|Year : 2018 | Volume
| Issue : 3 | Page : 119-124
T helper 1/T helper 2-associated chemokine and chemokine receptor expression in immune thrombocytopenia
Nadia I Sewelam1, Hanan Al-Wakeel1, Zainab El Saadany1, Rania Magdy2, Nevin Fouad1
1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Pediatrics, Faculty of Medicine, Cairo University, Cairo, Egypt
|Date of Submission||22-Mar-2018|
|Date of Acceptance||03-Apr-2018|
|Date of Web Publication||3-Dec-2018|
Nadia I Sewelam
50 Mekias Street, Manial Alrouda, Cairo, 11451
Source of Support: None, Conflict of Interest: None
Background Immune thrombocytopenia (ITP) is an acquired immune disorder. Chemokines have complicated role in different autoimmune disorders including ITP. C-C motif chemokine ligand 2 (CCL2) chemokine represents a T helper (Th)2 polarizing chemokine, whereas C-X-C motif chemokine receptor 3 (CXCR3) and C-C motif chemokine receptor 2 (CCR2) represent chemokine members with Th1 polarization effect on the immune system. ITP is associated with an imbalance in Th1/Th2 ratio and favors Th1 polarization. This study aimed to explore the role of CCL2 and its receptor CCR2 in addition to CXCR3 receptor gene expression in the pathogenesis and severity of ITP.
Participants and methods Expression of CCL2, CCR2, and CXCR3 was assayed using real-time quantitative polymerase chain reaction in peripheral blood mononuclear cells of 21 normal healthy participants and 68 patients with ITP: 24 acute cases, 25 chronic responder cases, and 19 chronic non-responder cases.
Results Acute ITP group showed a 1.85 median fold change in CCL2 gene expression from the control group. CCR2 and CXCR3 showed a higher median fold change from the control group in acute ITP (7.36 and 5.42, respectively) and in chronic non-responder patients (3.38 and 4.32, respectively), whereas the chronic responder patients showed the least changes (1.63 and 2.35, respectively). There was no significant difference in chemokine or chemokine receptors gene expression between different ITP groups (P>0.05). Statistically significant positive correlations were detected between CCL2 and CCR2 (r=0.453, P<0.001) and between CXCR3 and CCR2 (r=0.583, P<0.001) among patients with ITP.
Conclusion CCR2 and CXCR3 but not CCL2 may have a role in ITP pathogenesis. Further studies investigating the role of the complicated chemokine network may help better understanding of ITP pathogenesis.
Keywords: CCL2, CCR2, chemokines, CXCR3, immune thrombocytopenia, real-time PCR
|How to cite this article:|
Sewelam NI, Al-Wakeel H, El Saadany Z, Magdy R, Fouad N. T helper 1/T helper 2-associated chemokine and chemokine receptor expression in immune thrombocytopenia. Egypt J Haematol 2018;43:119-24
|How to cite this URL:|
Sewelam NI, Al-Wakeel H, El Saadany Z, Magdy R, Fouad N. T helper 1/T helper 2-associated chemokine and chemokine receptor expression in immune thrombocytopenia. Egypt J Haematol [serial online] 2018 [cited 2020 Sep 19];43:119-24. Available from: http://www.ehj.eg.net/text.asp?2018/43/3/119/246774
| Introduction|| |
Immune thrombocytopenia (ITP) is an acquired immune disorder characterized by isolated thrombocytopenia (peripheral blood platelet count <100×109/l) , and increased risk of bleeding . ITP may be due to antiplatelet antibodies production, T-cell-mediated platelet destruction , or defective megakaryopoiesis . Platelet-bound antibodies lead to Fcγ receptor-mediated clearance of platelets by phagocytes residing in the spleen and liver . Dysfunction of cellular immunity has a main role in understanding ITP pathogenesis ,. Several studies proposed that patients with ITP have disturbed T helper 1/T helper 2 ratio (Th1 and Th2)  and altered cytokine profiles . Based on their differential lymphokine production, Th cells are subdivided into Th1, producing IFNγ and lymphotoxin, and Th2, producing interleukin (IL)-4, IL-5, and IL-13 . Cytokines, present at the commencement of a T-cell response, will stimulate naïve T cells to differentiate into a particular Th-cell subset . Th1 and Th2 cells secrete cytokines that enhance cell-mediated and humoral immunity, respectively .
