- Email: [email protected]
Assessment of Interleukin 2 Cytokine Expression Levels After Renal Transplantation
Assessment of Interleukin 2 Cytokine Expression Levels After Renal Transplantation H._I. Karahana,b, M. Soyöza,b,*, M. Pehlivanc, E. Tatard, A. Uslue, B. Çerçi Gürbüza,b, _I. Pirima,b, and T. Kiliçaslan Aynaa,b a Medical Biology and Genetics Department, Faculty of Medicine, Izmir Katip Celebi University, Izmir, Turkey; bTissue Typing Laboratory, Health Science University Tepecik Education and Research Hospital, Izmir, Turkey; cVocational School of Health Services, Izmir Katip Celebi University Izmir, Turkey; dNephrology, Health Science University Bozyaka Education and Research Hospital, Izmir, Turkey; and e General Surgery, Health Science University Bozyaka Education and Research Hospital, Izmir, Turkey
ABSTRACT Aim. End-stage renal disease is a disease in which the kidney is not able to perform its functions. Kidney transplantation is the most effective treatment and cost-effective modality of renal replacement therapy for patients with end-stage renal disease. However, the most important problem in end-stage renal disease patients is the unpredictability of immunologic response after transplants. In this study, it was aimed to investigate the possible association between the interleukin 2 (IL-2) expression level and an organ rejection or rejection episode. Materials and Methods. Lymphocytes were isolated from peripheral blood obtained from 21 end-stage renal diseaseediagnosed patients prior to transplant and at the sixth month after transplant. CD4þ T cells were separated from lymphocytes by the magnetic cell-sorting method. The purity of these cells were controlled by a flow cytometer. After total RNA isolation from CD4þ T cells, IL-2 was examined by the real-time polymerase chain reaction (RT-PCR) method. Results. Among nonrejection patients (n ¼ 18), the IL-2 expression level decreased in 12 patients in post-transplant time, and 3 of these were statistically significant (P < .05). The level was the same in 1 of 18 patients; it increased in 5 patients, and 1 of them was significant (P < .05). The IL-2 expression level also increased in 3 patients who had a rejection episode, and the increase was statistically significant in 2 samples (P < .05). Conclusion. When the patients were evaluated individually, it was observed that there might be a relationship between IL-2 expression levels in CD4þ T cells and rejection episodes. The clinical data of the patients, the immunosuppressive therapies, and posttransplant evaluation of cytokines should be considered together.
D
ISRUPTION of the main functions of the kidney causes renal failure. Renal transplantation is the most valid method in the treatment of end-stage renal disease. However, the causes of subsequent complications, which occur after transplantations and lead to organ rejection, have not been discovered [1,2]. T cells, 1 of the lymphocyte population groups, lead to an immune response after transplantation. After activation and differentiation into different effector subtypes, CD4þ T cells play an important role in mediating the immune response via specific cytokines [3]. Cytokines regulate 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.02.008
1074
lymphocyte activation and differentiation. Interleukin 2 (IL-2) is a major cytokine molecule that plays a curious role in the immune response produced by CD4þ T cells. It is a This study was supported by the Scientific Research Projects Administration (project number 2017-TYL-SABE-0046) and the Teaching Assistant Education Program. *Address correspondence to Mustafa Soyöz, _Izmir Katip Celebi University, Izmir Tepecik Education and Research Hospital, Tissue Typing Lab, A Bloc, 1st Floor, 35110, Yenisehir-Konak, _Izmir, Turkey. Tel: (0232) 469 69 69-1709; Fax: (0232) 433 07 56. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1074e1077 (2019)
IL-2 CYTOKINE EXPRESSION LEVELS
