|Year : 2013 | Volume
| Issue : 1 | Page : 47-50
Role of T-helper 17 cells and interleukin-17 expression in patients with acute myeloid leukemia
Mona A Ismail1, Mohamed T.H. Sallam1, Walaa A El Salakawy2
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Clinical Hematology and Bone Marrow Transplant Unit, Internal Medicine Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||25-Sep-2012|
|Date of Acceptance||16-Oct-2012|
|Date of Web Publication||20-Jun-2014|
Mona A Ismail
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University Hospitals, Ramses St, Abbassia, Cairo 11566
Source of Support: None, Conflict of Interest: None
Several observations suggest that immunological events are important for antileukemic immune reactivity in acute myeloid leukemia (AML). T-helper type 17 (Th17) cells play an active role in inflammatory and autoimmune diseases; however, the nature of Th17 cells in hematological malignancies remains unknown.
The aim of this work was to assess the role of Th17 cells and its related cytokine interleukin-17 (IL-17) in the pathogenesis and prognosis of AML.
Participants and methods
The study was carried out on 40 adult AML patients and 20 age-matched and sex-matched controls. Patients were diagnosed and classified according to the WHO criteria for AML. Plasma levels of IL-17 in patients and controls were measured using the sandwich ELISA technique. Th17 cells were assessed using flow cytometry.
The percentage of Th17 cells and the concentration of IL-17 were significantly increased in untreated patients with AML compared with the healthy controls (10.2±2.2 vs. 4.38±0.16% and 20.15±2.9 vs. 8.2±1.3 pg/ml, respectively). In addition, the increased Th17 cells were reduced significantly when the patient achieved complete remission after chemotherapy, but the decrease was not significant in patients with incomplete remission.
These results suggest that Th17 cells play a role in the pathogenesis and response of therapy in AML patients through the secretion of IL-17 cytokine.
Keywords: acute myeloid leukemia, cytokines, T-helper type 17 cells
|How to cite this article:|
Ismail MA, Sallam MT, El Salakawy WA. Role of T-helper 17 cells and interleukin-17 expression in patients with acute myeloid leukemia. Egypt J Haematol 2013;38:47-50
|How to cite this URL:|
Ismail MA, Sallam MT, El Salakawy WA. Role of T-helper 17 cells and interleukin-17 expression in patients with acute myeloid leukemia. Egypt J Haematol [serial online] 2013 [cited 2020 Apr 10];38:47-50. Available from: http://www.ehj.eg.net/text.asp?2013/38/1/47/134803
| Introduction|| |
Acute myeloid leukemia (AML) is an aggressive disorder that results from a block in the differentiation of hematopoietic progenitor cells along with uncontrolled proliferation 1. The only curative treatment is intensive chemotherapy that can be combined with stem cell transplantation, especially in patients younger than 60 years of age 2. This treatment is followed by a period of severe pancytopenia including lymphopenia. Rapid lymphoid reconstitution after therapy is associated with increased AML-free survival, an observation strongly suggesting that immunological events early after chemotherapy are clinically important 3,4.
Circulating CD4 T cells are essential regulators of an antitumor immune response. They are divided traditionally into three major subsets: T-helper type 1 (Th1), Th2, and regulatory T cells (Treg) 5. More recently, a unique T cell subset named Th17 has been described 6. They play important roles in the pathogenesis of many diseases, including inflammatory diseases, autoimmune diseases, and cancers. A range of cytokines, such as interleukin-23 (IL-23), transforming growth factor-β (TGF-β), IL-1β, IL-6, IL-17, IL-22, and IL-21, have been found to be related to Th17 cells 7,8.
Previous studies have described increased levels of circulating immunosuppressive CD4+ CD25+ Treg cells in patients with untreated AML 9,10 and their involvement in the pathogenesis of solid tumors, but the role of Th17 cells in tumor immunity has been poorly defined 11,12.
Hence, the aim of this study was to determine the role of Th17 cells and its related cytokine IL-17 in the pathogenesis and prognosis of AML.
| Participants and methods|| |
The study was carried out on 40 patients with AML attending Ain Shams University Hospitals, ranging in age from 39 to 70 years, mean age 53.75±3.23 years. There were 23 men and 17 women, with a male to female ratio of 1.3/1. Twenty age-matched and sex-matched control individuals whose bone marrow (BM) samples were free of hematologic disease were used as a control group. Patients with inflammation, autoimmune diseases, or malignancy were excluded from the study.
