Galectin-1 in Stable Liver Transplant Recipients M.J. Garcíaa,b, F. Juradob,c, D. San Segundob,c, M. López-Hoyosb,c, P. Iruzubietaa,b, S. Llerenaa,b, F. Casafonta,b, M. Ariasa,b, Á. Puentea,b, J. Crespoa,b, and E. Fábregaa,b,* a Gastroenterology and Hepatology Unit, University Hospital “Marqués de Valdecilla”, Santander, Spain; bInstituto de Investigación Márqués de Valdecilla (IDIVAL), Santander, Spain; and cImmunology Unit, University Hospital “Marqués de Valdecilla”, Santander, Spain
ABSTRACT Introduction. The achievement of a state of tolerance and minimization of the immunosuppressive load form part of the “Holy Grail” in solid organ transplantation. Galectin-1 recently has been described to be involved in the maintenance of a tolerant environment, but there is no evidence of its role in human liver transplantation. The aim of our study was to measure the serum levels of galectin-1 in stable liver transplant recipients. Methods. Serum levels of galectin-1 were determined in 30 stable liver transplant recipients who had been free of rejection episodes for at least 8 years. Fifteen patients with an acute rejection episode and 34 healthy subjects were used as the control group. Results. The concentrations of galectin-1 were significantly higher in stable liver transplant recipients compared with healthy subjects and with the acute rejection group. Conclusions. These preliminary results indicate that galectin-1 is upregulated in stable liver transplant recipients. Thus, our results extend the recent findings that galectin-1 may play an immune-suppressive role in liver transplantation. It remains to be established whether it might help to induce tolerance in liver transplantation.
T
HE STUDY of mechanisms underlying the induction of immune suppression and tolerance in transplantation has placed little emphasis on the role of interaction between proteins and carbohydrates or lipids. Lectins are carbohydrate-binding proteins that recognize specific oligosaccharides on glycoprotein/lipids. Galectins constitute a still growing family of b-galactoside-binding lectins that have been highly conserved throughout evolution. One member of this family, galectin-1 (GAL-1), has been shown to participate in regulation of innate and adaptive immunity. Galectin-1 exerts anti-inflammatory effects by impairing leukocyte adhesion and transendothelial migration, and reducing the secretion of proinflammatory cytokines. In addition, GAL-1 participates in T-cell homeostasis and tolerance due to the ability of trigger apoptosis of Th1, Th17, and Tc1 cell subsets, but spares Th2 or regulatory FoxP3þ T cells and promotes the generation of tolerogenic dendritic cells [1]. The antiinflammatory role of recombinant GAL-1 has been demonstrated in different mouse models of autoimmunity. Recombinant GAL-1 is known to ameliorate experimental models of inflammatory Th1- and/or Th17-mediated diseases such as autoimmune uveitis [2], or collagen-induced ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 47, 93e96 (2015)
arthritis [3]. Offner et al. provided clinical and histopathological evidence that GAL-1 prevents the development of experimental autoimmune encephalomyelitis in rats [4]. Furthermore, administration of recombinant GAL-1 in murine models has been shown to inhibit graft-vs-host disease and to increase host survival after bone marrow transplantation [5]. In the present work, we measured the serum levels of GAL-1 in stable liver transplant recipients (LTR) with long-term functioning allograft. Additionally, we looked at the pretransplantation and early posttransplantation changes in serum levels of GAL-1 in patients suffering from acute rejection episode (ARE) and compared with those without ARE. METHODS We studied a cohort of 30 LTR who displayed stable function and had been free of rejection episodes for at least 8 years. All of them
