Alexandra F. Sharland

TLR4 activation mediates kidney ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) may activate innate immunity through the engagement of TLRs by endogenous ligands. TLR4 expressed within the kidney is a potential mediator of innate activation and inflammation. Using a mouse model of kidney IRI, we demonstrated a significant increase in TLR4 expression by tubular epithelial cells (TECs) and infiltrating leukocytes within the kidney following ischemia. TLR4 signaling through the MyD88-dependent pathway was required for the full development of kidney IRI, as both TLR4(-/-) and MyD88(-/-) mice were protected against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. In vitro, WT kidney TECs produced proinflammatory cytokines and chemokines and underwent apoptosis after ischemia. These effects were attenuated in TLR4(-/-) and MyD88(-/-) TECs. In addition, we demonstrated upregulation of the endogenous ligands high-mobility group box 1 (HMGB1), hyaluronan, and biglycan, providing circumstantial evidence that one or more of these ligands may be the source of TLR4 activation. To determine the relative contribution of TLR4 expression by parenchymal cells or leukocytes to kidney damage during IRI, we generated chimeric mice. TLR4(-/-) mice engrafted with WT hematopoietic cells had significantly lower serum creatinine and less tubular damage than WT mice reconstituted with TLR4(-/-) BM, suggesting that TLR4 signaling in intrinsic kidney cells plays the dominant role in mediating kidney damage.

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.

Evidence that apoptosis of activated T cells occurs in spontaneous tolerance of liver allografts and is blocked by manipulations which break tolerance

BACKGROUND Fully allogeneic liver grafts from piebald virol glaxo to dark agouti rats are spontaneously tolerated, whereas kidney transplants between these strains are rejected. Liver tolerance is broken by donor irradiation or peritransplant corticosteroid treatment of recipient rats, both of which interfere with the activation of recipient cells. METHODS In this study we used a combination of immunohistochemical staining, reverse transcription-polymerase chain reaction, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling and Annexin-V apoptosis assays to compare donor cell migration, cytokine profiles, and leukocyte apoptosis in grafts and lymphoid organs from tolerant liver and rejecting kidney recipients. We then examined the effect on apoptosis of treatments which abrogate liver tolerance. RESULTS Liver transplantation in this tolerant strain combination is accompanied by rapid migration of many passenger leukocytes to the recipient spleen and lymph node, concurrent with a marked but transient increase in the amount of mRNA for the cytokines interleukin-2 and interferon-gamma. Apoptotic cells appear promptly in the spleen, their numbers reaching a peak 2 days earlier than has been previously shown for the graft infiltrate. Both CD4+ and CD8+ T cells undergo apoptosis and apoptotic cells are most concentrated among CD25+ T cells. In contrast, renal transplant rejection is associated with limited donor cell migration to lymphoid tissues and significantly less up-regulation of interleukin-2 and interferon-gamma in the spleen. Few apoptotic cells are detected in spleen or graft infiltrate during rejection, whereas apoptotic renal tubular and glomerular cells are found from day 5. Either recipient steroid treatment or donor irradiation significantly reduced the number of apoptotic cells in liver graft infiltrates and recipient spleen. CONCLUSIONS Taken together, these findings suggest that a mechanism akin to activation-induced cell death, with apoptosis of alloreactive recipient cells may be responsible for the induction of spontaneous liver transplant tolerance.

Molecular pathogenesis of liver disease: an approach to hepatic inflammation, cirrhosis and liver transplant tolerance

Acknowledgments:

Spontaneous acceptance of liver transplants in rodents: Evidence that liver leucocytes induce recipient T-cell death by neglect

In many animal models transplanted livers are not rejected, even when there is a complete MHC mismatch between the donor and recipient and the recipient is not immunosuppressed. This distinguishes liver transplants from other organs, such as kidneys and hearts, which are rapidly rejected in mismatched individuals. Acceptance of transplanted livers in a rat model is not due to the absence of an immune response to the liver and there is a rapid, abortive response that is ultimately exhausted. Donor leucocytes transferred with the liver appear to be responsible for both liver acceptance and the abortive activation of the recipients T cells. The immune mechanism of liver transplant acceptance appears to be due to ‘death by neglect’ in which T cells are activated to express IL‐2 and IFN‐γ mRNA in the recipient lymphoid tissues, but not at adequate levels within the graft. Subsequently the activated T cells die leading to specific clonal deletion of liver donor‐reactive T cells. These findings have important implications for liver transplant patients as immunosuppressive drugs that are given to prevent rejection can also interfere with this form of tolerance. In addition, it might be possible to modify the immunosuppressive drug treatment of transplant patients to promote the process of death by neglect of recipient alloreactive T cells.

