- Email: [email protected]
Distinct Cytokine Patterns in Different States of Kidney Allograft Function
Distinct Cytokine Patterns in Different States of Kidney Allograft Function M. Karczewski, J. Karczewski, B. Poniedzialek, K. Wiktorowicz, M. Smietanska, and M. Glyda ABSTRACT Cytokines are crucial inflammatory mediators involved in the development of immune response leading to allograft rejection. We investigated the cytokine patterns in patients sera from cases of acute rejection episodes (ARE), chronic rejection (CR), and long-term stable courses (STABLE). The project included 20 patients with ARE, 20 with CR, and 15 with at least a 5-year stable course. Serum samples collected at the time of rejection diagnosis were cytometrically tested for concentrations of interleukin (IL) 2, IL-4, IL-6, IL-10, interferon (IFN) ␥, and tumor necrosis factor ␣. No significant differences between investigated groups were observed before transplantation (P ⬎ .05). Significant differences were observed among the groups in serum levels of IFN-␥, IL-4, IL-6, and IL-10. Our data suggested that distinct serum cytokine patterns were present among various states of kidney allograft function. ARE was characterized by a mixed cytokine pattern with elevated IL-10 and IFN-␥ compared with the STABLE patients. The cytokine pattern in CR patients, in turn, was characterized by elevated levels of IL-4, IL-6, and IL-10 and decreased levels of IFN- ␥ compared with both STABLE and ARE subjects. Our results suggested that the TH2 response may contribute to the initiation and/or maintenance of CR, because IL-4, IL-6, and IL-10 serve as growth and differentiation factors for B cells to increase antibody production. We also observed up-regulated production of IFN-␥ and down-regulation of TH2 cytokines among patients with stable long- term graft function. IDNEY TRANSPLANTATION is a well established method of treatment for end-stage renal disease. Cytokines are crucial inflammatory mediators involved in development of immune responses leading to allograft rejection.1,2 These small soluble proteins may be categorized into two major groups: TH1-type cytokines, including interleukin (IL) 2, interferon (IFN) ␥, and tumor necrosis factor (TNF) ␣, which mediate cellular responses, and TH2-type cytokines, including IL-4, IL-5, and IL-10, which mediate the humoral response.3,4 Importantly, TH1-type cytokines may down-regulate TH2 cytokines, and vice versa.5 A body of evidence suggests that cytokines are involved in allograft rejection. TH1 cytokines are believed to be associated with rejection, TH2 cytokines with graft acceptance.6,7 Various studies, however, have also demonstrated the involvement of TH2 cytokines in acute and/or chronic kidney rejection.8,9 In a previous study, we observed higher levels of pre- and post-transplant IFN-␥ and IL- 10 in the sera of patients with diagnosed acute rejection episodes (ARE).10 To further explore the role of these mediators in the process of kidney rejection, we investigated the cytokine
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patterns in sera of patients with diagnosed ARE, chronic rejection (CR), and long-term stable graft function (STABLE). PATIENTS, MATERIALS, AND METHODS Approval for this study was obtained from the local Ethics Committee. It included 20 patients with diagnosed ARE, 20 with CR, and 15 with at least 5-year stable graft function. Rejection diagnoses were based on biopsy results applying the Banff classification. Investigated patients were treated with standard doses of the following immunosuppressive protocols: 1) cyclosporine ⫹ mycophenolate mofetil ⫹ glucocorticoids; 2) tacrolimus ⫹ rapamycin ⫹ glucocorticoids; or 3) tacrolimus ⫹ mycophenolate mofetil ⫹ glucocorticoids. We examined demographic characteristics of ARE, CR, and STABLE patients including age, gender, race, time on dialysis, serum creatinine and urea concentrations, cold and From the Department of Transplantology and General Surgery, District Hospital, Pozhan, Poland. Address reprint requests to Dr Marek Karczewski, District Hospital, Juraszow 7/19, Poznan, wikp. 60-479, Poland. E-mail: [email protected]
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.08.067
Transplantation Proceedings, 41, 4147– 4149 (2009)
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warm ischemic time, degree of HLA match, percentage plasma senin activity, and type of immunosuppression therapy. Serum samples collected at the time of rejection diagnosis were cytometrically tested for concentrations of IL-2, IL-4, IL-6, IL-10, IFN-␥, and TNF-␣, using the Human TH1/TH2 Cytokine Cytometric Bead Array Kit (CBA; BD Biosciences Pharmingen) according to the manufacturer’s instructions. Data were analyzed by Mann-Whitney U, Wilcoxon signed rank test, and 2 test using SPSS version 15. Values were considered to be significant when P ⬍ .05.