*Address correspondence to Emilio Fábrega, MD, Gastroenterology and Hepatology Unit, Faculty of Medicine, University Hospital “Marqués de Valdecilla”, Avenida Valdecilla s/n, Santander 39008, Cantabria, Spain. E-mail: [email protected] 0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.12.001
93
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Table 1. Comparison of Demographic Features and Clinical Events in Stable Liver Transplant Recipient (LTR) and the Acute Rejection Group (ARG)
Age (y) Gender (male/female) Etiology of cirrhosis (alcohol, hepatitis C virus, others) Hepatocellular carcinoma (n, %) Biliary complications (strictures and leaks) (n, %) Hepatic artery thrombosis (n, %) Cardiac complications (cardiomyopathy, acute myocardial ischemic/infarction) (n, %) Acute ischemic stroke (n, %) Cytomegalovirus disease (n, %) Renal in sufficiency (serum creatinine > 1.5 mg/dL) (n, %) De novo neoplasms (n, %) Diabetes mellitus (n, %) Arterial hypertension (n, %)
LTR (n ¼ 30)
ARG (n ¼ 15)
P Value
58.7 (38e73) 20/10 24, 6, 0 (2, 6.7) (8, 26.7) (0, 0) (7, 23.3)
56 (24e64) 13/2 10, 1, 4 (2, 13.3) (6, 40) (0, 0) (0, 0)
NS NS NS NS NS NS 0,042
(4, 13.3) (1, 3.3) (15, 50) (6, 20) (11, 36.7) (21, 70)
(1, (5, (7, (2, (9, (7,
6.7) 33.3) 46.7) 13.3) 60) 46.7)
NS 0,005 NS NS NS NS
Abbreviation: NS, not significant.
were subjected to an equivalent immunosuppression therapy with either cyclosporine (n ¼ 16) or tacrolimus (n ¼ 14). In addition, we analyzed 50 LTR who were divided according to the occurrence of ARE confirmed by biopsy in the early postoperative period: ARE Yes (n ¼ 15) vs ARE No (n ¼ 35). Blood samples for GAL-1 measurements were collected at 1, 7, and 15 days after liver transplantation (LT) in the non-ARE group. In the rejection group, the blood samples were drawn on days 1 and 7 and between days 12 and 18 after LT (on the day of liver biopsy). Thirty-four age-matched healthy subjects were used as control subjects. Serum GAL-1 was measured using a commercial enzyme-linked immunoassay (QAYEE-BIO, Shanghai, China) according to the manufacturer’s instructions. The study protocol was performed in accordance with the Helsinki Declaration of 1975 (as revised in 1983). Approval by the hospital ethics committee and informed consent were obtained from each patient. Data were expressed as median values and interquartile ranges. Mann-Whitney U and Kruskal-Wallis tests were used for comparisons between patients and control subjects and
Fig 1. Serum levels of galectin-1 in stable liver transplant recipients (LTR) compared with acute rejection group (ARG) and healthy subjects (HS). Data are represented as a box plot showing the median and the interquartile range.
within the patient group, respectively. Graft survival was assessed by the Kaplan-Meier method. Values of P < .05 were considered statistically significant.
RESULTS
The demographic and clinical events in the stable LTR and rejection groups are summarized in Table 1. Regarding the first part of the study in long-term recipients, we observed differences in the serum levels of GAL-1 among healthy subjects, the rejection group, and the stable LTR group. As shown in Fig 1, the levels of GAL-1 were significantly higher in the LTR compared with the healthy and acute rejection groups (P ¼ .005). On the other hand, in the early postoperative group of patients, the concentration of GAL-1 was lower (although not statistically significant) in the ARE vs the non-ARE group during the entire postoperative period (P ¼ not significant; Fig 2). Moreover, we also observed a progressive decrease in serum levels of GAL-1 from day 1 to day 15 in the ARE group. The whole transplant group, including those with stable graft function, had higher serum
Fig 2. Time course of serum levels of galectin-1 after liver transplantation. Data are presented as mean standard deviation. ARE, acute rejection episode; NS, not significant.