133-amino acid residual polypeptide chain and is mainly produced by T cells [4,5]. It is thought that the determination of the IL-2 cytokine level will play a role in elucidating the immune response after transplantation. The aims of this study were to investigate the effect of IL2 on rejection in early post-transplant time and to predict the rejection episode in the long term. MATERIAL AND METHODS Peripheral blood samples were obtained from 21 patients who were diagnosed with end-stage renal disease according to the international study group criteria prior to the transplants and at the sixth month after transplantation. Inclusion criteria were ABO blood group compatibility, human immunodeficiency virus negativity, and cross-match negativity. Three of the patients were diagnosed with a rejection episode by the clinicians. All patients received anti-thymocyte globulin (ATG) and corticosteroids (prednisolone) for induction immunosuppression. Rabbit-ATG (Fresenius) was administered to all patients on the zeroth and first days in doses of 4 mg/kg and 2 mg/kg, respectively. In cases of delayed graft function, ATG was administered at doses of 1 to 2 mg/kg every other day until the serum creatinine value was reduced to 3 mg/dL. Corticosteroid protocol was administered on the zeroth, first, and second days in doses of 1000 mg/day, 250 mg/ day, and 100 mg/day, respectively. Subsequently, prednisolone was decreased 20 mg each day until 20 mg/day, and patients were discharged with 0.3 mg/kg/day. The maintenance immunosuppressive protocol included an antimetabolite (mycophenolate mofetil or mycophenolic acid), a calcineurin inhibitor (tacrolimus or cyclosporin), and a corticosteroid. The whole blood concentration targets of tacrolimus and cyclosporine within the first 3 months after transplantation were 8 to 12 ng/mL, C0 200 to 400 ng/mL, and C2 1400 to 1600 ng/mL, respectively. In this study, the pre- and post-transplant expression levels of the cytokine IL-2 gene (Homo sapiens chromosome 4, GRCh38.p7 Primary Assembly NCBI RefSeqNC_000004.12) were compared. Lymphocytes were isolated from the peripheral blood of the patients using the density centrifugation separation method with Ficoll (Biocoll, Biochrom, Berlin, Germany). CD4þ T cells were separated by magnetic-activated cell sorting (Miltenyi Biotec, Madrid, Spain). The purity of the CD4þ T cells were controlled by a flow cytometer (BD Facs Flow, BD Biosciences, San Jose, Calif, United States). CD4þ T cells with 98% purity were accepted. Total RNA was isolated from CD4þ T cells using RNeasy Plus Mini Kit (Qiagen USA, Valencia, Calif, United States). RNA purity was verified by OD260/OD280 nm absorption ratio. cDNA synthesis was achieved by using a commercial kit (cDNA synthesis RT2 HT First Strand Kit [96], Qiagen USA). The RT-PCR protocol was performed according to the manufacturer’s instructions (Qiagen USA). The appropriate design for the IL-2 cytokine gene was performed by the company (UniGene no: Hs.89679, Refseq Accession No:NM_000586.3, USA), and b-Actin gene-specific primers (Refseq Accession No: NM_001101, USA) were used as controls. RT-PCR was performed by the RT2 SYBR Green Mastermix kit (Qiagen USA) using the Rotor-Gene Q cycling device. All experiments were done in triplicate. The expression values of the IL-2 cytokine gene were determined by the Qiagen website (https://www.qiagen.com/tr/shop/genes-andpathways/data-analysis-center-overview-page/rt2-primer-assay-dataanalysis-center/?instrument¼R, September 2018). The results were statistically compared using IBM SPSS Statistics 25.0 (IBM,
1075 Armonk, NY, United States). The results were compared by the c2 test. P < .05 was accepted as statistically significant. This study was performed according to the principles expressed by the Declaration of Helsinki, and the study was approved by the Izmir Katip Celebi University Ethics Committee according to issue number 55 on June 8, 2017.