RESULTS
No significant differences were observed in demographic characteristics of STABLE, ARE, and CR patients (P ⬎ .05), suggesting that the patient populations were similar. Significant differences were observed in serum levels of IFN-␥, IL-4, IL-6, and IL-10 among the groups. The mean serum levels of IFN-␥ in STABLE, ARE, and CR patients were 8.85 ⫾ 2.6, 6.91 ⫾ 1.4, and 4.95 ⫾ 1.7 pg/mL, respectively (Fig 1). IL-10 serum levels were 4.40 ⫾ 1.9 pg/mL in STABLE, 6.67 ⫾ 2.0 pg/mL in ARE, and 8.39 ⫾ 2.5 pg/mL in CR patients (Fig 2). IL-4 serum levels were 1.80 ⫾ 1.9 pg/mL in STABLE, 2.25 ⫾ 0.8 pg/mL in ARE, and 4.31 ⫾ 1.3 pg/mL in CR patients (Fig 3). The cytometric analysis also showed differences in IL-6 serum levels: 0.42 ⫾ 0.3 pg/mL in STABLE, 0.93 ⫾ 1.7 pg/mL in ARE, and 1.64 ⫾ 0.8 pg/mL in CR patients (Fig 4). No significant differences were observed in serum levels of IL-2 and TNF-␣ among the investigated groups (P ⬎ .05).
Fig 2. Mean concentrations of interleukin (IL) 10 in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
Our data suggested that there were distinct serum cytokine patterns among different states of kidney allograft function. ARE was characterized by a mixed cytokine pattern with elevated IFN-␥ and IL-10 compared with STABLE patients. The role of cytokines, particularly IFN-␥, in the ARE process has been well documented.7,11 A similar serum cytokine pattern in ARE patients was observed in our previous study.10 The selective production of IFN-␥, not
accompanied by elevations of IL-2 and TNF-␣, suggests an ongoing nonspecific TH1 immune response involving activated natural killer cells and macrophages. In contrast, the observed elevated concentrations of IL-10 probably resulted from monocyte activation rather than from regulatory/suppressor T cells, owing to the deficiency/dysfunction of cell subpopulations in ARE patients (unpublished data).12 The cytokine pattern in CR patients, in turn, was characterized by elevated levels of TH2 cytokines IL-4, IL-6, and IL-10 and decreased levels of IFN-␥ compared with both STABLE and ARE patients. Although the etiology of CR is multifactorial, immunologic factors play an essential role. Our results suggested that the TH2 response may contribute to the initiation and/or maintenance of CR, because IL-4, IL-6, and IL-10 serve as growth and differentiation factors for B cells to increase antibody production. The potential role of TH2 cytokines in chronic kidney rejection has been previously reported by some authors.13,14 In contrast, the
Fig 1. Mean concentrations of interferon (IFN) gamma in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
Fig 3. Mean concentrations of interleukin (IL) 4 in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
DISCUSSION
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Fig 4. Medium concentrations of interleukin (IL) 6 in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
elevated levels of TH2 cytokines observed may be a byproduct of other immune processes underlying CR. In the present study, we also observed up-regulated production of IFN-␥ among patients with stable long-term graft function, an observation consistent with earlier reports by Sadeghi et al.6,15 Higher concentrations of IFN-␥ in this group may result from the up-regulation of regulatory/ suppressor T cells owing to the constant allogeneic stimulation. It is believed that transient production of IFN-␥ by induced regulatory/suppressor T cells is important for their functional activity on APCs and other T cells.15,16 We demonstrated distinct serum cytokine patterns among various states of kidney allograft function. It appears that the predominance of serum TH2 cytokines IL-4, IL-6, and IL-10, was related to chronic kidney rejection, and up-regulation of IFN-␥ and down-regulation of TH2 cytokines to long-term graft acceptance. Acute rejection, in turn, showed a mixed cytokine profile with up-regulated levels of IFN-␥ and IL-10. In this study, we also observed that the TH1/TH2 CBA technique, which permits simultaneous measurement of multiple biomarkers in a single serum sample, is a quick, efficient, and safe method to screen for diagnosis and to monitor renal transplant injuries.