The balance between Th1/Th2 regulates the immune system and is known to be defective in autoimmune diseases. Wang et al.  reported a significantly higher Th1/Th2 ratio in patients with active primary ITP and adult chronic primary ITP, which reverted to a normal range when the patients achieved sustained remission. Moreover, Panitsas et al.  supported the concept that chronic adult ITP is a result of a type-1 polarized immune response. Th1 and Th2 express a variety of chemokine receptors that dictate the site of their migration in response to a specific chemokine . Chemokines are small chemotactic cytokines that play a crucial role in immune functions such as angiogenesis, hematopoiesis, leukocyte trafficking, and T-cell differentiation ,. Chemokines have a major role in Th1-cell and Th2-cell polarizations and their mediated immune responses; thus their receptors could be used as a Th1 versus Th2 response marker . Deregulation of expression of the chemokines and their receptors plays an important role in autoimmune disorders; however, their role in ITP is still controversial.
Chemokines have been classified according to their structure into four major subfamilies, namely CC, CXC, CX3C, and XC chemokines ,. Monocyte chemoattractant protein-1 (MCP-1/CCL2, C-C motif chemokine ligand 2) can stimulate IL-4 production, and its overexpression is associated with defects in cell-mediated immunity, suggesting its role in Th2 polarization . The chemokine receptor C-C motif chemokine receptor 2 (CCR2) is the only known receptor for CCL2. In contrast to the action of CCL2, its chemokine receptor CCR2 has a positive effect on Th1 polarization based on the results from CCR2 knockout mice . C-X-C motif chemokine receptor 3 (CXCR3) is a transmembrane chemokine receptor that is activated by the three IFN-γ-inducible chemokines of the CXC family: CXCL9, CXCL10, and CXCL11 . It is expressed on activated T cells, especially the Th1 subset , and is thus associated with a Th1 phenotype ,,.
The present study aimed to explore the probable role of CCL2 and its receptor CCR2 in addition to CXCR3 gene expression in the pathogenesis and severity of ITP. To our knowledge, CCL2 chemokine and its receptor CCR2 in addition to CXCR3 receptor in childhood-onset ITP have not been extensively investigated.
| Participants and methods|| |
The study included 68 children with ITP (40 females and 28 males, median age: 7 years, range: 0.8–17 years) who visited hematological outpatient clinic or admitted in Cairo University Specialized Pediatric Hospital (CUSPH) and Cairo University Children Hospital El-Mounira (Hematology Clinic). ITP was diagnosed in accordance with the guidelines of the American Society of Hematology . Patients with connective tissue diseases such as systemic lupus erythematosus (SLE) were excluded from the study. Among the enrolled patients, 24 were patients with acute newly diagnosed ITP and did not receive any glucocorticoid and/or immunosuppressive treatments before sampling. Twenty-five patients were chronic responder ITP cases who were clinically stable on corticosteroids and/or immunosuppressive agents. Nineteen patients were chronic non-responder ITP cases. They were pretreated with different types of immunosuppressive therapies and showed no clinical response. Clinical characteristics of different ITP groups are shown in [Table 1]. Clinical parameters were collected from patients’ medical records.
|Table 1 Clinical characteristics of patients with immune thrombocytopenia|
Click here to view
A total of 21 healthy children (eight females and 13 males; median age: 5.5 years, range: 1–12 years) were enrolled as a normal control group. Their platelet counts ranged between 150 and 510×109/l, with a median count of 344.5×109/l. The ethical committee of the Faculty of Medicine, Cairo University, approved this study. Consents were taken from parents/guardians of all participants before being involved in this study.
