Nephrol Dial Transplant (2014) 29: 1020–1028 doi: 10.1093/ndt/gft495 Advance Access publication 18 December 2013
Invariant natural killer T cells are depleted in renal impairment and recover after kidney transplantation Konrad Peukert1, Gerhard Wingender2, Margret Patecki1, Stephan Wagner3, Roland Schmitt1, Shuwang Ge1, Anke Schwarz1, Mitchell Kronenberg2, Hermann Haller1 and Sibylle von Vietinghoff1 1
Division of Nephrology and Hypertension, Department of Medicine, Hannover Medical School, Hannover, Germany,
2
Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA and
3
Georg Haas Dialysis Centre, Giessen, Germany
ORIGINAL ARTICLE
Correspondence and offprint requests to: Sibylle von Vietinghoff; E-mail: [email protected]
A B S T R AC T
INTRODUCTION
Background. Altered immune function in patients with renal failure results in both susceptibility to infection and increased inflammatory response. Invariant natural killer T (iNKT) cells are a conserved, immunoregulatory T lymphocyte subset that responds to lipid antigens with near-immediate cytokine production and cytotoxicity. iNKT cells are required for the antibacterial host response. Whether renal failure and renal replacement therapy alter iNKT cell abundance or phenotype has not been investigated. Methods. iNKT cells were studied by flow cytometry in the peripheral blood of patients with acute renal failure, chronic haemo- and peritoneal dialysis (PD), chronic kidney disease and after renal transplantation. Results. A very marked reduction in iNKT lymphocytes was found in acute renal failure before the first haemodialysis (HD) session. iNKT cells were depleted in end-stage renal disease patients receiving either HD or PD. iNKT cell depletion was accentuated after an HD session. Lesser degrees were observed in patients with non-dialysis-dependent chronic kidney disease. CD56 and CD161 NK cell marker expression was decreased in renal impairment. CD56+ and CD161+ iNKT cells produced more interferon-γ than negative cells of the same donor. Within the first year after kidney transplantation, the decrease in iNKT cells and their NK cell markers was reverted. Conclusions. We describe for the first time that iNKT lymphocytes are reduced in end-stage renal disease and further depleted by HD. iNKT cells are important for early host response including activation of other immune cells and their depletion may contribute to immune dysfunction in renal disease.
End-stage renal disease is associated with profound immune system alterations. Clinically, high rates of infection in the presence of elevated levels of systemic inflammatory markers [1] suggest an altered, and less efficient host response and at the same time detrimental inflammation, e.g. in atherosclerosis [2, 3]. Among peripheral blood leucocytes in renal impairment, neutrophilic granulocytes were increased, monocyte counts and subsets altered [4] and lymphocyte counts decreased [5, 6]. Peripheral blood neutrophilia and lymphopenia correlated with increased mortality in the general population and also in end-stage renal disease [7]. This was shown in several independent studies in patients receiving both haemodialysis (HD) [6, 8–10] and peritoneal dialysis (PD) [11] for renal replacement. Lymphopaenia was already apparent in patients with chronic kidney disease (CKD) before renal replacement therapy was required [12]. Decreases in T-cell number and function [13] were observed with the current HD regimens in the majority [14, 15] albeit not in all cohorts [16]. A study comparing patients with end-stage renal disease before and after the first HD treatment found no significant difference [17] suggesting T-cell depletion to be due to uraemia rather than a specific treatment modality. Among T-cells, CD4+ T-cell concentrations were decreased in patients receiving HD [14–18]. However, this was not observed in PD patients or patients before the start of renal replacement therapy [15]. Naïve CD4+ T cells [16–18] and also CD4+CD25+ and Foxp3+ regulatory T cells were depleted [19, 20]. Antigen-specific classical T cell responses were only partially impaired to HBsAg, the frequency of CMV reactive CD4+CD28− T cells was even increased in aged HD patients [21].
Keywords: dialysis, end-stage renal disease, natural killer T cells, specific immunity
© The Author 2013. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
1020
iNKT cells in renal failure
M AT E R I A L S A N D M E T H O D S Patients and probands Venous blood was drawn after local ethics board approval (MHH 2010/807, KfH20110006, LIAI #VD-057) and informed consent according to the Declaration of Helsinki. Blood from HD patients was collected after a long interval. Peripheral blood counts and clinical chemistry were assessed in the clinical laboratory at Hannover Medical School. Cell isolation, stimulation, staining and flow cytometry Peripheral blood mononuclear cells were isolated by density gradient centrifugation as described [43]. Cells were counted in a haemocytometer, viability was assessed by trypan blue exclusion. Ex vivo re-stimulation was for 4 h with PMA/ ionomycin (both Sigma-Aldrich) and Golgi-Stop (BD PharMingen, San Jose, CA, USA). The following anti-human antibodies were used in flow cytometry: iVα24Jα18 (6B11), CD3 (HIT3a), CD4 (OKT4), CD19 (HIB19), CD56 (NCAM16.2), CD161 (HP-3G10), IFNγ (4S.B3 and B27) and IL-4 (8D4-8). Antibodies were purchased from Abcam (Cambridge, MA), BD Biosciences (San Diego, CA), BioLegend (San Diego, CA), eBioscience (San Diego, CA), or Invitrogen (Carlsbad, CA). BD-Fix-Perm (BD PharMingen) and near infra-red LIVE/ DEAD® Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) were used according to the manufacturer’s instructions. Flow cytometry analysis was performed on a Becton-Dickinson FACSCanto or LSRII. Data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR). Statistical analysis Two-tailed t-test with correction for unequal variances, if applicable, or ANOVA with appropriate post hoc test was used as indicated in figure legends, P-values of