RESULTS
In this study, the mean ages of female and male patients were 45.14 6.362 (n ¼ 7) and 44.9 12.982 (n ¼ 14), respectively. Of the patients, 52.4% were transplanted from deceased donors, whereas 47.6% were transplanted from a living kidney donor. The mean ages of the living kidney donor and deceased donors were 42.8 14.047 (n ¼ 10) and 40.09 18.245 (n ¼ 11), respectively. There was no infection in the patients during the collection of the samples. Pretransplant panel reactive antibody screening test results were negative. IL-2 expression level increased in 50% and 27.28% of the patients who were transplanted from living kidney donor and deceased donors, whereas it decreased in 40% and 72.72%, respectively. IL-2 expression levels increased in 66.6% of the patients with a rejection episode. Among patients who did not have a rejection episode (n ¼ 18), the level of IL-2 expression decreased in 12 patients, increased in 5 patients, and did not change in 1 patient. The decreases in samples 3, 5, and 15 and the increase in sample 18 were statistically significant (P < .05) (Table 1). Rejection and rejection episodes were detected in 3 samples (respectively 21 and 13, 16), and all of them were biopsy proved. Increases of the expression levels of samples 13 and 21 were statistically significant (P < .05) (Table 1). In sample 13, an increase in IL-2 expression levels was detected at the sixth month, and a renal biopsy was performed at the eighth month after transplantation because of the rejection episode. T cell mediated rejection was reported; after induction therapy, graft function became stable. This result may support the use of the IL-2 expression level as a biomarker to predict the rejection. However, further studies are required. The IL-2 expression level of sample 16 increased (P > .05) at the sixth month after transplantation. The patient had a rejection episode, which was a biopsyproven antibody mediated rejection. The patient had a donor-specific antibodies post-transplant. The graft was stable, but the patient died because of the infection at the eighth month after transplantation. The allograft rejection was observed at the first month, and the IL-2 expression level increased at the sixth month after transplantation in sample 21. The patient had a biopsy-proven antibody mediated rejection, and graft function was lost. In this patient, the post-transplant panel-reactive antibody result was negative. This strong immune response may be due to the patient’s previous transplantation history and may be associated with the increase of the IL-2 expression levels. Comparison of IL-2 expression status for cold ischemia time, donor source, baseline serum creatinine range, peak serum creatinine at 6 months, % CD4þ, CD4/min3 values
1076
KARAHAN, SOYÖZ, PEHLIVAN ET AL Table 1. Demographic Characteristics of the Patients and IL-2 Expression Level Alterations
Patient No Patient Sex Patient Age Donor Source Donor Sex Donor Age
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Male Male Female Female Female Male Male Male Male Female Male Female Male Male Male Male Female Male Male Female Male
30 25 51 37 52 55 49 65 42 41 51 40 61 52 47 49 52 21 35 43 47
Living Living Deceased Deceased Deceased Living Living Living Deceased Living Deceased Deceased Living Living Deceased Deceased Deceased Living Living Deceased Deceased
Male Male Male Male Male Female Female Male Male Male Male Female Female Female Male Male Male Female Female Male Male
58 29 57 57 64 54 29 37 27 48 58 58 33 32 21 18 25 48 60 28 28
Histocompatibility
Rejection Episode
1A,1B,1DR,1DQB1,1DQA 1A,1B,2DR 1A,1DR 1DR 1A,1B,2DR 1A,1B,1DR,1DQB1,1DQA 1B,1DQB,1DQA 1A,1B,1DR,1DQB1,1DQA 1A,2B,2DR 2A,2B,2C,2DR,2DQB2DQA 1B,1DR 1B,1DR 1DQB,1DQA (Eighth month) 1A,1B,1C,1DR,1DQA,1DQB 1A,2B,1DR 1A,1DR (Seventh month) 1DR 1A,1B,1C,1DR,1DQA,1DQB 1A,1B,1C,1DR,1DQA,1DQB 2DR 1DR þ (first month)
IL-2 Expression Level
P
3.6553 1.2983 48.2793 3.6217 16.8733 1 17.7942 1.2058 3.6893 2.3027 2.9214 1.923 2817.1096 31.7056 2.8613 835.5988 3.3404 22.5752 1.4406 7.3445 17.188
.09 .90 .05 .25 .04 0 .12 .65 .27 .21 .35 .30 .02 .09 .03 .07 .09 .03 .27 .09 .037
Abbreviation: IL-2, interleukin 2.