Posttransplant Administration of Donor Leukocytes Induces Long-Term Acceptance of Kidney or Liver Transplants by an Activation-Associated Immune Mechanism

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mismatched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain skin grafts. Acceptance was associated with early activation of recipient T cells in the spleen, demonstrated by a rapid increase in IL-2 and IFN-γ at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-γ mRNA were not increased in the spleens of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid graft destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class II barrier, a finding with direct clinical application.

Approaching the promise of operational tolerance in clinical transplantation.

Long-term acceptance of transplanted organs without requirement for indefinite immunosuppression remains the ultimate goal of transplant clinicians and scientists. This clinical state of allograft acceptance termed “operational tolerance” has been elusive in routine practice. However, there are published reports of recipients where immunosuppression has been discontinued, by intention or patient noncompliance, in which the outcome is a nondestructive immune response and normal function. The question now arises how clinical operational tolerance might be achieved in the majority of recipients. This review provides an overview of current approaches to achieve operational tolerance, including the use of donor bone marrow and depletion of recipient T cells and the resistance of liver transplants to rejection. It also describes the key role of clinical immune monitoring and future approaches to tolerance induction including inhibition of T-cell signaling, manipulation of costimulatory pathways, and expansion of regulatory T cells. The principles of these experimental approaches may ultimately be extended to provide safe and effective control of transplant rejection and induction of clinical operational tolerance.

Genetically modified HLA class I molecules able to inhibit human NK cells without provoking alloreactive CD8+ CTLs.

Human NK cells are likely to be important effectors of xenograft rejection. Expression of HLA class I molecules by transfected porcine cells can protect them from human NK cell-mediated lysis; however, this strategy has the potential to augment the anti-graft response by recipient CD8+ T cells recognizing foreign pig peptides presented by HLA. In this study we show that the introduction of a mutation (D227K) in the α3 domain of HLA-Cw3 abrogates its recognition by CD8-dependent T cells but leaves intact its ability to function as an inhibitory ligand for NK cells. Such genetically modified molecules may have potential therapeutic applications in the prevention of delayed xenograft rejection and in the facilitation of allogeneic and xenogeneic bone marrow engraftment.

Infiltrating Foxp3(+) regulatory T cells from spontaneously tolerant kidney allografts demonstrate donor-specific tolerance.

Foxp3+ regulatory T cells (Tregs) have an essential role in immune and allograft tolerance. However, in both kidney and liver transplantation in humans, FOXP3+ Tregs have been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In the studies outlined, we demonstrated that Foxp3+ Tregs were expanded in tolerant kidney allografts and in draining lymph nodes in the DBA/2 (H‐2d) to C57BL/6 (H‐2b) mouse spontaneous kidney allograft tolerance model. Kidney allograft tolerance was abrogated after deletion of Foxp3+ Tregs in DEpletion of REGulatory T cells (DEREG) mice. Kidney allograft infiltrating Foxp3+ Tregs (K‐Tregs) expressed elevated levels of TGF‐β, IL‐10, interferon gamma (IFN‐γ), the transcriptional repressor B lymphocyte‐induced maturation protein‐1 (Blimp‐1) and chemokine receptor 3 (Cxcr3). These K‐Tregs had the capacity to transfer dominant tolerance and demonstrate donor alloantigen‐specific tolerance to skin allografts. This study demonstrated the crucial role, potency and specificity of graft infiltrating Foxp3+ Tregs in the maintenance of spontaneously induced kidney allograft tolerance.

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments.

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8:CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG-/- donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.