Serum Level of Soluble Fibrinogen-Like Protein 2 in Renal Allograft Recipients With Acute Rejection: A Preliminary Study Z. Zhao, C. Yang, Q. Tang, T. Zhao, Y. Jia, Z. Ma, R. Rong, M. Xu, and T. Zhu ABSTRACT Background. Soluble fibrinogen-like protein 2 (sfgl2), which is mainly secreted by T cells, is a novel effector of regulatory T cells with immunosuppressive functions. The aim of this study was to investigate serum levels of sfgl2 among renal allograft recipients. Methods. From November 2010 to August 2011 we retrospectively divided 47 renal allograft recipients into an acute rejection (n ϭ 19) versus a stable group (n ϭ 28) according to allograft biopsy results, using the Banff 2007 classification. The acute rejection group was subdivided into grade I (n ϭ 8) versus grade II T-cell–mediated (n ϭ 6) or antibody-mediated rejection episodes (n ϭ 5). Peripheral blood samples were collected at the time of biopsy. Fourteen healthy volunteers were included as normal group controls. Serum levels of sfgl2 were analyzed by enzyme-linked immunosorbent assay. Results. Serum levels of sfgl2 were increased among renal allograft recipients suffering from biopsy-proven acute rejection episodes (61.91 Ϯ 45.68 ng/mL), versus those with stable allografts (38.59 Ϯ 19.92 ng/mL, P Ͻ .05) or healthy volunteers (29.10 Ϯ 18.08 ng/mL, P Ͻ .05). The sfgl2 level was significantly higher among patients with antibody- mediated (118.48 Ϯ 55.54 ng/mL) than T-cell–mediated acute rejection episodes (41.71 Ϯ 16.44 ng/mL, P Ͻ .01). Serum sfgl2 levels were remarkably elevated in patients with grade II (51.87 Ϯ 19.13 ng/mL) versus grade I T-cell–mediated rejection (34.10 Ϯ 9.26 ng/mL, P Ͻ .05). Conclusions. Serum sfgl2 levels were increased among renal allograft recipients with acute rejection episodes to an extent dependent upon the pathological type and severity of the response. IDNEY TRANSPLANTATION SUCCESS is limited Soluble fgl2 (sfgl2) has been recently identified as an- K by acute rejection episodes miphros toxicity, malig- other form of fgl2. It is mainly secreted by CD4ϩ and CD8ϩ nancy, and cardiovascular complications.1 Despite the im- T cells, especially regulatory T cells (Tregs).8 sfgl2 serves as provements in immunosuppressants, acute rejection, an an effector of Tregs, demonstrating immunoregulatory unsolved problem, contributes to morbidity and mortality functions.9 sfgl2 inhibits dendritic cell maturation and in- after kidney transplantation. T cells and antibodies are duces B cell apoptosis in vitro, while down-regulation of major factors involved in acute rejection, employing mech- sfgl2 improves T-cell proliferation, promotes Th1 cell po- anisms that remain not fully understood. Fibrinogen-like protein 2 (fgl2/fibroleukin), a member of the fibrinogen-related protein superfamily, has a membrane- From the Department of Urology, Zhongshan Hospital, Fudan protein form, fgl2 prothrombinase, that is mostly expressed on University (Z.Z., C.Y., Q.T., Y.J., R.R., T.Z.); Shanghai Key reticuloendothelial cells, playing procoagulant activity in im- Laboratory of Organ Transplantation (C.Y., T.Z., M.X., T.Z.); and mune-associated coagulation.2 It is up-regulated in several Clinical Laboratory, Zhongshan Hospital, Fudan University (Z.M.), Shanghai, PR China. inflammatory disorders such as viral hepatitis,3 graft rejec- 4 5 Grants provided by the National Nature Science Foundation of tion, and autoimmune glomerulonephritis. In organ trans- China (No. 81070595); Science and Technology Commission of 6,7 4 plant settings in both animals and humans , high expres- Shanghai Municipality (No. 10DZ2212000). sion of fgl2 prothrombinase is seen in grafts experiencing Address reprint requests to Tongyu Zhu, 180 Fenglin Road, acute rejection responses. Shanghai, 200032, China. E-mail: [email protected] 0041-1345/12/$–see front matter Crown Copyright © 2012 Published by Elseiver Inc. All rights reserved. http://dx.doi.org/10.1016/j.transproceed.2012.05.082 360 Park Avenue South, New York, NY 10010-1710 2982 Transplantation Proceedings, 44, 2982–2985 (2012) SERUM LEVEL OF SOLUBLE FIBRINOGEN-LIKE PROTEIN 2 2983 larization, and inhibits Treg activity.10 The changes in sfgl2 tistics 19.0 (International Business Machines Corp, New York, NY, expression during acute rejection episodes after kidney USA) was used for data analysis. Student t test was employed to transplantation, however, remain unclear. This study sought compare continuous variables between groups, while Pearson ␹2 Ͻ to investigate serum levels of sfgl2 among renal transplant -test for categorical data. A two-tailed P .05 was considered recipients with biopsy-proven T-cell– or antibody-mediated statistically significant. acute rejection episodes, compared with those among re- cipients with stable allografts or healthy volunteers. RESULTS Baseline Characteristics of Groups MATERIALS AND METHODS Baseline characteristics of each group are shown in Table 1. Demographics There was no significant difference among the groups in the From November 2010 to August 2011 we enrolled 47 renal values of mean age, sex, induction therapy, cold ischemia transplant