1. Noronha IL, Eberlein-Gonska M, Stephens S, Cameron JS, Waldherr R: In situ expression of tumor necrosis factor-alpha, interferon-gamma, and interleukin-2 receptors in renal allograft biopsies. Transplantation 54:1017, 1992 2. Dallman MJ: Cytokines and transplantation: TH1/TH2 regulation of the immune response to solid organ transplantation in the adult. Curr Opin Immunol 7:632, 1995 3. Mosmann TR, Coffman R: TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145, 1989 4. O’Garra A: Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275, 1998 5. Dallman MJ: Cytokines as mediators of organ graft rejection and tolerance. Curr Opin Immunol 5:788, 1993 6. Sadeghi M, Daniel V, Weimar W, Hergesell O, Opelz G: Pre-transplant TH1 and post-transplant TH2 cytokine patterns are associated with early acute rejection in renal transplant recipients. Clin Transplant 17:151, 2003 7. Amirzargar A, Lessanpezeshki M, Fathi A, et al: TH1/TH2 cytokine analysis in Iranian renal transplants recipients. Transplant Proc 37:2985, 2005 8. Berber I, Yigit B, Isitmangil G, et al: Evaluation of pretransplant serum cytokine levels in renal transplant recipients. Transplant Proc 40:92, 2008 9. Merville P, Lambert C, Durand I, et al: High frequency of IL-10 secreting CD4⫹ graft-infiltrating T lymphocytes in promptly rejected kidney allografts. Transplantation 59:1113, 1995 10. Karczewski J, Karczewski M, Glyda M, Wiktorowicz K: Role of TH1/TH2 cytokines in kidney allograft rejection. Transplant Proc 40:3390, 2008 11. Jimenez R, Ramirez R, Carracedo J, et al: Cytometric bead array (CBA) for the measurement of cytokines in urine and plasma of patients undergoing renal rejection. Cytokine 32:45, 2005 12. Karczewski M, Karczewski J, Kostrzewa A, Wiktorowicz K, Glyda M: The role of Foxp3⫹ regulatory T cells in kidney transplantation. Transplant Proc 41:1527, 2009 13. Baan CC, Weimar W: Intragraft cytokine gene expression: implications for clinical transplantation. Transplant Int 11:169, 1998 14. Loong CC, Chen A, Lui WY, King KL, Lin CY: Expression of cytokines, growth factors and adhesion molecules in rejecting human renal allograft. Transplant Proc 28:1445, 1996 15. Sadeghi M, Daniel V, Naujokat C, Mehrabi A, Zeier M, Opelz G: Evidence for IFN-gamma up-and IL-4 down-regulation late post-transplant in patients with good kidney graft outcome. Clin Transplant 21:449, 2007 16. Wood KJ, Sawitzki B: Interferon gamma: a crucial role in the function of induced regulatory T cells in vivo. Trends Immunol 27:183, 2006
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patterns in sera of patients with diagnosed ARE, chronic rejection (CR), and long-term stable graft function (STABLE). PATIENTS, MATERIALS, AND METHODS Approval for this study was obtained from the local Ethics Committee. It included 20 patients with diagnosed ARE, 20 with CR, and 15 with at least 5-year stable graft function. Rejection diagnoses were based on biopsy results applying the Banff classification. Investigated patients were treated with standard doses of the following immunosuppressive protocols: 1) cyclosporine ⫹ mycophenolate mofetil ⫹ glucocorticoids; 2) tacrolimus ⫹ rapamycin ⫹ glucocorticoids; or 3) tacrolimus ⫹ mycophenolate mofetil ⫹ glucocorticoids. We examined demographic characteristics of ARE, CR, and STABLE patients including age, gender, race, time on dialysis, serum creatinine and urea concentrations, cold and From the Department of Transplantology and General Surgery, District Hospital, Pozhan, Poland. Address reprint requests to Dr Marek Karczewski, District Hospital, Juraszow 7/19, Poznan, wikp. 60-479, Poland. E-mail: [email protected]
© 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.08.067
Transplantation Proceedings, 41, 4147– 4149 (2009)
4147
4148
KARCZEWSKI ET AL
warm ischemic time, degree of HLA match, percentage plasma senin activity, and type of immunosuppression therapy. Serum samples collected at the time of rejection diagnosis were cytometrically tested for concentrations of IL-2, IL-4, IL-6, IL-10, IFN-␥, and TNF-␣, using the Human TH1/TH2 Cytokine Cytometric Bead Array Kit (CBA; BD Biosciences Pharmingen) according to the manufacturer’s instructions. Data were analyzed by Mann-Whitney U, Wilcoxon signed rank test, and 2 test using SPSS version 15. Values were considered to be significant when P ⬍ .05.
