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Circulating and Intragraft TH17 Cells Are Not Increased at the Early Stage of Chronic Allograft Nephropathy
Circulating and Intragraft TH17 Cells Are Not Increased at the Early Stage of Chronic Allograft Nephropathy C. Tinel, M. Samson, L. Martin, B. Bonnotte, and C. Mousson ABSTRACT Since TH17 cells could play a role in the pathogenesis of allograft nephropathy, we investigated them in peripheral blood and kidney allograft infiltrates. We compared percentages of TH17 cells and IL17A in peripheral blood of 14 kidney allograft recipients and 8 healthy volunteers. Allograft recipients experiencing graft dysfunction and kidney biopsy specimens showing chronic allograft nephropathy (CAN) were distinguished from a “control group,” both of which were tested for TH17 and CD15⫹ staining. Allograft recipients displayed a significantly lower percentage of TH17 cells and IL17A blood levels than healthy volunteers, suggesting effects of the immunosuppressive regimen. No difference in these values was observed between the CAN group and the control group. On kidney allograft biopsies, CD15⫹ infiltrate was significantly higher in the CAN group than in the control group. In CAN, IL17 secretion might play a chemoattractant role for neutrophils. However, these preliminary data have to be confirmed in larger studies. AIVE CD4⫹ T cells are traditionally believed to differentiate into TH1 and TH2 elements.1 Recently, a subset of interleukin (IL)-17–producing T cells (TH17) has been described to be distinct from TH1 or TH2 cells. Characterized by their specific transcription factor ROR-␥t (retinoic-related orphan receptor gamma t), TH17 cells have been shown to play a role in the induction of several autoimmune diseases.2 Their precise role in the pathogenesis of allograft rejection remains to be determined.3– 6 In addition to drug toxicity and impaired initial fraction, long-term graft survival is limited by chronic allograft nephropathy (CAN). So far, cellular chronic allograft rejection is thought to be mediated by TH1 effectors. A recent report by Deteix et al7 correlated shorter graft survival with TH17 infiltration in detransplanted kidneys displaying terminal failure.7 We investigated whether a local or systemic TH17 signature could be observed at an earlier stage of CAN.
N
MATERIALS AND METHODS Kidney Samples Among renal transplant recipients at our center, 14 subjects underwent a biopsy for graft dysfunction between January and June 2011. Upon histological analysis according to the Banff classification, biopsy specimens were classified as CAN or “controls.” CAN was defined by the presence of interstitial fibrosis and tubular atrophy (IF/TA) associated with glomerulitis and/or tubulitis. The control group was defined as either recurrence of the initial
dysfunction or calcineurin inhibitor toxicity or no pathological lesion. In addition to routine examination using Masson’s trichrome hematoxylin and eosin (H&E)-Safran, periodic acid Schiff, and silver staining, we performed immunohistochemistry stains for IL-17 (R&D Systems, Lille, France) and CD15 (Immunotech, Luminy, France) primary antibodies, as revealed by biotinylated secondary antibodies ⫹ Streptavidine-Peroxidase and DAB substrate (Ventana Medical, Tucson, Ariz).