are shown in Table 2. According to these results, there is no statistically significant interaction between expression levels of IL-2 and these clinical values and cell properties. DISCUSSION
In allografts, the neutrophils and lymphocytes secrete cytokines after they are stimulated by antigen-presenting
cells. The leading cytokines are IL-2, tumor necrosis factor a, IFN-gamma, and IL-1b. The recipient’s T cells are stimulated by the antigenic structure of the allograft via these cytokines. To better understand the immune response after transplantation and to determine the rejection, the cytokine profile should be identified [6]. This study was designed based on studies that examined the relationship between organ rejection and cytokine after
Table 2. Comparison of IL-2 Expression Status for CIT, Donor Source, Baseline SCR, Peak SCR at 6 Months, % CD4D, CD4/min3 Patient No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Donor Source
IL-2 Expression Level
Baseline SCR
SCR at 6 Months
%CD4þ Pre-Tx
%CD4þ min3 Pre-Tx
%CD4þ Post-Tx
%CD4þ min3 Post-Tx
CIT (hours)
Living Living Deceased Deceased Deceased Living Living Living Deceased Living Deceased Deceased Living Living Deceased Deceased Deceased Living Living Deceased Deceased
3.6553 1.2983 48.2793 3.6217 16.8733 1 17.7942 1.2058 3.6893 2.3027 2.9214 1.923 2817.1096 31.7056 2.8613 835.5988 3.3404 22.5752 1.4406 7.3445 17.188
6.0 9.7 3.8 2.3 10.9 5.98 4.3 6.16 7.4 5.3 10.7 6.1 4.5 7.4 7.1 8.3 5.8 8.4 8.8 8.5 10.8
1.43 1.14 1.25 1.28 1.02 1.52 0.97 0.94 2.1 1.19 2.19 1.22 2.29 1.29 1.16 2.26 1.02 1.72 1.84 0.92 8.18
97.1 96.2 97.4 98.1 97.8 96.7 95.8 97.7 96.8 98.1 98.4 97.9 98.3 97.7 98.2 96.8 97.4 98.1 97.6 98.5 97.9
1.36 1.38 0.65 0.63 1.25 1.80 3.25 2.23 0.13 1.78 1.75 1.81 2.39 2.10 1.71 2.14 0.19 1.93 1.68 2.18 1.52
97.4 93.5 98.2 94.4 97.6 98.6 98.5 97.6 95.3 97.2 94.6 98.5 96.6 97.7 96.9 98.9 97.1 97.3 97.5 97.6 95.5
1.81 1.87 1.04 0.71 1.21 1.80 2.75 0.81 1.23 1.17 0.30 1.76 1.64 0.80 2.03 0.13 1.00 1.18 1.92 2.00 0.74
1 1 18 18 14.5 1 19 1 15 1 11 15 1.5 1 13 11 11 1 1 18 13
Abbreviations: CIT, cold ischemia time; IL-2, interleukin 2; SCR, serum creatinine range; Tx, transplant.
IL-2 CYTOKINE EXPRESSION LEVELS
transplantation. In this study, we investigated the effects of the pre- and post-transplant immunologic status of the renal function of candidates. The expression level of IL-2 decreased as expected in patients who did not have any rejection. According to our results, there was a significant association between IL-2 expression levels and rejection episodes. We observed that IL-2 expression increased at the sixth month and the patient had a rejection episode at the eighth month. With further studies containing larger study cohorts, the rejection episodes can be predicted according to the expression levels at a certain time, and this can support the use of the changes in expression levels as a biomarker for rejection. Furthermore, an increase of the expression levels without a rejection episode have been inferred us to follow the clinical conditions of these patients. Distinct studies have showed that IL-2 expression levels in serum samples can be associated with rejection [7e10]. However, the indication of the expression level changes in cellular processes can be more sensitive for long-term graft survival. Despite of the rejection at the first month, the IL-2 expression level increased at the sixth month significantly in patient 21. Previous transplant history may lead to strong immune response. The level of IL-2 expression increased in 1 patient (sample 18), although there was no rejection episode; continuous follow-up of the immunologic status of this patient may be beneficial for the studies that focus on expression profiles of the cytokines. Our study is limited by the fact that the protein levels have not been determined. Further studies with a larger sample size and for longer duration is required to validate the results. CONCLUSION
The determination of the IL-2 cytokine level may play a role in elucidating the immune response after transplantation. When the patients were evaluated individually, it was observed that there might be a relationship between IL-2 expression levels in CD4þ T cells and rejection after renal
1077
transplantation. The clinical data of the patients, the immunosuppressive therapies, and post-transplant evaluation of cytokines should be considered, which is important in the immune response. We believe that periodic posttransplant follow-up of the IL-2 gene expression level of patients may help to predict the rejection episodes in advance and to select the appropriate treatment. ACKNOWLEDGMENTS We thank the staff of the Tepecik Education and Research Hospital Tissue Typing Laboratory and Health Sciences University Izmir Bozyaka Education and Research Hospital Organ Transplantation Unit for their efforts.