GALECTIN-1 IN STABLE LT
95
Fig 3. The frequency distribution of galectin-1 in liver transplant recipients.
levels of GAL-1 than the control subjects (231 ng/mL; range: 164 to 246 ng/mL; P ¼ .05) at any time after the early LT period. Fig 3 shows the frequency distribution of GAL-1 in LTR. In addition, we have performed receiver-operator characteristic curve analysis to differentiate the LTR and ARE groups (Fig 4), and we have performed receiveroperator characteristic curve analysis to differentiate the LTR and ARE groups (Fig 4). Fig 5 shows the graft survival curves from patients with LT, with or without ARE. DISCUSSION
Galectins are expressed in immune-privileged sites. For example, GAL-1 is expressed in placenta, brain, and reproductive organs, suggesting that it might trigger the death of infiltrating T cells and protect these sites from tissue damage induced by activated bystander T cells. In addition, GAL-1 can induce the differentiation of dendritic cells with regulatory function involving interleukin (IL)-27 and IL-10, which promote T-cell tolerance [1]. In our study, we observed for the first time that GAL-1 levels were higher in LTR than in healthy subjects and LTR who had experienced acute rejection. Furthermore, we observed that GAL-1 levels were lower in the rejection group than in the nonrejection group during the entire postoperative period. These results are in agreement with arguments derived from experimental models showing that administration of recombinant GAL-1 prolongs renal allograft survival by inhibiting CD8þ T cell
Fig 4. Receiver-operator characteristic curve to differentiate stable liver transplant recipient (LTR) and acute rejection episode (ARE) groups. P ¼ .002. Area under curve ¼ 79.5%. Points that better discriminate between LTR and ARE group patients ¼ 312.5 ng/mL with sensitivity of 83.3% and specificity of 64.5%.
96
GARCÍA, JURADO, SAN SEGUNDO ET AL
Fig 5. Kaplan-Meier survival curves from patients with liver transplant (LTR), with or without acute rejection episode (ARE).
responses and delays allograft liver rejection through the enhancement of T-cell apoptosis in the graft and spleen, diminished Th1 and Th17 cytokine profiles, and increased IL-10 within the grafts [6e8]. Conversely, in the absence of GAL-1, CD8þ T cells, showing enhanced interferon g and IL-17 production, accelerate rejection of skin grafts in mice [9]. The present study, together with previous references, point to a possible role of GAL-1 in the promotion of alloimmune tolerance in LT, and could be of help in protocols of operational tolerance or at least in weaning off of immunosuppression. REFERENCES [1] Ilarregui JM, Croci DO, Bianco GA, et al. Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1driven immunoregulatory circuit involving interleukin 27 and interleukin 10. Nat Immunol 2009;10:981e91. [2] Toscano MA, Commodaro AG, llarregui JM, et al. Galectin-1 suppresses autoimmune retinal disease by promoting
concomitant Th2- and T regulatory-mediated anti-inflammatory responses. J Immunol 2006;176:6323e32. [3] Rabinovich GA, Daly G, Dreja H, et al. Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T-cell apoptosis. J Exp Med 1999;190:385e98. [4] Offner H, Colnik B, Bringman TS, et al. Recombinant human beta-galactoside binding lectin suppresses clinical and histological signs of experimental autoimmune encephalomyelitis. J Neuroimmunol 1990;28:177e84. [5] Baum LG, Blackall DP, Arias-Magallano S, et al. Amelioration of graft vs host disease by galectin-1. Clin Immunol 2003;109:295e307. [6] Xu G, Tu W, Xu C. Immunological tolerance induced by galectin-1 in rat allogeneic renal transplantation. Int Immunopharmacol 2010;10:643e7. [7] Ye Y, Yan S, Jiang G, et al. Galectin-1 prolongs survival of mouse liver allografts from Flt3L-pretreated donors. Am J Transplant 2013;13:569e79. [8] Fábrega E, López-Hoyos M, San Segundo D, et al. Changes in the serum levels of interleukin-17/interleukin-23 during acute rejection in liver transplantation. Liver Transpl 2009;15:629e33. [9] Moreau A, Noble A, Ratnasothy K, et al. Absence of galectin-1 accelerates CD8þ T cell-mediated graft rejection. Eur J Immunol 2012;42:2881e8.