Gene expression analysis using quantitative real-time polymerase chain reaction
Peripheral blood mononuclear cells (PBMNCs) were isolated from 2-ml EDTA anticoagulated venous blood sample, withdrawn from each participant included in the study, by density gradient using 1.077 g/ml Ficoll–Hypaque (St. Louis, Missouri, USA). Total RNA was extracted from PBMNCs with QIAamp RNA blood mini kit (QIAGEN, Austin, Texas, USA), according to the manufacturer’s instructions. Total RNA was reversely transcribed in a total volume of 20 µl reaction using high-capacity cDNA reverse transcription kit (Applied Biosystems, USA). PCR amplification for CCL2 and its receptor CCR2, in addition to CXCR3 mRNA was performed by Real-Time PCR (7300; Applied Biosystems, Foster City, California) using QuantiTect SYBR Green Master Mix (Applied Biosystems, Warrington, UK). β-Actin (housekeeping gene) was used to normalize RNA. Primers used in this assay are shown in [Table 2] .
Amplification of CCL2 and its receptor CCR2, CXCR3, in addition to the housekeeping gene was done in separate reactions. Total volume of each reaction was 25 µl containing 2.5 µl cDNA, 12.5 µl 2× QuantiTect SYBR Green PCR Master Mix, 0.75 µl of each of the forward and reverse primers (300 nmol/l), and 8.5 µl of RNAase free water. The program consisted of an initial step for 15 min at 95°C followed by 45 cycles, with each cycle consisting of 15 s at 94°C, and 1 min at 60°C. Melting curve analysis of amplification products was performed at the end of each PCR reaction by changing temperature from 60 to 95°C for 15 s, then 60°C for 1 min, then 95°C for 30 s, and finally 60°C for 15 s, to distinguish genuine products from nonspecific products and primer dimers. The relative expression of each of the target genes was determined using the ΔΔCt method, as previously described . A comparative threshold cycle (Ct) was used to determine the gene expression relative to a normal control. Each sample of either patient or control was normalized for the expression of β-Actin using the formula ΔCt=(Ct target–Ct β-actin). The mean expression of the control samples was then chosen as a normal calibrator, and relative target expression for every sample was calculated using 2–ΔΔCt formula, where ΔΔCt=ΔCt target–ΔCt calibrator. The final result of this method is presented as the fold change of the target gene expression in the patient’s group relative to the control group. The relative gene expression is usually set to 1 for control group because ΔΔCt is equal to 0 and therefore 20 is equal to 1.
Data were analyzed using IBM SPSS advanced statistics version 20 (SPSS Inc., Chicago, Illinois, USA). χ2-Test (Fisher’s exact test) was used to examine the relation between qualitative variables. For not normally distributed quantitative data, comparison between two groups was done using Mann–Whitney test (nonparametric t-test). Comparison between three groups was done using Kruskal–Wallis test (nonparametric analysis of variance) and then pair-wise comparison based on Kruskal–Wallis test. Spearman’ ρ method was used to test correlation between numerical variables. A P value less than 0.05 was considered significant.
| Results|| |
CCL2 and its receptor CCR2 relative gene expression
Acute ITP group showed a 1.85 median fold change in CCL2 gene expression from the control group. On the contrary, chronic responder and chronic non-responder groups’ CCL2 gene expression was less than the control group, 0.80 and 0.87, respectively. CCL2 expression levels among the three ITP groups did not differ significantly (P>0.05; [Table 3]).
|Table 3 CCL2, CCR2, and CXCR3 gene expression patterns in patients with immune thrombocytopenia|
Click here to view
Compared with the control group, CCR2 showed a higher median fold change in patients with acute ITP (7.36) than chronic non-responder patients (3.38), whereas chronic responder patients showed the least change (1.63). Comparisons between the three ITP groups did not yield a significant difference in relative gene expression (P>0.05%; [Table 3]).