RESULTS
No significant differences were observed in demographic characteristics of STABLE, ARE, and CR patients (P ⬎ .05), suggesting that the patient populations were similar. Significant differences were observed in serum levels of IFN-␥, IL-4, IL-6, and IL-10 among the groups. The mean serum levels of IFN-␥ in STABLE, ARE, and CR patients were 8.85 ⫾ 2.6, 6.91 ⫾ 1.4, and 4.95 ⫾ 1.7 pg/mL, respectively (Fig 1). IL-10 serum levels were 4.40 ⫾ 1.9 pg/mL in STABLE, 6.67 ⫾ 2.0 pg/mL in ARE, and 8.39 ⫾ 2.5 pg/mL in CR patients (Fig 2). IL-4 serum levels were 1.80 ⫾ 1.9 pg/mL in STABLE, 2.25 ⫾ 0.8 pg/mL in ARE, and 4.31 ⫾ 1.3 pg/mL in CR patients (Fig 3). The cytometric analysis also showed differences in IL-6 serum levels: 0.42 ⫾ 0.3 pg/mL in STABLE, 0.93 ⫾ 1.7 pg/mL in ARE, and 1.64 ⫾ 0.8 pg/mL in CR patients (Fig 4). No significant differences were observed in serum levels of IL-2 and TNF-␣ among the investigated groups (P ⬎ .05).
Fig 2. Mean concentrations of interleukin (IL) 10 in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
Our data suggested that there were distinct serum cytokine patterns among different states of kidney allograft function. ARE was characterized by a mixed cytokine pattern with elevated IFN-␥ and IL-10 compared with STABLE patients. The role of cytokines, particularly IFN-␥, in the ARE process has been well documented.7,11 A similar serum cytokine pattern in ARE patients was observed in our previous study.10 The selective production of IFN-␥, not
accompanied by elevations of IL-2 and TNF-␣, suggests an ongoing nonspecific TH1 immune response involving activated natural killer cells and macrophages. In contrast, the observed elevated concentrations of IL-10 probably resulted from monocyte activation rather than from regulatory/suppressor T cells, owing to the deficiency/dysfunction of cell subpopulations in ARE patients (unpublished data).12 The cytokine pattern in CR patients, in turn, was characterized by elevated levels of TH2 cytokines IL-4, IL-6, and IL-10 and decreased levels of IFN-␥ compared with both STABLE and ARE patients. Although the etiology of CR is multifactorial, immunologic factors play an essential role. Our results suggested that the TH2 response may contribute to the initiation and/or maintenance of CR, because IL-4, IL-6, and IL-10 serve as growth and differentiation factors for B cells to increase antibody production. The potential role of TH2 cytokines in chronic kidney rejection has been previously reported by some authors.13,14 In contrast, the
Fig 1. Mean concentrations of interferon (IFN) gamma in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
Fig 3. Mean concentrations of interleukin (IL) 4 in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
DISCUSSION
CYTOKINES IN KIDNEY ALLOGRAFT DYSFUNCTION
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REFERENCES
Fig 4. Medium concentrations of interleukin (IL) 6 in serum of patients with long-term stable graft function (STABLE), patients with acute rejection (AR), and patients with chronic rejection (CR).