ABSTRACT Introduction. The achievement of a state of tolerance and minimization of the immunosuppressive load form part of the “Holy Grail” in solid organ transplantation. Galectin-1 recently has been described to be involved in the maintenance of a tolerant environment, but there is no evidence of its role in human liver transplantation. The aim of our study was to measure the serum levels of galectin-1 in stable liver transplant recipients. Methods. Serum levels of galectin-1 were determined in 30 stable liver transplant recipients who had been free of rejection episodes for at least 8 years. Fifteen patients with an acute rejection episode and 34 healthy subjects were used as the control group. Results. The concentrations of galectin-1 were significantly higher in stable liver transplant recipients compared with healthy subjects and with the acute rejection group. Conclusions. These preliminary results indicate that galectin-1 is upregulated in stable liver transplant recipients. Thus, our results extend the recent findings that galectin-1 may play an immune-suppressive role in liver transplantation. It remains to be established whether it might help to induce tolerance in liver transplantation.
T
HE STUDY of mechanisms underlying the induction of immune suppression and tolerance in transplantation has placed little emphasis on the role of interaction between proteins and carbohydrates or lipids. Lectins are carbohydrate-binding proteins that recognize specific oligosaccharides on glycoprotein/lipids. Galectins constitute a still growing family of b-galactoside-binding lectins that have been highly conserved throughout evolution. One member of this family, galectin-1 (GAL-1), has been shown to participate in regulation of innate and adaptive immunity. Galectin-1 exerts anti-inflammatory effects by impairing leukocyte adhesion and transendothelial migration, and reducing the secretion of proinflammatory cytokines. In addition, GAL-1 participates in T-cell homeostasis and tolerance due to the ability of trigger apoptosis of Th1, Th17, and Tc1 cell subsets, but spares Th2 or regulatory FoxP3þ T cells and promotes the generation of tolerogenic dendritic cells [1]. The antiinflammatory role of recombinant GAL-1 has been demonstrated in different mouse models of autoimmunity. Recombinant GAL-1 is known to ameliorate experimental models of inflammatory Th1- and/or Th17-mediated diseases such as autoimmune uveitis [2], or collagen-induced ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 47, 93e96 (2015)
arthritis [3]. Offner et al. provided clinical and histopathological evidence that GAL-1 prevents the development of experimental autoimmune encephalomyelitis in rats [4]. Furthermore, administration of recombinant GAL-1 in murine models has been shown to inhibit graft-vs-host disease and to increase host survival after bone marrow transplantation [5]. In the present work, we measured the serum levels of GAL-1 in stable liver transplant recipients (LTR) with long-term functioning allograft. Additionally, we looked at the pretransplantation and early posttransplantation changes in serum levels of GAL-1 in patients suffering from acute rejection episode (ARE) and compared with those without ARE. METHODS We studied a cohort of 30 LTR who displayed stable function and had been free of rejection episodes for at least 8 years. All of them
*Address correspondence to Emilio Fábrega, MD, Gastroenterology and Hepatology Unit, Faculty of Medicine, University Hospital “Marqués de Valdecilla”, Avenida Valdecilla s/n, Santander 39008, Cantabria, Spain. E-mail: [email protected] 0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.12.001
93
94
GARCÍA, JURADO, SAN SEGUNDO ET AL
Table 1. Comparison of Demographic Features and Clinical Events in Stable Liver Transplant Recipient (LTR) and the Acute Rejection Group (ARG)
Age (y) Gender (male/female) Etiology of cirrhosis (alcohol, hepatitis C virus, others) Hepatocellular carcinoma (n, %) Biliary complications (strictures and leaks) (n, %) Hepatic artery thrombosis (n, %) Cardiac complications (cardiomyopathy, acute myocardial ischemic/infarction) (n, %) Acute ischemic stroke (n, %) Cytomegalovirus disease (n, %) Renal in sufficiency (serum creatinine > 1.5 mg/dL) (n, %) De novo neoplasms (n, %) Diabetes mellitus (n, %) Arterial hypertension (n, %)
LTR (n ¼ 30)
ARG (n ¼ 15)
P Value
58.7 (38e73) 20/10 24, 6, 0 (2, 6.7) (8, 26.7) (0, 0) (7, 23.3)
56 (24e64) 13/2 10, 1, 4 (2, 13.3) (6, 40) (0, 0) (0, 0)
NS NS NS NS NS NS 0,042
(4, 13.3) (1, 3.3) (15, 50) (6, 20) (11, 36.7) (21, 70)
(1, (5, (7, (2, (9, (7,
6.7) 33.3) 46.7) 13.3) 60) 46.7)
NS 0,005 NS NS NS NS
Abbreviation: NS, not significant.
