Spontaneous tolerance: experience with the rat liver transplant model

Abstract

Soluble major histocompatibility complex (MHC) class I antigens released from hepatocytes and the passenger leukocyte population of the liver allograft have both been considered as important contributors for spontaneous liver tolerance upon allogeneic transplantation into fully MHC-mismatched hosts. This study was conducted to delineate the role of “passenger leukocytes” (PL) as well as local intra-graft defence mechanisms of long-term accepted liver allografts in more detail. Orthotopic liver transplantation was performed in male inbred rats as follows ( n = 4–6): (i) Lewis (LEW; RT1. l) → LEW; (ii) DA (RT1. av1) → DA; (iii) DA → LEW; (iv) LEW → DA; (v) LEW (10-Gy whole body irridiation [WBI], d-7) → DA; (vi) LEW (10-Gy WBI, d-7) → LEW; (vii) LEW (10-Gy WBI, d-7) → LEW (parked for 36 hours) → DA; and (viii) LEW (10-Gy WBI, d-7) → DA (parked for 36 hours) → DA. The model specifically investigated the role of PLs as potential contributors to liver tolerance as WBI destroys this nonresident liver population. Characterization of Fas/FasL expression and the frequency of apoptotic cell death was performed by immunohistochemistry and TUNEL staining. Reverse transcriptase—polymerase chain reaction, by the use of Fas and FasL-specific cDNA probes, was performed on isolated hepatocytes of tolerized and rejected livers at various time points after transplantation. Animal survival in the various experimental groups was calculated using Kaplan-Meier survival statistics and related log-rank statistics as follows: (i) < 100 days; (ii) ≥ 100 days; (iii) > 100 days; (iv) 11.3 ± 1.0 day; (v) 9.0 ± 0.5 days; (vi) > 100 days; (vii) 5/6 > 100 days; and (viii) 8.0 ± 1.5 days. Immunohistochemistry revealed high numbers of proliferating cells in tolerized liver allografts. Apoptotic cell death of hepatocytes could be detected in both rejecting and to a lower extent in tolerized animals. Conversely, only tolerized but not rejected liver allografts revealed upregulation of FasL-expression on hepatic parenchymal cells from day 3 onwards. Irridiated LEW livers, in turn, loose their ability to survive in allogeneic DA hosts (group v) whereas they survive in syngeneic hosts (group vi), indicating that irridiation itself does not destroy the liver parenchyma. Reconstitution of irridiated LEW livers with syngeneic (group vii) but not with allogeneic (group viii) PLs restored tolerance induction. The underlying mechanisms of immune-privilege observed with liver allografts appear to share characteristics of clonal exhaustion suggesting that alloreactive lymphocytes are depleted by AICD via the FasL/Fas signal transduction pathway. The high frequency of apoptotic lymphocytes found in the portal tract of tolerized (but not rejected) LEW grafts supposes that functional FasL expression on graft hepatocytes mediates specific elimination of graft-directed effector lymphocytes. This mechanism constitutes peripheral deletion as one of the possible tolerogenic mechanisms involved. Chimerical liver grafts consisting of donor (LEW) parenchyma and host (DA) passenger leukocytes loose their tolerogenic capacity. In contrast, syngeneic reconstitution with LEW-PLs, restores liver graft acceptance upon transplantation into allogeneic DA hosts. This phenomenon is not relying on the induction of micro- or macrochimeric hosts, as no LEW PLs were found in spleen, thymus or the blood compartment from long-term surviving DA rats. Thus, non-resident liver cells contribute significantly to liver graft acceptance. Subsequently, liver tolerance appears to be mainly induced in the graft itself.