REFERENCES [1] Galliford J, Game DS. Modern renal transplantation: present challenges and future prospects. Postgrad Med J 2009;85: 91e101. [2] Ganji MR, Haririan A. Chronic allograft dysfunction major contributing factors. Iran J Kidney Dis 2012;6:88e93. [3] Luckheeram RV, Zhou R, Verma AD, Xia B. CD4þ T Cells: differentiation and functions. Clin Dev Immunol 2012:925135. [4] Khan MM. Immunopharmacology. 2nd ed. Boston: Springer; 2016. p. 442. [5] Gaffen SL, Liu KD. Overview of interleukin-2 function, production and clinical applications. Cytokine 2004;28:109e23. [6] Ertosun MG, Özkan Ö, Özkan Ö. Transplantations which might shed light on organ rejections: composite tissue transplantations. Akd Tıp D 2015;2:77e84. [7] Hu H, Knetchle SJ. Elevation of multiple cytokines/chemokines in urine of human renal transplant recipients with acute and chronic injuries: potential usage for diagnosis and monitoring. Transpl Rev 2006;20:165e71. [8] Ates¸ K, Aylı D, Duranay M, et al. The value of serum soluble interleukin-2 receptor levels in diagnosis of acute rejection in renal transplant recipients. Turkish J Nephrol Dial Transpl 1995;1:21e4. [9] Lessan-Pezeshki M, Amirzargar A, Fathi A. Value of pretransplantation cytokine profiles for predicting acute rejection in renal transplant recipients. Transplant Proc 2005;37:2982e4. [10] Millán O, Rafael-Valdivia L, San Segundo D. Should IFNg, IL-17 and IL-2 be considered predictive biomarkers of acute rejection in liver and kidney transplant? Results of a multicentric study. Clin Immunol 2014;154:141e54.
ABSTRACT Aim. End-stage renal disease is a disease in which the kidney is not able to perform its functions. Kidney transplantation is the most effective treatment and cost-effective modality of renal replacement therapy for patients with end-stage renal disease. However, the most important problem in end-stage renal disease patients is the unpredictability of immunologic response after transplants. In this study, it was aimed to investigate the possible association between the interleukin 2 (IL-2) expression level and an organ rejection or rejection episode. Materials and Methods. Lymphocytes were isolated from peripheral blood obtained from 21 end-stage renal diseaseediagnosed patients prior to transplant and at the sixth month after transplant. CD4þ T cells were separated from lymphocytes by the magnetic cell-sorting method. The purity of these cells were controlled by a flow cytometer. After total RNA isolation from CD4þ T cells, IL-2 was examined by the real-time polymerase chain reaction (RT-PCR) method. Results. Among nonrejection patients (n ¼ 18), the IL-2 expression level decreased in 12 patients in post-transplant time, and 3 of these were statistically significant (P < .05). The level was the same in 1 of 18 patients; it increased in 5 patients, and 1 of them was significant (P < .05). The IL-2 expression level also increased in 3 patients who had a rejection episode, and the increase was statistically significant in 2 samples (P < .05). Conclusion. When the patients were evaluated individually, it was observed that there might be a relationship between IL-2 expression levels in CD4þ T cells and rejection episodes. The clinical data of the patients, the immunosuppressive therapies, and posttransplant evaluation of cytokines should be considered together.
D
ISRUPTION of the main functions of the kidney causes renal failure. Renal transplantation is the most valid method in the treatment of end-stage renal disease. However, the causes of subsequent complications, which occur after transplantations and lead to organ rejection, have not been discovered [1,2]. T cells, 1 of the lymphocyte population groups, lead to an immune response after transplantation. After activation and differentiation into different effector subtypes, CD4þ T cells play an important role in mediating the immune response via specific cytokines [3]. Cytokines regulate 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.02.008
1074
