Lin-Fu Zhu

Changes in the Transcriptome in Allograft Rejection: IFN-γ-Induced Transcripts in Mouse Kidney Allografts

We used Affymetrix Microarrays to define interferon‐γ (IFN‐γ)‐dependent, rejection‐induced transcripts (GRITs) in mouse kidney allografts. The algorithm included inducibility by recombinant IFN‐γ in kidneys of three normal mouse strains, increase in kidney allografts in three strain combinations and less induction in IFN‐γ‐deficient allografts. We identified 40 transcripts, which were highly IFN‐γ inducible (e.g. Cxcl9, ubiquitin D, MHC), and 168 less sensitive to IFN‐γ in normal kidney. In allografts, expression of GRITs was intense and consistent at all time points (day 3 through 42). These transcripts were partially dependent on donor IFN‐γ receptors (IFN‐γrs): receptor‐deficient allografts manifested up to 76% less expression, but some transcripts were highly dependent (ubiquitin D) and others relatively independent (Cxcl9). Kidneys of hosts rejecting allografts showed expression similar to that observed with IFN‐γ injections. Many GRITs showed transient IFN‐γ‐dependent increase in isografts, peaking at day 4–5. GRITs were increased in heart allografts, indicating them as generalized feature of alloresponse. Thus, expression of rejection‐induced transcripts is robust and consistent in allografts, reflecting the IFN‐γ produced by the alloresponse locally and systemically, acting via host and donor IFN‐γr, as well as local IFN‐γ production induced by post‐operative stress.

IFN-γ Alters the Pathology of Graft Rejection: Protection from Early Necrosis

We studied the effect of host IFN-γ on the pathology of acute rejection of vascularized mouse heart and kidney allografts. Organs from CBA donors (H-2k) were transplanted into BALB/c (H-2d) hosts with wild-type (WT) or disrupted (GKO, BALB/c mice with disrupted IFN-γ genes) IFN-γ genes. In WT hosts, rejecting hearts and kidneys showed mononuclear cell infiltration, intense induction of donor MHC products, but little parenchymal necrosis at day 7. Rejecting allografts in GKO recipients showed infiltrate but little or no induction of donor MHC and developed extensive necrosis despite patent large vessels. The necrosis was immunologically mediated, since it developed during rejection, was absent in isografts, and was prevented by immunosuppressing the recipient with cyclosporine or mycophenolate mofetil. Rejecting kidneys in GKO hosts showed increased mRNA for heme oxygenase 1, and decreased mRNA for NO synthase 2 and monokine inducible by IFN-γ (MIG). The mRNA levels for CTL genes (perforin, granzyme B, and Fas ligand) were similar in rejecting kidneys in WT and GKO hosts, and the host Ab responses were similar. The administration of recombinant IFN-γ to GKO hosts reduced but did not fully prevent the effects of IFN-γ deficiency: MHC was induced, but the prevention of necrosis and induction of MIG were incomplete compared with WT hosts. Thus, IFN-γ has unique effects in vascularized allografts, including induction of MHC and MIG, and protection against parenchymal necrosis, probably at the level of the microcirculation. This is probably a local action of IFN-γ produced in large quantities in the allograft.

Expression of CTL Associated Transcripts Precedes the Development of Tubulitis in T-Cell Mediated Kidney Graft Rejection

The usual phenotype of clinical kidney allograft rejection is infiltration by lymphocytes and macrophages and evolution of histologic Banff lesions, particularly tubulitis, which indicate parenchymal injury. Using Affymetrix microarrays, we evaluated the relationship between the evolution of pathologic lesions and the transcriptome. We studied CBA/J into C57Bl/6 mouse kidney allografts in which one host kidney is left in place to permit observation of lesion development. Histology was dominated by early infiltration by mononuclear cells from day 3 and slower evolution of tubulitis after day 7. We defined a set of cytotoxic T lymphocyte‐associated transcripts (CATs) on the basis of expression in purified cytotoxic T lymphocytes (CTL) and in a mixed lymphocyte culture, and absence in normal kidney. CATs were detectable by day 3 and highly expressed by day 5 in rejecting kidneys, with a median signal 14% of that in CTL, compared to 4% in isografts and normal kidneys, and persisted through day 42. Lack of mature B cells had little effect on CAT expression, confirming that CATs reflect T‐cell‐mediated rejection. Expression of CATs was established before diagnostic lesions and remained remarkably consistent through day 42 despite massive alterations in the pathology, and probably reflects T cells recruited to the graft.

Effects of Donor Age and Cell Senescence on Kidney Allograft Survival

The biological processes responsible for somatic cell senescence contribute to organ aging and progression of chronic diseases, and this may contribute to kidney transplant outcomes. We examined the effect of pre‐existing donor aging on the performance of kidney transplants, comparing mouse kidney isografts and allografts from old versus young donors. Before transplantation, old kidneys were histologically normal, but displayed an increased expression of senescence marker p16INK4a. Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16INK4a. Similar but much milder changes occurred in old isografts. These changes were absent in young allografts at day 7, but emerged by day 21. The expression of p16INK4a remained low in young kidney allografts at day 7, but increased with severe rejection at day 21. Isografts from young donors showed no epithelial changes and no increase in p16INK4a. The measurements of the alloimmune response—infiltrate, cytology, expression of perforin, granzyme B, IFN‐γ and MHC—were not increased in old allografts. Thus, old donor kidneys display abnormal parenchymal susceptibility to transplant stresses and enhanced induction of senescence marker p16INK4a, but were not more immunogenic. These data are compatible with a key role of somatic cell senescence mechanisms in kidney transplant outcomes by contributing to donor aging, being accelerated by transplant stresses, and imposing limits on the capacity of the tissue to proliferate.