All patients were subjected to a thorough history taking and clinical examination, complete blood picture (using LH750 Coulter; Beckman, Hialeah, Florida, USA), and flow cytometric (FCM) immunophenotyping for the estimation of CD3, CD4, and Th17 (using Epics XL flow cytometer; Coulter Electronics, Hialeah, Florida, USA); in addition, IL-17 was determined using an enzyme-linked immunosorbent assay (R&D Systems Inc., Minneapolis, Minnesota, USA).
The diagnosis and classification of leukemic cases were based on morphology and cytochemistry according to the French American British classification and fulfilled the WHO criteria for AML, together with immunophenotyping results 13.
Three milliliters of venous blood was withdrawn aseptically into vials containing EDTA: 1 ml for FCM immunophenotyping and 2 ml for measurement of the plasma level of IL-17.
Flow cytometric immunophenotyping
FCM immunophenotyping was carried out using the whole-blood lysis method. Cells were incubated with FITC-labeled anti-CD4 and PC-5-labeled CD3 (BD Biosciences, Mountain View, California, USA) for 30 min at 4°C in the dark. After washing, the cells were fixed with a fixation solution and incubated for 20 min at room temperature in the dark. After permeabilization, cells were incubated with a PE-labeled anti-human cytokine antibody (for Th17 detection) for 20 min at room temperature in the dark. An appropriate isotype control IgG1 was used in all cases to determine the background fluorescence intensity.
The lymphocytes were identified according to their forward and side scatter and analyzed using flow cytometry. The frequency of Th17 cells in peripheral blood (PB) of AML patients and controls was assessed.
Enzyme-linked immunosorbent assay for interleukin-17
To determine the level of IL-17, blood samples were centrifuged at 2000g for 20 min. Plasma fractions were aspirated and transferred to a plastic tube and stored at −20°C until analyzed for the measurement of IL-17 (eBioscience, San Diego, California, USA).
Assessment of remission
Morphologic complete remission (CR) was defined by (i) PB neutrophil count greater than 1.5×109/l and platelet count greater than100×109/l, (ii) absence of blasts in PB, (iii) less than 5% blasts with no detectable Auer rods in the BM sample showing more than 20% cellularity with maturation of all cell lines, and (iv) absence of extramedullary leukemia. Incomplete remission (IR) was defined as 5–10% blasts or less than 5% blasts in the presence of Auer rods in the BM with adequate cellularity and PB without leukemic cells 14.
Data were analyzed using the statistical package for social science (SPSS version 15; Chicago, Illinois, USA). Quantitative data were described in the form of mean±SD, whereas qualitative data were described in the form of number and percentage. Comparisons between paired or unpaired groups were carried out using the appropriate paired t-test. A P value less than 0.05 was considered significant and 0.01 as highly significant.
| Results|| |
The results of the present study are presented in [Table 1], [Table 2] and [Table 3] and [Figure 1] and [Figure 2].
|Table 1: Frequency of T-helper type 17 cells and plasma level of interleukin-17 in acute myeloid leukemia patients compared with controls|
Click here to view
|Table 2: Frequency of T-helper type 17 cells and plasma level of interleukin-17 in response to therapy|
Click here to view
|Table 3: Frequency of T-helper type 17 cells in acute myeloid leukemia patients before and after treatment|
Click here to view
|Figure 1: Flow cytometric analysis of control. (a) FS and SS showing gating on peripheral blood lymphocytes; (b) CD3+ cells; (c) expression of CD4+ and Th17+ cells. FS, forward scatter; SS, side scatter; Th17, T-helper type 17.|
Click here to view
|Figure 2: Flow cytometric analysis of an acute myeloid leukemia patient. (a) FS and SS showing gating on peripheral blood lymphocytes; (b) CD3 cells; (c) expression of CD4+ and Th17+ cells. FS, forward scatter; SS, side scatter; Th17, T-helper type 17.|
Click here to view
The Th17 cells in CD3+ CD4+ T cells were increased in AML patients compared with healthy volunteers (10.2±2.2 vs. 4.38±0.16%, respectively), and this difference was statistically significant with a P value less than 0.05 [Table 1].
In terms of the concentration of plasma IL-17, the level was increased in AML patients compared with the controls (20.15±2.9 vs. 8.2±1.3 pg/ml, respectively). This increase was highly significant with a P value less than 0.01.
Patients were followed up for 15 months to determine patient outcome. Twenty-four patients (60%) achieved CR, whereas 16 patients (40%) achieved IR (two patients died before the first assessment of remission).