were subjected to an equivalent immunosuppression therapy with either cyclosporine (n ¼ 16) or tacrolimus (n ¼ 14). In addition, we analyzed 50 LTR who were divided according to the occurrence of ARE confirmed by biopsy in the early postoperative period: ARE Yes (n ¼ 15) vs ARE No (n ¼ 35). Blood samples for GAL-1 measurements were collected at 1, 7, and 15 days after liver transplantation (LT) in the non-ARE group. In the rejection group, the blood samples were drawn on days 1 and 7 and between days 12 and 18 after LT (on the day of liver biopsy). Thirty-four age-matched healthy subjects were used as control subjects. Serum GAL-1 was measured using a commercial enzyme-linked immunoassay (QAYEE-BIO, Shanghai, China) according to the manufacturer’s instructions. The study protocol was performed in accordance with the Helsinki Declaration of 1975 (as revised in 1983). Approval by the hospital ethics committee and informed consent were obtained from each patient. Data were expressed as median values and interquartile ranges. Mann-Whitney U and Kruskal-Wallis tests were used for comparisons between patients and control subjects and
Fig 1. Serum levels of galectin-1 in stable liver transplant recipients (LTR) compared with acute rejection group (ARG) and healthy subjects (HS). Data are represented as a box plot showing the median and the interquartile range.
within the patient group, respectively. Graft survival was assessed by the Kaplan-Meier method. Values of P < .05 were considered statistically significant.
RESULTS
The demographic and clinical events in the stable LTR and rejection groups are summarized in Table 1. Regarding the first part of the study in long-term recipients, we observed differences in the serum levels of GAL-1 among healthy subjects, the rejection group, and the stable LTR group. As shown in Fig 1, the levels of GAL-1 were significantly higher in the LTR compared with the healthy and acute rejection groups (P ¼ .005). On the other hand, in the early postoperative group of patients, the concentration of GAL-1 was lower (although not statistically significant) in the ARE vs the non-ARE group during the entire postoperative period (P ¼ not significant; Fig 2). Moreover, we also observed a progressive decrease in serum levels of GAL-1 from day 1 to day 15 in the ARE group. The whole transplant group, including those with stable graft function, had higher serum
Fig 2. Time course of serum levels of galectin-1 after liver transplantation. Data are presented as mean standard deviation. ARE, acute rejection episode; NS, not significant.