recipients who underwent allograft biopsies in this study. time, immunosuppressant, or posttransplantation time. 19 recipients showed acute rejection episodes based upon the Banff 2007 classification system,11 including 14 with T-cell– and five with Serum Levels of sfgl2 antibody-mediated rejection. No rejection was discovered among the other 28 recipients, who maintained stable renal function for at Serum levels of sfgl2 were significantly higher among least 6 months. All patients administered cyclosporine or tacroli- patients with acute rejection episodes (61.91 Ϯ 45.68 ng/ mus plus mycophenolate mofetil and prednisone. Patients were mL, n ϭ 19) versus those with stable allografts (38.59 Ϯ excluded if they had undergone a multiorgan transplantation, were 19.92 ng/mL, n ϭ 28, P Ͻ .05) or healthy volunteers (29.10 Ϯ experiencing a generalized infection at the time of transplantation, 18.08 ng/mL, n ϭ 14, P Ͻ .05) (Fig 1). There was no had a history of malignancy, or were positive for HIV, hepatitis C virus antibody, or hepatitis B surface antigen. Also, 14 healthy significant difference between the stable and the normal volunteers were included as a normal group. Approval for this groups. Serum sfgl2 levels were significantly higher among study was obtained from our ethics committee. subjects experiencing antibody-mediated (118.48 Ϯ 55.54 ng/mL, n ϭ 5) versus T-cell–mediated rejection episodes Serum Levels of sfgl2 (41.71 Ϯ 16.44 ng/mL, n ϭ 14, P Ͻ .01) according to the Peripheral blood samples were obtained from the patients at the Banff 2007 criteria. Among the patients with T-cell–medi- time of biopsy and from healthy volunteers. Serum levels of sfgl2 ated acute rejections, serum sfgl2 was remarkably higher in were measured using enzyme-linked immunosorbent assay the grade II (51.87 Ϯ 19.13 ng/mL, n ϭ 6) versus the grade (ELISA) kits (Biolegend, San Diego, Calif, USA) according to the I subset (34.10 Ϯ 9.26 ng/mL, n ϭ 8, P Ͻ .05). manufacturer’s instructions. Briefly, serum samples (30 ␮L) were added to each well of a plate precoated with anti-human fgl2 DISCUSSION antibodies. Following 2-hour incubation at room temperature and four washes with buffer, 100 ␮L of human fgl2 detection antibody Acute rejection remains a formidable problem in kidney was added to each well for a 1-hour incubation at room tempera- transplantation, impairing allograft function and recipient ture. The plate was washed again and treated with secondary survival. It is of great importance to understand the mech- horseradish peroxidase-conjugated antibodies. After another five anisms of acute rejection seeking critical molecules in this washes, 100 ␮L of substrate solution were then added for a process. sfgl2, a novel effector secreted by Tregs, exerts 20-minute incubation in the dark. After the reaction was stopped, absorbance was measured at 450 nm using an ELISA plate reader. immunoregulatory functions. Current research on sfgl2 is The concentration was determined using a standard curve accord- mostly limited to in vitro experiments and animal models, ing to the kits instructions. rather than transplantations. In this study, we first demon- strated that serum sfgl2 levels were remarkably elevated Statistics among renal allograft recipients suffering acute rejection Baseline characteristics and data were presented as mean values Ϯ episodes, compared with these displaying stable allografts standard deviations or frequencies (percentages). IBM SPSS Sta- or healthy volunteers. Furthermore, serum sfgl2 levels were Table 1. Baseline Characteristics All Groups Normal Group Stable Group Acute Rejection Group P Value Number 61 14 28 19 — Age (y)a 46.0 Ϯ 16.9 61.6 Ϯ 12.4 46.4 Ϯ 15.2 43.9 Ϯ 15.5 Ͼ.05 Sex (M/F)b 47 (77%)/14 (23%) 8 (57%)/6 (43%) 23 (82%)/5 (18%) 16 (84%)/3 (16%) Ͼ.05 Induction therapy (CD25 mAb/ATG)b — — 26 (93%)/2 (7%) 18 (95%)/1 (5%) Ͼ.05 Cold ischemia time (h)a — — 7.4 Ϯ 2.1 7.8 Ϯ 2.3 Ͼ.05 Immunosuppressant (MMF ϩ CsA ϩ — — 15 (54%)/13 (46%) 10 (53%)/9 (47%) Ͼ.05 Pre/MMF ϩ FK506 ϩ Pre)b Posttransplantation time (y)a — — 3.7 Ϯ 3.1 3.5 Ϯ 3.2 Ͼ.05 mAb, monoclonal antibody; ATG, antithymocyte globulin; MMF, mycophenolate mofetil; CsA, cyclosporine; FK506, tacrolimus; Pre, prednisone. aMean Ϯ standard deviations. bFrequence (percentage). 2984 ZHAO, YANG, TANG ET AL significantly higher in antibody-mediated versus T-cell– mediated rejection episodes as well as among grade II versus grade I rejections. Tregs are believed to play an important role in the induction and maintenance of immune tolerance.12 In the same way, Tregs can also be recruited into the graft during acute rejection in order to restrain the inflammation.13 Recent findings in experimental transplant models have shown Tregs to increase during acute rejection to diminish inflammation and associated lesions, thereby ameliorating acute rejection.14,15 Moreover, enhanced numbers and functions of Tregs have been shown in peripheral blood during aggressive acute rejection responses.16 These obser- vations are consistent with our findings of increased sfgl2 expression in renal allograft recipients experiencing acute Fig 2.