Blood Samples Following informed consent we collected blood from 14 patients at the time of their biopsy as well as from 8 healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated using density gradient centrifugation. Regulatory T cells (Treg) were detected by cell surface staining using flow cytometric analysis employing PECy5-CD4, PE-CD25, and intracellular eFluor450FoxP3. For CD4 T-cell analysis, PBMC were stimulated for 5 hours with ionomycin (1 g/mL), phorbol 12-myristate 13-acetate (PMA; 0.1 g/mL), and Brefeldin A prior to surface staining with PECy7CD3 and Pacific Blue-CD8 as well as intracellular staining with From the Departments of Nephrology-Transplantation (C.T., C.M.), and Pathology (L.M.), University Hospital, and the INSERM U1098, Faculty of Medicine (C.T., M.S., B.B.), Dijon, France. Address reprint requests to Christiane Mousson, MD, PhD, Department of Nephrology-Transplantation, University Hospital, 14 rue Gaffarel, 21079 Dijon, France. E-mail: christiane.mousson@ chu-dijon.fr
© 2012 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.09.092
Transplantation Proceedings, 44, 2827–2828 (2012)
2827
2828
TINEL, SAMSON, MARTIN ET AL
B NS
NS
p = 0.02
10
5
0
Number of positive cells per field
15
15
IL-17A pg/mL
% IL-17A - CD4+ T cells
A Fig 1. (A) TH17 cell percentage was higher in healthy volunteer blood than in transplant recipients. (B) TH17 and neutrophils cells on kidney allograft biopsy specimens; CD15⫹ infiltrate is significantly higher in the CAN group (CIR) than in the control group.
10
5
0
CIR group
Control group
Healthy volunteers
PE-IL-17, APC-IFN-␥, and FITC-IL-4. Analysis was performed using a LSR II Flow Cytometer (BD Biosciences) and Flow Jo software (Tree Star Inc.). Serum IL-17A levels were measured with an ELISA kit (eBioscience) according to the manufacturer’s instructions.
RESULTS
The time from transplantation to biopsy was 82 ⫾ 64 months for the 7 patients in the CAN group and 43 ⫾ 30 months for the 7 patients in the control group (P ⫽ .17). Measured glomerular filtration rate at biopsy was 29 mL/ min⫺1 in the CAN group versus 49 mL/min⫺1 in the control group (P ⬍ .05) (Fig 1). TH17 cell percentage (Fig 1A) was lower among transplant recipients than healthy volunteers (1.76% vs 7.18%; P ⫽ .05). Kidney allograft recipients revealed no significant difference between the CAN and the control group (1.17% vs 0.54%; P ⫽ .33). Mean IL-17 blood level was 5.44 pg/mL in healthy volunteers, whereas it was undetectable in transplant recipients (detection threshold of 4 pg/mL). Finally, analysis of kidney sections did not show significant difference in the IL-17–positive cell infiltrate between the CAN and the control group: number of IL-17⫹ cells per field 5.83 vs 4.71 (P ⫽ .40). In contrast, the number of CD15⫹ cells (neutrophils) was significantly higher (Fig 1B) in the CAN as compared with the control group: 8.62 vs 5.44 cells/field (P ⫽ .05). DISCUSSION
We observed a TH17 signature neither in peripheral lymphocytes nor among serum cytokines of kidney allograft recipients experiencing CAN. In peripheral blood the percentage of TH17 lymphocytes as well as the IL-17 levels were higher among healthy volunteers, suggesting a possible role of the immunosuppressive regimen. Moreover, CAN may be a local phenomenon within the graft itself
CIR group
Control group
Healthy volunteers
10
p = 0.40
p 0.05 CIR group Control group
8 6 4 2 0
IL-17
CD15
without any systemic reflection.8 In this study, we did not observe any difference in the TH17 cell infiltrate. Significantly more neutrophils infiltrated the grafts in CAN. IL-17 has been shown to be a strong chemo-attractant for neutrophils9; thus, these neutrophils might have been recruited via IL-17 secretion by TH17 cells in CAN. In conclusion, these results need to be confirmed with more patients and controlled by an automated count. The precise role of TH17 cells among the full T-helper orchestra remains to be determined in allograft rejection. REFERENCES 1. Mosmann TR, Cherwinski H, Bond MW, et al: Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136:2348, 