lymphocyte activation and differentiation. Interleukin 2 (IL-2) is a major cytokine molecule that plays a curious role in the immune response produced by CD4þ T cells. It is a This study was supported by the Scientific Research Projects Administration (project number 2017-TYL-SABE-0046) and the Teaching Assistant Education Program. *Address correspondence to Mustafa Soyöz, _Izmir Katip Celebi University, Izmir Tepecik Education and Research Hospital, Tissue Typing Lab, A Bloc, 1st Floor, 35110, Yenisehir-Konak, _Izmir, Turkey. Tel: (0232) 469 69 69-1709; Fax: (0232) 433 07 56. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1074e1077 (2019)
IL-2 CYTOKINE EXPRESSION LEVELS
133-amino acid residual polypeptide chain and is mainly produced by T cells [4,5]. It is thought that the determination of the IL-2 cytokine level will play a role in elucidating the immune response after transplantation. The aims of this study were to investigate the effect of IL2 on rejection in early post-transplant time and to predict the rejection episode in the long term. MATERIAL AND METHODS Peripheral blood samples were obtained from 21 patients who were diagnosed with end-stage renal disease according to the international study group criteria prior to the transplants and at the sixth month after transplantation. Inclusion criteria were ABO blood group compatibility, human immunodeficiency virus negativity, and cross-match negativity. Three of the patients were diagnosed with a rejection episode by the clinicians. All patients received anti-thymocyte globulin (ATG) and corticosteroids (prednisolone) for induction immunosuppression. Rabbit-ATG (Fresenius) was administered to all patients on the zeroth and first days in doses of 4 mg/kg and 2 mg/kg, respectively. In cases of delayed graft function, ATG was administered at doses of 1 to 2 mg/kg every other day until the serum creatinine value was reduced to 3 mg/dL. Corticosteroid protocol was administered on the zeroth, first, and second days in doses of 1000 mg/day, 250 mg/ day, and 100 mg/day, respectively. Subsequently, prednisolone was decreased 20 mg each day until 20 mg/day, and patients were discharged with 0.3 mg/kg/day. The maintenance immunosuppressive protocol included an antimetabolite (mycophenolate mofetil or mycophenolic acid), a calcineurin inhibitor (tacrolimus or cyclosporin), and a corticosteroid. The whole blood concentration targets of tacrolimus and cyclosporine within the first 3 months after transplantation were 8 to 12 ng/mL, C0 200 to 400 ng/mL, and C2 1400 to 1600 ng/mL, respectively. In this study, the pre- and post-transplant expression levels of the cytokine IL-2 gene (Homo sapiens chromosome 4, GRCh38.p7 Primary Assembly NCBI RefSeqNC_000004.12) were compared. Lymphocytes were isolated from the peripheral blood of the patients using the density centrifugation separation method with Ficoll (Biocoll, Biochrom, Berlin, Germany). CD4þ T cells were separated by magnetic-activated cell sorting (Miltenyi Biotec, Madrid, Spain). The purity of the CD4þ T cells were controlled by a flow cytometer (BD Facs Flow, BD Biosciences, San Jose, Calif, United States). CD4þ T cells with 98% purity were accepted. Total RNA was isolated from CD4þ T cells using RNeasy Plus Mini Kit (Qiagen USA, Valencia, Calif, United States). RNA purity was verified by OD260/OD280 nm absorption ratio. cDNA synthesis was achieved by using a commercial kit (cDNA synthesis RT2 HT First Strand Kit [96], Qiagen USA). The RT-PCR protocol was performed according to the manufacturer’s instructions (Qiagen USA). The appropriate design for the IL-2 cytokine gene was performed by the company (UniGene no: Hs.89679, Refseq Accession No:NM_000586.3, USA), and b-Actin gene-specific primers (Refseq Accession No: NM_001101, USA) were used as controls. RT-PCR was performed by the RT2 SYBR Green Mastermix kit (Qiagen USA) using the Rotor-Gene Q cycling device. All experiments were done in triplicate. The expression values of the IL-2 cytokine gene were determined by the Qiagen website (https://www.qiagen.com/tr/shop/genes-andpathways/data-analysis-center-overview-page/rt2-primer-assay-dataanalysis-center/?instrument¼R, September 2018). The results were statistically compared using IBM SPSS Statistics 25.0 (IBM,
1075 Armonk, NY, United States). The results were compared by the c2 test. P < .05 was accepted as statistically significant. This study was performed according to the principles expressed by the Declaration of Helsinki, and the study was approved by the Izmir Katip Celebi University Ethics Committee according to issue number 55 on June 8, 2017.