GALECTIN-1 IN STABLE LT
95
Fig 3. The frequency distribution of galectin-1 in liver transplant recipients.
levels of GAL-1 than the control subjects (231 ng/mL; range: 164 to 246 ng/mL; P ¼ .05) at any time after the early LT period. Fig 3 shows the frequency distribution of GAL-1 in LTR. In addition, we have performed receiver-operator characteristic curve analysis to differentiate the LTR and ARE groups (Fig 4), and we have performed receiveroperator characteristic curve analysis to differentiate the LTR and ARE groups (Fig 4). Fig 5 shows the graft survival curves from patients with LT, with or without ARE. DISCUSSION
Galectins are expressed in immune-privileged sites. For example, GAL-1 is expressed in placenta, brain, and reproductive organs, suggesting that it might trigger the death of infiltrating T cells and protect these sites from tissue damage induced by activated bystander T cells. In addition, GAL-1 can induce the differentiation of dendritic cells with regulatory function involving interleukin (IL)-27 and IL-10, which promote T-cell tolerance [1]. In our study, we observed for the first time that GAL-1 levels were higher in LTR than in healthy subjects and LTR who had experienced acute rejection. Furthermore, we observed that GAL-1 levels were lower in the rejection group than in the nonrejection group during the entire postoperative period. These results are in agreement with arguments derived from experimental models showing that administration of recombinant GAL-1 prolongs renal allograft survival by inhibiting CD8þ T cell
Fig 4. Receiver-operator characteristic curve to differentiate stable liver transplant recipient (LTR) and acute rejection episode (ARE) groups. P ¼ .002. Area under curve ¼ 79.5%. Points that better discriminate between LTR and ARE group patients ¼ 312.5 ng/mL with sensitivity of 83.3% and specificity of 64.5%.
96
GARCÍA, JURADO, SAN SEGUNDO ET AL
Fig 5. Kaplan-Meier survival curves from patients with liver transplant (LTR), with or without acute rejection episode (ARE).
responses and delays allograft liver rejection through the enhancement of T-cell apoptosis in the graft and spleen, diminished Th1 and Th17 cytokine profiles, and increased IL-10 within the grafts [6e8]. Conversely, in the absence of GAL-1, CD8þ T cells, showing enhanced interferon g and IL-17 production, accelerate rejection of skin grafts in mice [9]. The present study, together with previous references, point to a possible role of GAL-1 in the promotion of alloimmune tolerance in LT, and could be of help in protocols of operational tolerance or at least in weaning off of immunosuppression. REFERENCES [1] Ilarregui JM, Croci DO, Bianco GA, et al. Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1driven immunoregulatory circuit involving interleukin 27 and interleukin 10. Nat Immunol 2009;10:981e91. [2] Toscano MA, Commodaro AG, llarregui JM, et al. Galectin-1 suppresses autoimmune retinal disease by promoting
concomitant Th2- and T regulatory-mediated anti-inflammatory responses. J Immunol 2006;176:6323e32. [3] Rabinovich GA, Daly G, Dreja H, et al. Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T-cell apoptosis. J Exp Med 1999;190:385e98. [4] Offner H, Colnik B, Bringman TS, et al. Recombinant human beta-galactoside binding lectin suppresses clinical and histological signs of experimental autoimmune encephalomyelitis. J Neuroimmunol 1990;28:177e84. [5] Baum LG, Blackall DP, Arias-Magallano S, et al. Amelioration of graft vs host disease by galectin-1. Clin Immunol 2003;109:295e307. [6] Xu G, Tu W, Xu C. Immunological tolerance induced by galectin-1 in rat allogeneic renal transplantation. Int Immunopharmacol 2010;10:643e7. [7] Ye Y, Yan S, Jiang G, et al. Galectin-1 prolongs survival of mouse liver allografts from Flt3L-pretreated donors. Am J Transplant 2013;13:569e79. [8] Fábrega E, López-Hoyos M, San Segundo D, et al. Changes in the serum levels of interleukin-17/interleukin-23 during acute rejection in liver transplantation. Liver Transpl 2009;15:629e33. [9] Moreau A, Noble A, Ratnasothy K, et al. Absence of galectin-1 accelerates CD8þ T cell-mediated graft rejection. Eur J Immunol 2012;42:2881e8.