CXCR3 relative gene expression
Acute ITP and chronic non-responder patients showed higher levels of CXCR3 gene expression (5.42 and 4.32 median fold change) in comparison with the control group whereas the chronic responder group showed relatively lower level (2.35 median fold change) in relation to the latter groups. However, on comparing CXCR3 relative gene expression levels between the three studied ITP groups, there was no statistically significant difference (P>0.05; [Table 3]).
Relationship of target gene expression levels and different laboratory parameters
Statistically significant positive correlations were detected between CCL2 and CCR2 (r=0.453, P<0.001) and between CXCR3 and CCR2 (r=0.583, P<0.001; [Figure 1]) among all patients with ITP. Moreover, a statistically significant positive correlation was found between absolute lymphocytic counts and CCR2 level (r=0.459, P=0.028; [Figure 2]) within the acute group, with no significant correlations among other studied groups.
|Figure 1 Correlation between CXCR3 and CCR2 among all patients with immune thrombocytopenia.|
Click here to view
|Figure 2 Correlation between CCR2 and absolute lymphocytic count in acute immune thrombocytopenia.|
Click here to view
Platelet counts were not significantly correlated with the expression levels of any of our target genes expression levels in all studied groups (P>0.05).
Follow-up of acute cases after 6 months revealed cure in 62.5% of cases and chronicity in 37.5%. On comparing the relative gene expression levels of assayed chemokines in cured and chronic cases, no significant difference in expression levels was detected. with P values greater than 0.05.
| Discussion|| |
Owing to the emerging evidence that deregulation of the chemokine pathways plays an important role in autoimmunity, several studies were directed toward a better understanding of the role of chemokines in autoimmune disorders . Leukocyte subsets express different receptors, which determine the range of chemokine actions .
In the present study, acute ITP group showed a 1.85 median fold change in CCL2 gene expression from the control group; however, the chronic responder and chronic non-responder groups’ gene expression levels were lower than the control. Similarly, CCL2 relative gene expression in the study by Gu et al.  was higher in PBMNCs and splenocytes of patients with active adult ITP than PBMNCs of the control group, yet this level showed no statistically significant difference (P>0.05). Moreover they measured CCL2 levels by enzyme-linked immunosorbent assay, which also did not differ significantly in their examined groups. The role of CCL2 in other autoimmune disorders is controversial. Some studies have shown its implication in some autoimmune disorders. Liou et al. , demonstrated a significant higher level of CCL2 in patients with rheumatoid arthritis than in healthy controls where they considered plasma CCL2 as one of the best indicators of clinical arthritic activity awaiting for confirmation by further studies on different racial and ethnic groups. In another study by Abujam et al. , urinary and serum CCL2 levels were found to be higher in patients with active SLE compared with inactive SLE. On the contrary, other studies failed to find a role for CCL2 in other autoimmune disorders such as type-1 diabetes and chronic autoimmune thyroiditis ,. Further studies on specific T-cell subsets and in different tissues could help better eliciting the role of CCL2 in autoimmune disorders.
Regarding CCR2 in acute and chronic non-responder groups, it showed 7.36 and 3.38 median fold change in gene expression from the control group, respectively. The chronic responder group showed a lower level of expression than the latter groups with 1.63 median fold change from the control group. These findings are consistent with previous results showing the positive effect of CCR2, but not CCL2, on Th1 polarization studied in CCR2 knockout mice ,. Our results were not consistent with Gu et al.  who found a significant decrease in CCR2 expression level in PBMNCs of patients with active ITP when compared with healthy controls; however, they found a significantly higher CCR2 level in the spleen of patients with ITP when compared with PBMNCs. Furthermore, the present study showed a positive significant correlation between CXCR3 and CCR2 gene expression, thus our results reinforce the concept that ITP is accompanied by a Th1 immune response. In fact, the exact role of CCR2 in autoimmune diseases and especially in ITP is still controversial, and more studies on a larger scale are needed to clarify its significance in ITP pathogenesis along with other autoimmune disorders.