RESULTS
In this study, the mean ages of female and male patients were 45.14 6.362 (n ¼ 7) and 44.9 12.982 (n ¼ 14), respectively. Of the patients, 52.4% were transplanted from deceased donors, whereas 47.6% were transplanted from a living kidney donor. The mean ages of the living kidney donor and deceased donors were 42.8 14.047 (n ¼ 10) and 40.09 18.245 (n ¼ 11), respectively. There was no infection in the patients during the collection of the samples. Pretransplant panel reactive antibody screening test results were negative. IL-2 expression level increased in 50% and 27.28% of the patients who were transplanted from living kidney donor and deceased donors, whereas it decreased in 40% and 72.72%, respectively. IL-2 expression levels increased in 66.6% of the patients with a rejection episode. Among patients who did not have a rejection episode (n ¼ 18), the level of IL-2 expression decreased in 12 patients, increased in 5 patients, and did not change in 1 patient. The decreases in samples 3, 5, and 15 and the increase in sample 18 were statistically significant (P < .05) (Table 1). Rejection and rejection episodes were detected in 3 samples (respectively 21 and 13, 16), and all of them were biopsy proved. Increases of the expression levels of samples 13 and 21 were statistically significant (P < .05) (Table 1). In sample 13, an increase in IL-2 expression levels was detected at the sixth month, and a renal biopsy was performed at the eighth month after transplantation because of the rejection episode. T cell mediated rejection was reported; after induction therapy, graft function became stable. This result may support the use of the IL-2 expression level as a biomarker to predict the rejection. However, further studies are required. The IL-2 expression level of sample 16 increased (P > .05) at the sixth month after transplantation. The patient had a rejection episode, which was a biopsyproven antibody mediated rejection. The patient had a donor-specific antibodies post-transplant. The graft was stable, but the patient died because of the infection at the eighth month after transplantation. The allograft rejection was observed at the first month, and the IL-2 expression level increased at the sixth month after transplantation in sample 21. The patient had a biopsy-proven antibody mediated rejection, and graft function was lost. In this patient, the post-transplant panel-reactive antibody result was negative. This strong immune response may be due to the patient’s previous transplantation history and may be associated with the increase of the IL-2 expression levels. Comparison of IL-2 expression status for cold ischemia time, donor source, baseline serum creatinine range, peak serum creatinine at 6 months, % CD4þ, CD4/min3 values
1076
KARAHAN, SOYÖZ, PEHLIVAN ET AL Table 1. Demographic Characteristics of the Patients and IL-2 Expression Level Alterations
Patient No Patient Sex Patient Age Donor Source Donor Sex Donor Age
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Male Male Female Female Female Male Male Male Male Female Male Female Male Male Male Male Female Male Male Female Male
30 25 51 37 52 55 49 65 42 41 51 40 61 52 47 49 52 21 35 43 47
Living Living Deceased Deceased Deceased Living Living Living Deceased Living Deceased Deceased Living Living Deceased Deceased Deceased Living Living Deceased Deceased
Male Male Male Male Male Female Female Male Male Male Male Female Female Female Male Male Male Female Female Male Male
58 29 57 57 64 54 29 37 27 48 58 58 33 32 21 18 25 48 60 28 28
Histocompatibility
Rejection Episode
1A,1B,1DR,1DQB1,1DQA 1A,1B,2DR 1A,1DR 1DR 1A,1B,2DR 1A,1B,1DR,1DQB1,1DQA 1B,1DQB,1DQA 1A,1B,1DR,1DQB1,1DQA 1A,2B,2DR 2A,2B,2C,2DR,2DQB2DQA 1B,1DR 1B,1DR 1DQB,1DQA (Eighth month) 1A,1B,1C,1DR,1DQA,1DQB 1A,2B,1DR 1A,1DR (Seventh month) 1DR 1A,1B,1C,1DR,1DQA,1DQB 1A,1B,1C,1DR,1DQA,1DQB 2DR 1DR þ (first month)
IL-2 Expression Level
P
3.6553 1.2983 48.2793 3.6217 16.8733 1 17.7942 1.2058 3.6893 2.3027 2.9214 1.923 2817.1096 31.7056 2.8613 835.5988 3.3404 22.5752 1.4406 7.3445 17.188
.09 .90 .05 .25 .04 0 .12 .65 .27 .21 .35 .30 .02 .09 .03 .07 .09 .03 .27 .09 .037
Abbreviation: IL-2, interleukin 2.