1986 2. Miossec P, Korn T, Kuchroo VK: Interleukin-17 and type 17 helper T cells. N Engl J Med 361:888, 2009 3. Antonysamy MA, Fanslow WC, Fu F, et al: Evidence for a role of IL-17 in organ allograft rejection: IL-17 promotes the functional differentiation of dendritic cell progenitors. J Immunol 162:577, 1999 4. Yuan X, Paez-Cortez J, Schmitt-Knosalla I, et al: A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy. J Exp Med 205:3133, 2008 5. Chadha R, Heidt S, Jones ND, et al: Th17: contributors to allograft rejection and a barrier to the induction of transplantation tolerance? Transplantation 91:939, 2011 6. Abadja F, Sarraj B, Ansari MJ: Significance of T helper 17 immunity in transplantation. Curr Opin Organ Transplant 17:8, 2012 7. Deteix C, Attuil-Audenis V, Duthey A, et al: Intragraft Th17 infiltrate promotes lymphoid neogenesis and hastens clinical chronic rejection. J Immunol 184:5344, 2010 8. Thaunat O, Field AC, Dai J, et al: Lymphoid neogenesis in chronic rejection: evidence for a local humoral alloimmune response. Proc Natl Acad Sci U S A 102:14723, 2005 9. Laan M, Cui ZH, Hoshino H, et al: Neutrophil recruitment by human IL-17 via CXC chemokine release in the airways. J Immunol 162:2347, 1999
N
MATERIALS AND METHODS Kidney Samples Among renal transplant recipients at our center, 14 subjects underwent a biopsy for graft dysfunction between January and June 2011. Upon histological analysis according to the Banff classification, biopsy specimens were classified as CAN or “controls.” CAN was defined by the presence of interstitial fibrosis and tubular atrophy (IF/TA) associated with glomerulitis and/or tubulitis. The control group was defined as either recurrence of the initial
dysfunction or calcineurin inhibitor toxicity or no pathological lesion. In addition to routine examination using Masson’s trichrome hematoxylin and eosin (H&E)-Safran, periodic acid Schiff, and silver staining, we performed immunohistochemistry stains for IL-17 (R&D Systems, Lille, France) and CD15 (Immunotech, Luminy, France) primary antibodies, as revealed by biotinylated secondary antibodies ⫹ Streptavidine-Peroxidase and DAB substrate (Ventana Medical, Tucson, Ariz).
Blood Samples Following informed consent we collected blood from 14 patients at the time of their biopsy as well as from 8 healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated using density gradient centrifugation. Regulatory T cells (Treg) were detected by cell surface staining using flow cytometric analysis employing PECy5-CD4, PE-CD25, and intracellular eFluor450FoxP3. For CD4 T-cell analysis, PBMC were stimulated for 5 hours with ionomycin (1 g/mL), phorbol 12-myristate 13-acetate (PMA; 0.1 g/mL), and Brefeldin A prior to surface staining with PECy7CD3 and Pacific Blue-CD8 as well as intracellular staining with From the Departments of Nephrology-Transplantation (C.T., C.M.), and Pathology (L.M.), University Hospital, and the INSERM U1098, Faculty of Medicine (C.T., M.S., B.B.), Dijon, France. Address reprint requests to Christiane Mousson, MD, PhD, Department of Nephrology-Transplantation, University Hospital, 14 rue Gaffarel, 21079 Dijon, France. E-mail: christiane.mousson@ chu-dijon.fr
© 2012 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.09.092
Transplantation Proceedings, 44, 2827–2828 (2012)
2827
2828
TINEL, SAMSON, MARTIN ET AL
B NS
NS
p = 0.02
10
5
0
Number of positive cells per field
15
15
IL-17A pg/mL
% IL-17A - CD4+ T cells
A Fig 1. (A) TH17 cell percentage was higher in healthy volunteer blood than in transplant recipients. (B) TH17 and neutrophils cells on kidney allograft biopsy specimens; CD15⫹ infiltrate is significantly higher in the CAN group (CIR) than in the control group.
10
5
0
CIR group
Control group
Healthy volunteers
PE-IL-17, APC-IFN-␥, and FITC-IL-4. Analysis was performed using a LSR II Flow Cytometer (BD Biosciences) and Flow Jo software (Tree Star Inc.). Serum IL-17A levels were measured with an ELISA kit (eBioscience) according to the manufacturer’s instructions.