The percentage of Th17 cells and the level of IL-17 were higher in patients with IR (11.5±3.5% and 22.5±1.1 pg/ml) compared with patients with CR (5.8±1.2% and 15.3±3.2 pg/ml), and this difference was statistically significant (P<0.01 and P<0.05) [Table 2].
The percentage of Th17 cells was reduced significantly in CR patients (1.9±1.2%) compared with the same patients before treatment (5.8±1.2%), with a P value less than 0.05. In patients with IR, the frequency of Th17 cells (9.8±3.3%) was reduced compared with the same patients before treatment (11.5±3.5%), but this decrease was statistically insignificant (P>0.05) [Table 3].
| Discussion|| |
AML is characterized by distorted proliferation and development of myeloid cells and their precursors in blood and BM. Although impressive biologic advances have advanced our understanding of leukemogenesis, little is known about the pathogenic events that lead to the initiation and progression of AML 8,15.
Th17 cells and their effector cytokines are increasingly being recognized as important mediators in inflammatory autoimmune diseases, but relatively little is known about their specific roles in human tumor immunity. Human Th17 cells release the proinflammatory cytokine IL-17; one important function of IL-17 seems to be the coordination and regulation of local inflammation through upregulation of other proinflammatory cytokines and chemokines 16.
The present study was carried out on 40 patients with AML and 20 controls with the aim of determining the role of Th17 cells and the concentration of the related cytokine (IL-17) in the pathogenesis and prognosis of AML.
Assessment of Th17 cells and IL-17 expression showed higher expression in AML patients compared with the control group, thus suggesting a tumorogenesis role of Th17 through the secretion of IL-17 that may promote the development or progression of AML patients. These results were in agreement with other studies that found that the frequency of Th17 cells and the concentrations of IL-17 were higher in untreated patients than those in controls 17.
In addition, similar results were obtained from patients with solid tumors 18, 19, and some studies in animals have shown that IL-17 may promote angiogenesis and tumor growth 20,21.
However, these results were not consistent with other studies that found that the frequency of Th17 cells and the serum level of IL-17 were not elevated in AML patients compared with controls. This contradiction in the results could be because of several factors including patients’ selection, the small number of samples (10 samples) used from healthy volunteers, which may not have been adequate to show a difference, and the differences in the treatment regimens used for the patients 22,23.
In terms of response to therapy, patients were followed up for 15 months. The percentage of Th17 cells and the level of IL-17 were higher in patients with IR compared with patients with CR. This was in agreement with Wu et al. 17, who found that the frequency of Th17 cells and the concentration of related cytokines (IL-17, IL-6, and TGF-β) were higher in AML patients compared with controls and that IL-6 was critical for human Th17 cell differentiation, whereas the role of TGF-β needs to be clarified.
Moreover, the percentage of Th17 cells were reduced significantly in CR patients compared with the same patients before treatment. Similar results were obtained by Fan et al. 24, who found a decrease in Th17 cells in untreated patients when those patients achieved CR after chemotherapy. This was also in agreement with Wu et al. 17, who reported an increase in circulating Th17 cells and IL-17 plasma levels in untreated AML patients, but in contrast to Treg, these levels were normalized when complete hematological remission was achieved.
These results may suggest that pretherapeutic and post-therapeutic assessment of Th17 cell frequency may be valuable as an evaluation of the therapeutic effect 17 and that the number of Th17 cells may relate to tumor burden, which is an important prognostic factor in a variety of tumor types 25.
In patients with IR, the frequency of Th17 cells was not significantly reduced compared with the same patients before treatment. These results were in agreement with the results of other studies carried out by Wu et al. 17, who found a decrease in Th17 cells in patients with IR compared with the same patients before treatment, but this reduction was not statistically significant.