are shown in Table 2. According to these results, there is no statistically significant interaction between expression levels of IL-2 and these clinical values and cell properties. DISCUSSION
In allografts, the neutrophils and lymphocytes secrete cytokines after they are stimulated by antigen-presenting
cells. The leading cytokines are IL-2, tumor necrosis factor a, IFN-gamma, and IL-1b. The recipient’s T cells are stimulated by the antigenic structure of the allograft via these cytokines. To better understand the immune response after transplantation and to determine the rejection, the cytokine profile should be identified [6]. This study was designed based on studies that examined the relationship between organ rejection and cytokine after
Table 2. Comparison of IL-2 Expression Status for CIT, Donor Source, Baseline SCR, Peak SCR at 6 Months, % CD4D, CD4/min3 Patient No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Donor Source
IL-2 Expression Level
Baseline SCR
SCR at 6 Months
%CD4þ Pre-Tx
%CD4þ min3 Pre-Tx
%CD4þ Post-Tx
%CD4þ min3 Post-Tx
CIT (hours)
Living Living Deceased Deceased Deceased Living Living Living Deceased Living Deceased Deceased Living Living Deceased Deceased Deceased Living Living Deceased Deceased
3.6553 1.2983 48.2793 3.6217 16.8733 1 17.7942 1.2058 3.6893 2.3027 2.9214 1.923 2817.1096 31.7056 2.8613 835.5988 3.3404 22.5752 1.4406 7.3445 17.188
6.0 9.7 3.8 2.3 10.9 5.98 4.3 6.16 7.4 5.3 10.7 6.1 4.5 7.4 7.1 8.3 5.8 8.4 8.8 8.5 10.8
1.43 1.14 1.25 1.28 1.02 1.52 0.97 0.94 2.1 1.19 2.19 1.22 2.29 1.29 1.16 2.26 1.02 1.72 1.84 0.92 8.18
97.1 96.2 97.4 98.1 97.8 96.7 95.8 97.7 96.8 98.1 98.4 97.9 98.3 97.7 98.2 96.8 97.4 98.1 97.6 98.5 97.9
1.36 1.38 0.65 0.63 1.25 1.80 3.25 2.23 0.13 1.78 1.75 1.81 2.39 2.10 1.71 2.14 0.19 1.93 1.68 2.18 1.52
97.4 93.5 98.2 94.4 97.6 98.6 98.5 97.6 95.3 97.2 94.6 98.5 96.6 97.7 96.9 98.9 97.1 97.3 97.5 97.6 95.5
1.81 1.87 1.04 0.71 1.21 1.80 2.75 0.81 1.23 1.17 0.30 1.76 1.64 0.80 2.03 0.13 1.00 1.18 1.92 2.00 0.74
1 1 18 18 14.5 1 19 1 15 1 11 15 1.5 1 13 11 11 1 1 18 13
Abbreviations: CIT, cold ischemia time; IL-2, interleukin 2; SCR, serum creatinine range; Tx, transplant.
IL-2 CYTOKINE EXPRESSION LEVELS
transplantation. In this study, we investigated the effects of the pre- and post-transplant immunologic status of the renal function of candidates. The expression level of IL-2 decreased as expected in patients who did not have any rejection. According to our results, there was a significant association between IL-2 expression levels and rejection episodes. We observed that IL-2 expression increased at the sixth month and the patient had a rejection episode at the eighth month. With further studies containing larger study cohorts, the rejection episodes can be predicted according to the expression levels at a certain time, and this can support the use of the changes in expression levels as a biomarker for rejection. Furthermore, an increase of the expression levels without a rejection episode have been inferred us to follow the clinical conditions of these patients. Distinct studies have showed that IL-2 expression levels in serum samples can be associated with rejection [7e10]. However, the indication of the expression level changes in cellular processes can be more sensitive for long-term graft survival. Despite of the rejection at the first month, the IL-2 expression level increased at the sixth month significantly in patient 21. Previous transplant history may lead to strong immune response. The level of IL-2 expression increased in 1 patient (sample 18), although there was no rejection episode; continuous follow-up of the immunologic status of this patient may be beneficial for the studies that focus on expression profiles of the cytokines. Our study is limited by the fact that the protein levels have not been determined. Further studies with a larger sample size and for longer duration is required to validate the results. CONCLUSION
The determination of the IL-2 cytokine level may play a role in elucidating the immune response after transplantation. When the patients were evaluated individually, it was observed that there might be a relationship between IL-2 expression levels in CD4þ T cells and rejection after renal
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transplantation. The clinical data of the patients, the immunosuppressive therapies, and post-transplant evaluation of cytokines should be considered, which is important in the immune response. We believe that periodic posttransplant follow-up of the IL-2 gene expression level of patients may help to predict the rejection episodes in advance and to select the appropriate treatment. ACKNOWLEDGMENTS We thank the staff of the Tepecik Education and Research Hospital Tissue Typing Laboratory and Health Sciences University Izmir Bozyaka Education and Research Hospital Organ Transplantation Unit for their efforts.
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