Paradoxical early immune activation during acceptance of liver allografts compared with rejection of skin grafts in a rat model of transplantation.

Liver allografts in many animal models are often spontaneously accepted across a complete histocompatibility barrier without requirement for immunosuppression. In contrast, skin allografts are usually rejected, even across minor histocompatibility barriers. To identify the mechanism of liver allograft acceptance we have compared skin rejection with liver acceptance in DA rat strain recipients of PVG donors, a major histocompatibility complex (MHC) class I plus II mismatch. In spite of the established role of draining lymph nodes (LN) in induction of rejection of skin allografts, there was much greater involvement of LN after liver than after skin transplantation. Few donor cells migrated to these organs from transplanted skin but many cells migrated from transplanted liver. There was also a paradoxical increase in interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) mRNA in LN and spleen of liver allograft recipients that greatly exceeded their expression in skin allograft recipients. For example, there were 2. 7+/-1.6x104 molecules of IFN-gamma per 106 molecules of beta-actin mRNA in the LN draining liver allografts 1 day after transplantation compared with 2.0+/-0.3x103 molecules/106 beta-actin in LN draining skin allografts and 8.1+/-1.8x102 molecules/106 beta-actin in LN draining skin isografts. Examination of the graft showed that infiltration and cytokine mRNA up-regulation occurred more slowly in the transplanted skin than in liver but progressed inexorably in skin grafts until rejection. These results show that liver acceptance is associated with a paradoxical marked early activation then subsequent decline of the immune response.

Kinetics of intragraft cytokine expression, cellular infiltration, and cell death in rejection of renal allografts compared with acceptance of liver allografts in a rat model: early activation and apoptosis is associated with liver graft acceptance.

BACKGROUND: Liver transplants in the rat strain combination PVG-to-Dark Agouti are spontaneously tolerated, whereas kidney transplants in the same strain combination are rejected in 7-9 days. METHODS: To identify organ-specific differences that might yield further information about the mechanism of tolerance induction in this strain combination, liver or kidney grafts, spleen, and draining lymph nodes were harvested at days 1, 3, 5, and 7, and examined by immunohistochemistry, terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay, and reverse transcriptase-polymerase chain reaction for interferon-gamma, interleukin (IL)-2, IL-4, and IL-10. RESULTS: Renal allograft rejection was associated with the progressive development of an intense mononuclear cell infiltrate. Markers of lymphocyte activation and cytokine up-regulation appeared from day 3, and many apoptotic parenchymal cells were noted on days 5-7, at the peak of rejection. Conversely, liver allograft tolerance was associated with more rapid infiltration by activated T cells and earlier increases in cytokine expression, but with a more limited degree of cellular infiltration. Concurrent with the early activation, high levels of apoptosis were found in areas of leukocyte infiltrate, paralleling the disappearance of activated T cells from the graft between days 3 and 5. CONCLUSIONS: Apoptosis of infiltrating leukocytes in liver allografts may represent an important process in the induction of spontaneous liver transplant tolerance and may underlie the abortive nature of the effector response observed within tolerated livers. In contrast, activated cells in renal allografts in the same strain combination survive and proliferate, express high levels of cytokines, and are efficient in bringing about graft destruction.

Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method.

Reverse transcriptase-PCR (RT-PCR) amplification of mRNA is often the only technique able to detect expression of cytokine mRNA in small samples. The aim of this work was to investigate the utility of a non-competitive RT-PCR which used external standards to quantitate TNF-alpha mRNA in liver biopsy specimens from liver transplant patients. It involved removal of aliquots from the PCR reaction at successive cycles, followed by dot-blotting of the samples onto nylon membrane and hybridization with a radioactively-labelled internal probe. Phosphorimage analysis of the labelled membranes allowed quantitation of the relative amount of PCR product at successive cycles. Plots of log(counts) versus cycle number showed straight lines in the exponential phase of amplification. The slopes of these lines showed the efficiency of amplification, which ranged from 76 to 87% for liver biopsy samples. Estimation of liver biopsy levels of TNF-alpha in two separate PCR amplifications showed low inter-assay variability (r2 = 0.98). Comparison of two separate cDNA syntheses also showed good correlation (r2 = 0.81, P < 0.0001), although not as good as for the PCR alone. This shows that variation in efficiency of cDNA synthesis is likely to contribute as much or more to variability of the analysis as variations in PCR amplification.

Tolerance to rat liver allografts: IV. Acceptance depends on the quantity of donor tissue and on donor leukocytes.