elevated levels of TH2 cytokines observed may be a byproduct of other immune processes underlying CR. In the present study, we also observed up-regulated production of IFN-␥ among patients with stable long-term graft function, an observation consistent with earlier reports by Sadeghi et al.6,15 Higher concentrations of IFN-␥ in this group may result from the up-regulation of regulatory/ suppressor T cells owing to the constant allogeneic stimulation. It is believed that transient production of IFN-␥ by induced regulatory/suppressor T cells is important for their functional activity on APCs and other T cells.15,16 We demonstrated distinct serum cytokine patterns among various states of kidney allograft function. It appears that the predominance of serum TH2 cytokines IL-4, IL-6, and IL-10, was related to chronic kidney rejection, and up-regulation of IFN-␥ and down-regulation of TH2 cytokines to long-term graft acceptance. Acute rejection, in turn, showed a mixed cytokine profile with up-regulated levels of IFN-␥ and IL-10. In this study, we also observed that the TH1/TH2 CBA technique, which permits simultaneous measurement of multiple biomarkers in a single serum sample, is a quick, efficient, and safe method to screen for diagnosis and to monitor renal transplant injuries.
1. Noronha IL, Eberlein-Gonska M, Stephens S, Cameron JS, Waldherr R: In situ expression of tumor necrosis factor-alpha, interferon-gamma, and interleukin-2 receptors in renal allograft biopsies. Transplantation 54:1017, 1992 2. Dallman MJ: Cytokines and transplantation: TH1/TH2 regulation of the immune response to solid organ transplantation in the adult. Curr Opin Immunol 7:632, 1995 3. Mosmann TR, Coffman R: TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145, 1989 4. O’Garra A: Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8:275, 1998 5. Dallman MJ: Cytokines as mediators of organ graft rejection and tolerance. Curr Opin Immunol 5:788, 1993 6. Sadeghi M, Daniel V, Weimar W, Hergesell O, Opelz G: Pre-transplant TH1 and post-transplant TH2 cytokine patterns are associated with early acute rejection in renal transplant recipients. Clin Transplant 17:151, 2003 7. Amirzargar A, Lessanpezeshki M, Fathi A, et al: TH1/TH2 cytokine analysis in Iranian renal transplants recipients. Transplant Proc 37:2985, 2005 8. Berber I, Yigit B, Isitmangil G, et al: Evaluation of pretransplant serum cytokine levels in renal transplant recipients. Transplant Proc 40:92, 2008 9. Merville P, Lambert C, Durand I, et al: High frequency of IL-10 secreting CD4⫹ graft-infiltrating T lymphocytes in promptly rejected kidney allografts. Transplantation 59:1113, 1995 10. Karczewski J, Karczewski M, Glyda M, Wiktorowicz K: Role of TH1/TH2 cytokines in kidney allograft rejection. Transplant Proc 40:3390, 2008 11. Jimenez R, Ramirez R, Carracedo J, et al: Cytometric bead array (CBA) for the measurement of cytokines in urine and plasma of patients undergoing renal rejection. Cytokine 32:45, 2005 12. Karczewski M, Karczewski J, Kostrzewa A, Wiktorowicz K, Glyda M: The role of Foxp3⫹ regulatory T cells in kidney transplantation. Transplant Proc 41:1527, 2009 13. Baan CC, Weimar W: Intragraft cytokine gene expression: implications for clinical transplantation. Transplant Int 11:169, 1998 14. Loong CC, Chen A, Lui WY, King KL, Lin CY: Expression of cytokines, growth factors and adhesion molecules in rejecting human renal allograft. Transplant Proc 28:1445, 1996 15. Sadeghi M, Daniel V, Naujokat C, Mehrabi A, Zeier M, Opelz G: Evidence for IFN-gamma up-and IL-4 down-regulation late post-transplant in patients with good kidney graft outcome. Clin Transplant 21:449, 2007 16. Wood KJ, Sawitzki B: Interferon gamma: a crucial role in the function of induced regulatory T cells in vivo. Trends Immunol 27:183, 2006