Human renal allograft rejection: molecular characterization.

Nephrol Dial Transplant (1998) 13: [Suppl 1]: 21–24 Nephrology Dialysis Transplantation Human renal allograft rejection: molecular characterization Manikkam Suthanthiran Division of Nephrology, Department of Medicine, Department of Transplantation Medicine and Extracorporeal Therapy, and The Rogosin Institute, The New York Hospital-Cornell Medical Center, New York, NY USA Introduction Acute rejection of human renal allografts is the most frequent serious post-transplantation complication [1,2]. In the UNOS Scientific Renal Transplant Registry [2], first transplant recipients who were rejection-free at discharge had 80% one-year graft survival rate compared to 67% for those with one or more rejection episodes (P<0.001). Moreover, the estimated half-life of renal allografts without rejection was 8.6 years, compared to 7.4 years for those with one or more rejection episodes [2]. Acute rejection is also a risk factor for chronic rejection [3], the most common cause of failure of long-term allografts. A highly sensitive molecular technique, reverse transcriptase-polymerase chain reaction (RT-PCR), permits detection of low-abundance mRNA [4]. We [5,6 ] and others [7–10] have utilized this powerful molecular tool to explore mechanisms of rejection of human renal allografts. RT-PCR was utilized in this investigation to identify intrarenal expression of cytotoxic attack molecules (granzyme B and perforin), and that of immunoregulatory cytokines (IL-2, IL-4, IL-10, IFN-c and TGF-b ). Intrarenal mRNA display was 1 correlated with the histologically confirmed acute rejection and/or chronic rejection of human renal allografts. Subjects and methods One hundred twenty-seven renal allograft core tissue samples were obtained from 107 patients. Eighty-eight patients underwent one biopsy each; 18 recipients, two biopsies; and one patient underwent three renal biopsies. The biopsies were performed to determine the basis for graft dysfunction. The biopsy tissues included in this study of intragraft gene expression were classified on the basis of the Banff working classification criteria [11]. The histological classification of the biopsy specimen was made without the knowledge of the results of the molecular studies. Portions of renal biopsy tissue were snap-frozen in liquid nitrogen and stored at −70°C for later RNA extraction. The renal tissue was homogenized with a tissue tearer in 4 M Correspondence and offprint requests to: M. Suthanthiran, 525 East 68th Street, Box 3, New York, NY 10021, USA. guanidinium isothiocyanate solution and loaded on top of 5.7 M CsCl cushion. RNA was isolated by centrifugation at 20°C and 50 000 rpm for 16–20 h using a TLS 55 rotor in a Beckmann TL100 ultracentrifuge [5,6 ]. Reverse transcriptase PCR was done as previously described [5,6 ]. Results Rejection and intrarenal expression cytotoxic attack molecules (granzyme B mRNA and perforin) Donor specific cytotoxic T cells (CTL) have been identified in human renal allografts undergoing rejection [12]. Since granzyme B and perforin represent molecular mediators of the lytic program of the cytotoxic effector cells [13,14], the correlation between intragraft expression of granzyme B and/or perforin and rejection was investigated in our study. Seventy-six of the 127 biopsies were positive for intrarenal expression of granzyme B mRNA. Among these 76, 59 biopsies displayed histological features of acute rejection; among the 51 biopsies that were negative for granzyme B mRNA, 28 did not express features of acute rejection (Table 1). The correlation between intragraft granzyme B mRNA expression and acute rejection was significant at P=0.0002, by Fisher’s Exact Test. In contrast, intragraft perforin mRNA expression was not a correlate of acute rejection ( Table 1). Recent studies have emphasized the importance of an earlier acute rejection episode to subsequent occurrence of chronic rejection. The possibility thus exists that chronic rejection might be due to CTL-dependent cellular effector mechanism(s). We therefore determined whether intragraft expression of granzyme B mRNA or perforin mRNA is a correlate of chronic rejection. Thirty-six of the 127 renal allograft biopsies displayed features consistent with chronic rejection. Data summarized in Table 1 demonstrate that intragraft expression of granzyme B mRNA or perforin mRNA is not a correlate of chronic rejection. © 1998 European Renal Association–European Dialysis and Transplant Association 22 M. Suthanthiran Table 1. Correlation between intragraft mRNA expression and rejection Acute Rejectionb Intragraft mRNAa Granzyme B mRNA Perforin mRNA IL-2 mRNA IFNc mRNA IL-4 mRNA IL-10 mRNA TGF-b mRNA 1 +(76) −(51) +(49) −(77) +(10) −(117) +(56) −(66) +(2) −(85) +(55) −(72) +(69) −(58) + − 59 23 32 50 10 72 40 39 2 55 46 36 43 39 17 28 17 27 0 45 16 28 0 30 9 36 26 19 Chronic Rejectionb Pc 0.0002 0.999 0.014 0.185 0.542 0.0001 0.582 + − 19 17 17 19 2 34 15 19 0 22 12 24 26 10 57 34 32 58 8 83 41 47 2 63 43 48 43 48 Pc 0.32 0.31 0.72 0.84 0.61 0.16 0.01 aTotal RNA was isolated and reverse transcribed into cDNA and amplified by PCR with the sequence specific primer pairs reported earlier [6 ]. bThe histological diagnosis of acute or chronic rejection was based on Banff criteria [11]. cP value derived with Fisher’s Exact Test for 2×2 tables. Rejection and intrarenal expression of TH1 or TH2 cytokines Mossman and Coffman originally described that the murine CD4 helper T cell can be divided into two distinct and mutually exclusive subsets: TH1 cells that produce IL-2, IFN-c and lymphotoxin, and TH2 cells that secrete IL-4, IL-5, IL-6 and IL-10 [15]. This differential pattern of cytokine production has also been reported with human T cell clones and has significant implications for immunity to infections. An attractive hypothesis is that the activation of TH1 cells results in rejection and the activation of TH2 cells results in graft accommodation [16 ]. We have therefore determined whether intrarenal expression of mRNA encoding IL-2, IFN-c, IL-4 or IL-10 is a correlate of the histological status of the allograft. Among the 127 renal allograft biopsies examined for intragraft IL-2 mRNA expression, only 10 were positive for intrarenal IL-2 mRNA expression, and all 10 exhibited histological features of acute rejection. The correlation between intragraft IL-2 mRNA expression and acute rejection was significant at P=0.014 ( Table 1). Intragraft IL-2 mRNA expression, however, was not a significant correlate of chronic rejection ( Table 1). IFN-c, a secretory product of activated T cells, promotes the expression of HLA antigens that serve as the major stimulus for the initiation of, and subsequently as the target for, the anti-allograft response. That IFN-c can enhance not only graft immunogenicity but also the cytotoxic armamentarium of the graft recipient represented the rationale for our investigation of IFN-c mRNA expression. Also, Nast et al. [10] have (...truncated)