Compared with the control group, acute ITP and chronic non-responder patients showed higher levels of CXCR3 gene expression (5.42 and 4.32 median fold change) whereas the chronic responder group showed a relatively lower level (2.35 median fold change) in relation to the latter groups. Liu et al.  demonstrated an elevated level for both Th1 chemokine receptors (CCR5 and CXCR3) in patients with active ITP compared with the control. The CCR5 level was normalized in response to a high-dose dexamethasone, whereas the CXCR3 receptor level only decreased but remained higher than the control group. The latter study prompted regulation of Th1 polarization through pulsed high-dose dexamethasone therapy as a new approach for immunoregulation in ITP . Similarly, Zhang et al.  observed significant elevation in the expression of CXCR3 mRNA in patients with ITP, and these levels decreased after treatment; however, they remained higher than the control levels. Studies on other autoimmune disorders demonstrated that CXCL10 and its receptor CXCR3 have strong chemotactic activity and have a significant role in several autoimmune diseases such as SLE  and rheumatoid arthritis , multiple sclerosis . Although Gu et al.  did not find significant difference in CXCR3 expression levels in PBMNCs between patients with ITP and normal controls, they proved an elevated expression of CXCR3 in the spleen of active ITP than in PBMNCs. Moreover, Zhou et al.  assessed Th1-associated chemokine receptors CXCR3 and CCR5 together with Th2-associated chemokine receptors CCR3 in the spleen of patients with ITP and found a higher rate of expression of CXCR3 and CCR5, but a significantly reduced rate of expression of CCR3, suggesting that abnormal expression of Th1/Th2 chemokine receptors could be a reason for considering ITP as a splenic immune disorder. These data may indicate a probable role of CXCR3 expression in active disease process in patients with ITP.
Chemokines and their receptors represent a complicated control system of immune responses in different autoimmune diseases. In the present study, we demonstrated an elevated relative gene expression of CCR2 and CXCR3 in relation to the control group; supporting a Th1 polarization in childhood-onset ITP. Compared with the control, CCL2 (Th2 polarization-associated chemokine) gene expression levels were slightly elevated in patients with acute ITP but not in chronic responder or chronic non-responder groups. Taken together, our findings support the role of Th1 polarization in ITP pathogenesis. Further studies investigating the role of the complicated chemokine network and its receptors on a larger subset of childhood-onset ITP may help a better understanding of ITP pathogenesis in children.
The authors acknowledge the patients for their participation in the study and the Faculty of Medicine, Cairo University for funding this work.
Financial support and sponsorship
The study was done by a fund form Faculty of medicine Cairo University.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zufferey A, Kapur R, Semple J. Pathogenesis and therapeutic mechanisms in immune thrombocytopenia (ITP). J Clin Med
Lambert MP, Gernsheimer TB. Clinical updates in adult immune thrombocytopenia. Blood
Ji L, Zhan Y, Hua F, Li F, Zou S, Wang W et al.
The ratio of Treg/Th17 cells correlates with the disease activity of primary immune thrombocytopenia. PLoS One
Shulman NR, Marder VJ, Weinrach RS. Similarities between known antiplatelet antibodies and the factor responsible for thrombocytopenia in idiopathic purpura. Physiologic, serologic and isotopic studies. Ann N Y Acad Sci
Khodadi E, Asnafi AA, Shahrabi S, Shahjahani M, Saki N. Bone marrow niche in immune thrombocytopenia: a focus on megakaryopoiesis. Ann Hematol
Cines DB, Bussel JB, Leibman HA, Luning Prak ET. The ITP syndrome: pathogenic and clinical diversity. Blood
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
Ware RE, Howard TA. Phenotypic and clonal analysis of T lymphocytes in childhood immune thrombocytopenic purpura. Blood
Rocha AM, Souza C, Rocha GA, de Melo FF, Clementino NC, Marino MC et al.
