), sST2 was proven to be associated with an active disease state in patients. This was assessed by the level of anti-dsDNA antibodies and SLEDAI [28]. In ITP patients, our results showed that sST2 in plasma was significantly upregulated in the patients with active disease compared with the patients in remission and normal controls. Additionally, no significant difference was detected between the patients in remission and normal controls. mRNA expression levels in the ITP patients and healthy controls were measured to evaluate the pathogenesis of IL-33 down-regulation. No statistical significance was detected in IL-33 mRNA expression between the two groups. The sST2 mRNA was up-regulated in the ITP patients with active disease. These results suggest that there exists no genetic variants in the IL-33 gene. The overexpression of sST2 could be correlated with the up-regulation of sST2 in plasma, which further inhibits the function of IL-33. The precise functions of IL-33 and its producing cells
and receptor expression in ITP patients need to be elucidated to better understand the pathogenesis of ITP. T cells are involved in the production and secretion of antibodies by B cells. To evaluate whether IL-33 or sST2 expression is associated with severity of platelet destruction, we performed a correlation analysis between IL-33 or sST2 expression and platelet count in the active ITP patients. No obvious correlation was detected in our study. Previous reports noted that IL-33 and sST2 cooperate in the pathogenesis of diseases [42–44]. Understanding differences in the IL-33/sST2 ratio in ITP patients and normal controls would help us better understand the function of the IL-33/ST2 pathway. Therefore, we compared ratios of the plasma and mRNA levels of sST2/IL-33 in active ITP, ITP in remission and normal control groups. Consistent with the trend of IL-33 and sST2 expressions in plasma, the ratio of sST2/IL-33 in plasma was up-regulated in the patients with active ITP compared with the other two groups. However, it was not associated with platelet count. At the mRNA level, the ratio of sST2/IL-33 was up-regulated in the patients with active ITP compared with the normal controls. There was a no statistically significant difference detected between the patients with ITP in remission and normal controls, though the ratio was up-regulated in the patients with ITP in remission. In the future, we can verify this trend by increasing the number of samples. There were significant differences between the patients with active ITP and ITP in remission, but there were no significant differences between the patients with ITP in remission and normal controls. 5. Conclusion As a new family member of the IL-1 family, IL-33 has various functions depending on its target cells and microenvironment. Recently, IL-33 has been treated as a potential therapeutic target for immunemediated diseases such as atopic disease, hepatitis and tumors, in which it has been shown to have a protective role [35,41,45,46]. Further investigating the role of IL-33 in the pathogenesis of ITP may provide insight into the disease and provide a new therapeutic target for ITP therapy. Competing interests The authors declare that they have no competing interests. References
Fig. 5. Correlation between the ratio of sST2/IL-33 and platelet count of the active ITP patients. There was no significant correlation between the ratio of sST2/IL-33 in plasma and platelet count of the active ITP patients (P = 0.5502).
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