Invariant natural killer T cells are depleted in renal impairment and recover after kidney transplantation Konrad Peukert1, Gerhard Wingender2, Margret Patecki1, Stephan Wagner3, Roland Schmitt1, Shuwang Ge1, Anke Schwarz1, Mitchell Kronenberg2, Hermann Haller1 and Sibylle von Vietinghoff1 1
Division of Nephrology and Hypertension, Department of Medicine, Hannover Medical School, Hannover, Germany,
2
Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA and
3
Georg Haas Dialysis Centre, Giessen, Germany
ORIGINAL ARTICLE
Correspondence and offprint requests to: Sibylle von Vietinghoff; E-mail: [email protected]
A B S T R AC T
INTRODUCTION
Background. Altered immune function in patients with renal failure results in both susceptibility to infection and increased inflammatory response. Invariant natural killer T (iNKT) cells are a conserved, immunoregulatory T lymphocyte subset that responds to lipid antigens with near-immediate cytokine production and cytotoxicity. iNKT cells are required for the antibacterial host response. Whether renal failure and renal replacement therapy alter iNKT cell abundance or phenotype has not been investigated. Methods. iNKT cells were studied by flow cytometry in the peripheral blood of patients with acute renal failure, chronic haemo- and peritoneal dialysis (PD), chronic kidney disease and after renal transplantation. Results. A very marked reduction in iNKT lymphocytes was found in acute renal failure before the first haemodialysis (HD) session. iNKT cells were depleted in end-stage renal disease patients receiving either HD or PD. iNKT cell depletion was accentuated after an HD session. Lesser degrees were observed in patients with non-dialysis-dependent chronic kidney disease. CD56 and CD161 NK cell marker expression was decreased in renal impairment. CD56+ and CD161+ iNKT cells produced more interferon-γ than negative cells of the same donor. Within the first year after kidney transplantation, the decrease in iNKT cells and their NK cell markers was reverted. Conclusions. We describe for the first time that iNKT lymphocytes are reduced in end-stage renal disease and further depleted by HD. iNKT cells are important for early host response including activation of other immune cells and their depletion may contribute to immune dysfunction in renal disease.
End-stage renal disease is associated with profound immune system alterations. Clinically, high rates of infection in the presence of elevated levels of systemic inflammatory markers [1] suggest an altered, and less efficient host response and at the same time detrimental inflammation, e.g. in atherosclerosis [2, 3]. Among peripheral blood leucocytes in renal impairment, neutrophilic granulocytes were increased, monocyte counts and subsets altered [4] and lymphocyte counts decreased [5, 6]. Peripheral blood neutrophilia and lymphopenia correlated with increased mortality in the general population and also in end-stage renal disease [7]. This was shown in several independent studies in patients receiving both haemodialysis (HD) [6, 8–10] and peritoneal dialysis (PD) [11] for renal replacement. Lymphopaenia was already apparent in patients with chronic kidney disease (CKD) before renal replacement therapy was required [12]. Decreases in T-cell number and function [13] were observed with the current HD regimens in the majority [14, 15] albeit not in all cohorts [16]. A study comparing patients with end-stage renal disease before and after the first HD treatment found no significant difference [17] suggesting T-cell depletion to be due to uraemia rather than a specific treatment modality. Among T-cells, CD4+ T-cell concentrations were decreased in patients receiving HD [14–18]. However, this was not observed in PD patients or patients before the start of renal replacement therapy [15]. Naïve CD4+ T cells [16–18] and also CD4+CD25+ and Foxp3+ regulatory T cells were depleted [19, 20]. Antigen-specific classical T cell responses were only partially impaired to HBsAg, the frequency of CMV reactive CD4+CD28− T cells was even increased in aged HD patients [21].
Keywords: dialysis, end-stage renal disease, natural killer T cells, specific immunity
© The Author 2013. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
1020
iNKT cells in renal failure
M AT E R I A L S A N D M E T H O D S Patients and probands Venous blood was drawn after local ethics board approval (MHH 2010/807, KfH20110006, LIAI #VD-057) and informed consent according to the Declaration of Helsinki. Blood from HD patients was collected after a long interval. Peripheral blood counts and clinical chemistry were assessed in the clinical laboratory at Hannover Medical School. Cell isolation, stimulation, staining and flow cytometry Peripheral blood mononuclear cells were isolated by density gradient centrifugation as described [43]. Cells were counted in a haemocytometer, viability was assessed by trypan blue exclusion. Ex vivo re-stimulation was for 4 h with PMA/ ionomycin (both Sigma-Aldrich) and Golgi-Stop (BD PharMingen, San Jose, CA, USA). The following anti-human antibodies were used in flow cytometry: iVα24Jα18 (6B11), CD3 (HIT3a), CD4 (OKT4), CD19 (HIB19), CD56 (NCAM16.2), CD161 (HP-3G10), IFNγ (4S.B3 and B27) and IL-4 (8D4-8). Antibodies were purchased from Abcam (Cambridge, MA), BD Biosciences (San Diego, CA), BioLegend (San Diego, CA), eBioscience (San Diego, CA), or Invitrogen (Carlsbad, CA). BD-Fix-Perm (BD PharMingen) and near infra-red LIVE/ DEAD® Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) were used according to the manufacturer’s instructions. Flow cytometry analysis was performed on a Becton-Dickinson FACSCanto or LSRII. Data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR). Statistical analysis Two-tailed t-test with correction for unequal variances, if applicable, or ANOVA with appropriate post hoc test was used as indicated in figure legends, P-values of