From this study, it can be concluded that Th17 cells and their effector cytokine IL-17 can be used as indicators for therapeutic response and may play an important role in the occurrence and development of AML. However, the precise involvement of Th17 cells in tumor pathogenesis needs more research and clarification.
| References|| |
|1.||Gregory T, Wald D, Chen Y, Vermaat J, Xiong Y, Tse W. Molecular prognostic markers for adult acute myeloid leukemia with normal cytogenetics. J Hematol Oncol. 2009;2:23–30 |
|2.||Smith M, Barnett M, Bassan R, Gatta G, Tondini C, Kern W. Acute myeloid leukemia. Crit Rev Oncol Hematol. 2004;50:197–222 |
|3.||Williams K, Hakim F, Gress R. T cell immune reconstitution following lymphodepletion. Semin Immunol. 2007;19:318–330 |
|4.||Auletta J, Lazarus H. Immune restoration following hematopoietic stem cell transplantation: an evolving target. Bone Marrow Transplant. 2005;35:835–857 |
|5.||Murphy K, Reiner S. The lineage decisions of helper T cells. Nat Rev Immunol. 2002;22:933–944 |
|6.||Park H, Li Z, Yang X. A distinct lineage of CD4 T cells regulates tissue inflammation by producing IL-17. Nat Immunol. 2005;6:1133–1141 |
|7.||Volpe E, Servant N, Zollinger R. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human Th17 responses. Nat Immunol. 2008;9:650–657 |
|8.||Li P, Ji M, Park J, Bunting K, Ji C, Tse W. Th17 related cytokines in acute myeloid leukemia. Front Biosci. 2012;17:2284–2294 |
|9.||Szczepanski M, Szajnik M, Czystowska M, Mandapathil M, Strauss L, Welsh A, et al. Increased frequency and suppression by regulatory T cells in patients with acute myelogenous leukemia. Clin Cancer Res. 2009;15:3325–3332 |
|10.||Wang X, Zheng J, Liu J, Yao J, He Y, Li X, et al. Increased population of CD4+ CD25high, regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute leukemia patients. Eur J Haematol. 2005;75:468–476 |
|11.||Pages F, Berger A, Camus M. Effector memory T cells, early metastesis and survival in colorectal cancer. N Engl J Med. 2005;353:2654–2666 |
|12.||Ormandy L, Hillemann T, Wedemeyer H, Mannus M, Greten T, Korangy F. Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma. Cancer Res. 2005;65:2457–2464 |
|13.||Vardiman J, Thiele J, Arber D, Brunning R, Borowitz M, Porwit A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114:937–951 |
|14.||Cheson B, Bennett J, Kopecky K, Büchner T, Willman C, Estey E, et al. Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol. 2003;21:4642–4649 |
|15.||Peters B, Matthews J, Gompels M, Hartley J, Pincing A. Acute lymphoblastic leukemia in AIDS. AIDS. 1990;4:367–368 |
|16.||Park K, Li Z, Yang X, Chang S, Nurieva R, Wang Y, et al. A distinct lineage of CD4 T cells regulate tissue inflammation by producing interleukin 17. Nat Immunol. 2005;6:1133–1141 |
|17.||Wu C, Wang S, Wang F, Chen Q, Peng S, Zhang Y, et al. Increased frequencies of T helper type 17 cells in the peripheral blood of patients with acute myeloid leukemia. Clin Exp Immunol. 2009;158:199–204 |
|18.||Kryczek I, Wei S, Zou L. T helper 17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment. J Immunol. 2007;178:6730–6733 |
|19.||Zhang B, Rong G, Wei H. The prevalence of Th17 cells in patients with gastric cancer. Biochem Biophys Res Commun. 2008;374:533–537 |
|20.||Numasaki M, Fukushi J, Ono M. Interleukin 17 promotes angiogenesis and tumor growth. Blood. 2003;101:2620–2627 |
|21.||Numasaki M, Watanabe M, Suzuki T. IL-17 enhances the net angiogenic activity and in vivo growth of human non-small cell lung cancer in SCID mice through promoting CXCR2 dependent angiogenesis. J Immunol. 2005;175:6177–6189 |
|22.||Wrobel T, Mazur G, Jazwiec B, Kuliczkowski K. Interleukin-17 in acute myeloid leukemia. J Cell Mol Med. 2003;7:472–474 |
|23.||Ersvaer E, Liseth K, Skavland J, Giertsen B, Bruserud J. Intensive chemotherapy for acute myeloid leukemia differentially affects TC1, TH1, TH17 and TREG cells. BMC Immunol. 2010;11:38–45 |
|24.||Fan Z, Zhang L, Chai Y, Zeng P, Wu C. The prevalence of Th17 cells in patients with acute myeloid leukemia. Zhonghua Xue Ye Xue Za Zhi. 2010;31:617–620 |
|25.||Scarisbrick J, Whittaker S, Evans A. Prognostic significance of tumor burden in the blood of patients with erythrodermic primary cutaneous T cell lymphoma. Blood. 2001;97:624–630 |
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]