Liver allografts in some rat strains are often spontaneously accepted across a complete major histocompatibility barrier without the requirement for immunosuppression while other nonliver allografts are rejected. In previous studies, we have shown that spontaneous acceptance is dependent on liver passenger leukocytes. Depletion of passenger leukocytes by donor irradiation allows rejection, with DA recipients of irradiated PVG livers having a median survival time (MST) of 16 days. Here we show that, in this model, spontaneous acceptance is reconstituted by intravenous injection of donor leukocytes. Intravenous injection of 3-5x10(7) PVG liver leukocytes significantly prolonged DA survival time (MST=96 days, P=0.026), as did 5x10(7) spleen leukocytes (MST>100 days, P=0.002). Deletion of T cells from the reconstituting inoculum reduced survival time (MST=78 days, P=0.039), whereas deletion of B cells or monocytes/macrophages had no effect on survival time. In contrast, PVG hearts are regularly rejected by DA recipients, and PVG liver or spleen leukocytes, even at doses of greater than 3x10(8) cells/recipient, were unable to induce heart acceptance. To investigate the possibility that acceptance of the irradiated liver but not the heart might be due to the large mass of the liver, two kidneys and two hearts of PVG origin were transplanted to each DA recipient together with 1.5x10(8) PVG leukocytes. These organs survived for greater than 200 days, thereby showing that a large mass of donor tissue, in association with donor leukocytes, leads to acceptance of organs that are rejected if transplanted singly. It appears likely that spontaneous liver transplant tolerance is a high-dose or activation-associated immune phenomenon.

Tolerance to rat liver allografts. III. Donor cell migration and tolerance-associated cytokine production in peripheral lymphoid tissues.

The aim of this work was to investigate the mechanism of spontaneous rat liver allograft tolerance. Liver allografts from a LEW donor into DA recipient (LEW-->DA) or of PVG-->DA were spontaneously tolerated (TOL) across a complete MHC mismatch. In contrast, DA-->LEW or PVG-->LEW liver allografts were rejected in 10 to 15 days (REJ). We examined whether donor cell migration to recipient lymphoid tissues might be associated with TOL. Many donor cells were observed in draining (celiac) lymph nodes (LN) and spleen, reaching a peak on day 1 and then decreasing rapidly thereafter. Irradiation of liver donors, which we have previously shown to delete tolerance, significantly reduced the number of donor leukocytes in recipient lymphoid tissues. While this suggested an association between donor cell migration and tolerance, the number, distribution, and type of donor cells in recipient lymphoid tissues of REJ was similar to those of TOL. Expression of cytokine mRNA in LN and spleen showed an early increase in the expression of IL-2 and IFN-gamma mRNA on day 1 and then a rapid decrease to constitutive levels. Spleen and LN levels of IL-6, IL-10, TNF-alpha, or TGF-beta mRNA showed much less up-regulation than IL-2 or IFN-gamma. Paradoxically, there was greater expression of IL-2 and IFN-gamma mRNA in TOL lymphoid tissues than in REJ, and this superinduction was partially prevented by donor irradiation. Superinduction of IL-2 and IFN-gamma was, therefore, more closely associated with TOL than was donor cell migration. This was confirmed by treatment of TOL recipients with a short course of methylprednisolone, which reduced survival of subsequent donor strain skin grafts. This finding has implications for treatment of human liver transplants and is evidence for a novel pathway of transplant tolerance.

Intragraft cytokine mRNA levels in human liver allograft rejection analysed by reverse transcription and semiquantitative polymerase chain reaction amplification.

Cytokine gene expression was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of RNA from 27 human liver allograft specimens diagnosed as acute (n = 19) or chronic (n = 8) rejection and from 12 normal human livers. In initial screening experiments, mRNA for cytokines interleukin (IL)-1 beta, IL-6, IL-10 and gamma-interferon (IFN-gamma) was expressed in all normal livers and almost all allograft specimens tested. IL-2 mRNA was expressed at barely detectable levels in four of 12 normal livers screened and in 20 of 26 liver allograft specimens with rejection. This constitutive expression of cytokine mRNA required semiquantitative PCR analysis to differentiate levels of cytokine mRNA expression between specimens. Titration of cDNA prior to PCR amplification was initially used and showed significantly more IL-2 (p = 0.02) and IFN-gamma (p = 0.03) in acute rejection compared to normal liver. There was also significantly less IL-10 in chronic rejection compared to acute rejection (p = 0.02) or normal liver (p = 0.01) and less IL-6 in acute rejection compared to chronically rejecting liver (p = 0.05). IL-1 beta (p = 0.04) and IL-6 (p = 0.01) were reduced in acute rejection compared to normal liver. The slight increase of IL-2 in acute rejection and the slight decrease of IL-10 in chronic rejection was confirmed by a second semiquantitative analysis which involved removal of aliquots of PCR reaction at successive cycles followed by dot-blotting and hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)