RESULTS
The time from transplantation to biopsy was 82 ⫾ 64 months for the 7 patients in the CAN group and 43 ⫾ 30 months for the 7 patients in the control group (P ⫽ .17). Measured glomerular filtration rate at biopsy was 29 mL/ min⫺1 in the CAN group versus 49 mL/min⫺1 in the control group (P ⬍ .05) (Fig 1). TH17 cell percentage (Fig 1A) was lower among transplant recipients than healthy volunteers (1.76% vs 7.18%; P ⫽ .05). Kidney allograft recipients revealed no significant difference between the CAN and the control group (1.17% vs 0.54%; P ⫽ .33). Mean IL-17 blood level was 5.44 pg/mL in healthy volunteers, whereas it was undetectable in transplant recipients (detection threshold of 4 pg/mL). Finally, analysis of kidney sections did not show significant difference in the IL-17–positive cell infiltrate between the CAN and the control group: number of IL-17⫹ cells per field 5.83 vs 4.71 (P ⫽ .40). In contrast, the number of CD15⫹ cells (neutrophils) was significantly higher (Fig 1B) in the CAN as compared with the control group: 8.62 vs 5.44 cells/field (P ⫽ .05). DISCUSSION
We observed a TH17 signature neither in peripheral lymphocytes nor among serum cytokines of kidney allograft recipients experiencing CAN. In peripheral blood the percentage of TH17 lymphocytes as well as the IL-17 levels were higher among healthy volunteers, suggesting a possible role of the immunosuppressive regimen. Moreover, CAN may be a local phenomenon within the graft itself
CIR group
Control group
Healthy volunteers
10
p = 0.40
p 0.05 CIR group Control group
8 6 4 2 0
IL-17
CD15
without any systemic reflection.8 In this study, we did not observe any difference in the TH17 cell infiltrate. Significantly more neutrophils infiltrated the grafts in CAN. IL-17 has been shown to be a strong chemo-attractant for neutrophils9; thus, these neutrophils might have been recruited via IL-17 secretion by TH17 cells in CAN. In conclusion, these results need to be confirmed with more patients and controlled by an automated count. The precise role of TH17 cells among the full T-helper orchestra remains to be determined in allograft rejection. REFERENCES 1. Mosmann TR, Cherwinski H, Bond MW, et al: Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136:2348, 1986 2. Miossec P, Korn T, Kuchroo VK: Interleukin-17 and type 17 helper T cells. N Engl J Med 361:888, 2009 3. Antonysamy MA, Fanslow WC, Fu F, et al: Evidence for a role of IL-17 in organ allograft rejection: IL-17 promotes the functional differentiation of dendritic cell progenitors. J Immunol 162:577, 1999 4. Yuan X, Paez-Cortez J, Schmitt-Knosalla I, et al: A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy. J Exp Med 205:3133, 2008 5. Chadha R, Heidt S, Jones ND, et al: Th17: contributors to allograft rejection and a barrier to the induction of transplantation tolerance? Transplantation 91:939, 2011 6. Abadja F, Sarraj B, Ansari MJ: Significance of T helper 17 immunity in transplantation. Curr Opin Organ Transplant 17:8, 2012 7. Deteix C, Attuil-Audenis V, Duthey A, et al: Intragraft Th17 infiltrate promotes lymphoid neogenesis and hastens clinical chronic rejection. J Immunol 184:5344, 2010 8. Thaunat O, Field AC, Dai J, et al: Lymphoid neogenesis in chronic rejection: evidence for a local humoral alloimmune response. Proc Natl Acad Sci U S A 102:14723, 2005 9. Laan M, Cui ZH, Hoshino H, et al: Neutrophil recruitment by human IL-17 via CXC chemokine release in the airways. J Immunol 162:2347, 1999