The levels of IL-17A and of the cytokines involved in Th17 cell commitment are increased in patients with chronic immune thrombocytopenia. Haematologica
Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature
Rogge L, Barberis Maino L, Biffi M, Passini N, Presky DH, Gubler U. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J Exp Med
Gu L, Tseng S, Horner RM, Tam C, Loda M, Tollins BJ. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature
Wang T, Zhao H, Ren H, Guo J, Xu M, Yang R. Type 1 and Type 2 T-cell profiles in idiopathic thrombocytopenic purpura. Haematologica
Bonecchi R, Bianchi G, Bordignon PP, D’Ambrosio D, Lang R, Borsatti A et al.
Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells. J Exp Med
Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity
Wang J, Knaut H. Chemokine signaling in development and disease. Development
Lacotte S, Brun S, Muller S, Dumortier H. CXCR3, inflammation, and autoimmune diseases. Ann N Y Acad Sci
Loetscher P, Uguccioni M, Bordoli L, Baggiolini M, Moser B, Chizzolini C et al.
CCR5 is characteristic of Th1 lymphocytes. Nature
Imai T, Nagira M, Takagi S, Kakizaki M, Nishimura M, Wang J et al.
Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int Immunol
Sallusto BF, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med
George JN, Woolf SH, Raskob GE, Wasser JS, Aledort LM, Ballem PJ et al.
Idiopathic thrombocytopenic purpura: a practice guideline by explicit methods for the American Society of Hematology. Blood
Gu D, Chen Z, Zhao H, Du W, Xue F, Ge J et al.
Th1 (CXCL10) and Th2 (CCL2) chemokine expression in patients with immune thrombocytopenia. Hum Immunol
Bloch G, Toma DP, Robinson GE. Behavioral rhythmicity, age, division of labor and period expression in the honey bee brain. J Biol Rhythms
Baggiolini M, Dewald B, Moser B. Human chemokines: an update. Annu Rev Immunol
Liou LB, Tsai WP, Chang CJ, Chao WJ, Chen MH. Blood monocyte chemotactic protein-1 (MCP-1) and adapted disease activity score 28-MCP-1: favorable indicators for rheumatoid arthritis activity. PloS One
Abujam B, Cheekatla S, Aggarwal A. Urinary CXCL-10/IP-10 and MCP-1 as markers to assess activity of lupus nephritis. Lupus
Antonelli A, Fallahi P, Ferrari SM, Pupilli C, D’Annunzio G, Lorini R et al.
Serum Th1 (CXCL10) and Th2 (CCL2) chemokine levels in children with newly diagnosed type 1 diabetes: a longitudinal study. Diabet Med
Antonelli A, Rotondi M, Fallahi P, Romagnani P, Ferrari SM, Paolicchi A et al.
Increase of interferon-gamma inducible alpha chemokine CXCL10 but not beta chemokine CCL2 serum levels in chronic autoimmune thyroiditis. Eur J Endocrinol
Traynor TR, Herring AC, Dorf ME, Kuziel WA, Toews GB, Huffngale GB. Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity. J Immunol
Liu Z, Wang M, Zhou S, Ma J, Shi Y, Peng J et al.
Pulsed high dose dexamethasone modulates Th1/Th2 chemokine imbalance in immune thrombocytopenia. J Transl Med
Zhang X, Feng JM, Li WQ, Li JP, Chen SB, Han GX. The role of CXCR3 and its ligand I-TAC in the pathogenesis of immune thrombocytopenic purpura. Zhonghua Nei Ke Za Zhi
Narumi S, Takeuchi T, Kobayashi Y, Konishi K. Serum levels of INF-inducible PROTEIN-10 relating to the activity of systemic lupus erythematosus. Cytokines
Hanaoka R, Kasama T, Muramatsu M, Yajima N, Shiozawa F, Miwa Y et al.
A novel mechanism for the regulation of IFN-gamma inducible protein-10 expression in rheumatoid arthritis. Arthritis Res Ther
Sorensen TL, Tani M, Jensen J, Pierce V, Lucchinetti C, Folcik VA et al.
Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J Clin Invest
Zhou S, Ma J, Qu HT, Liu ZT, He WT, Wang JD et al.
Characterization of Th1- and Th2-associated chemokine receptor expression in spleens of patients with immune thrombocytopenia. J Clin Immunol
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]