Interferon-γ Acts Directly on Rejecting Renal Allografts to Prevent Graft Necrosis

In transplant rejection interferon (IFN)-γ regulates the recipient immune response but also acts directly on IFN-γ receptors in the graft. We investigated these direct actions by comparing rejecting kidneys from donors lacking IFN-γ receptors (GRKO mice) or control donors (129Sv/J) in CBA recipients. Beginning day 5, 129Sv/J kidneys displayed high major histocompatibility complex (MHC) expression, progressive infiltration by inflammatory cells, but no thrombosis and little necrosis, even at day 21. GRKO kidneys showed increasing fibrin thrombi in small veins, peritubular capillary congestion, hyaline casts, and patchy parenchymal necrosis, progressing to near total necrosis at day 10. Terminal dUTP nick-end labeling assays were positive only in the interstitial infiltrate, confirming that massive cell death in GRKO transplants was not apoptotic. Paradoxically, GRKO kidneys showed little donor MHC induction and less inflammatory infiltration. Both GRKO and 129Sv/J allografts evoked vigorous host immune responses including alloantibody and mRNA for cytotoxic T cell genes (perforin, granzyme B, Fas ligand), and displayed similar expression of complement inhibitors (CD46, CD55, CD59). GRKO kidneys displayed less mRNA for inducible nitric oxide synthase and monokine inducible by IFN-γ but increased heme oxygenase-1 mRNA. Thus IFN-γ acting on IFN-γ receptors in allografts promotes infiltration and MHC induction but prevents early thrombosis, congestion, and necrosis.

IFN-gamma is an absolute requirement for spontaneous acceptance of liver allografts.

Experimental liver allografts undergo spontaneous acceptance despite undergoing rejection during the first few weeks post transplant. We explored the role of interferon‐γ (IFN‐γ) in the spontaneous acceptance of mouse liver allografts. Strain of mouse (CBA) liver allografts transplanted into normal BALB/c mice developed histologic changes typical of rejection that spontaneously regressed, permitting long‐term survival of these allografts similar to that of syngeneic grafts. In contrast, CBA liver allografts in IFN‐γ‐deficient hosts manifested not only infiltration but also hemorrhage and necrosis, with no survival beyond 14 days. Despite differences in survival, local expression of cytotoxic T‐cell genes in the transplant was not increased in IFN‐γ‐deficient hosts, but livers in interferon‐γ‐deficient mice (GKO) hosts displayed much less induction of major histocompatibility complex (MHC) class I and II expression. To determine whether the difference in survival was secondary to the direct effects of IFN‐γ on the liver, we transplanted livers from IFN‐γ‐receptor‐deficient mice into normal hosts. Liver allografts lacking IFN‐γ receptors also developed hemorrhage and necrosis with minimal induction of MHC expression. Thus IFN‐γ mediates a direct effect on rejecting liver allografts that reduces hemorrhage and necrosis, induces MHC expression, and is absolutely required for spontaneous acceptance.

Tubulitis and Epithelial Cell Alterations in Mouse Kidney Transplant Rejection Are Independent of CD103, Perforin or Granzymes A/B

One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E‐cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E‐cadherin and Ksp‐cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT‐PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild‐type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme‐perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact‐independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis.

Lesions of T-Cell-Mediated Kidney Allograft Rejection in Mice Do Not Require Perforin or Granzymes A and B

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection – interstitial infiltration, tubulitis, and endothelial arteritis – are T‐cell‐dependent and antibody‐independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T‐cell‐mediated lesions of mouse kidney transplant rejection. By real‐time RT‐PCR, allografts rejecting in wild‐type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T‐cell‐mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule‐associated cytotoxic mechanisms of T cells are not the effectors of T‐cell‐mediated allograft rejection.

IFN-γ Prevents Early Perforin-Granzyme-Mediated Destruction of Kidney Allografts by Inducing Donor Class I Products in the Kidney

Interferon‐γ (Ifng) protects organ allografts: mouse kidney allografts lacking Ifng receptors rapidly fail with massive ischemic necrosis around days 5 to 7, reflecting microcirculation failure. We hypothesized that Ifng protects the graft by preventing perforin‐granzyme‐mediated cytotoxic damage to the microcirculation by inducing class Ia and/or Ib products. We transplanted kidney allografts lacking Ifng receptors into various knockout hosts. The necrosis/congestion phenotype did not require host B cells or IL‐4 and IL‐13 receptors, but required the T‐cell alloresponse: it did not occur if the hosts were syngeneic or T‐cell deficient. However, host perforin‐granzyme mechanisms were required: no necrosis developed if hosts lacked either perforin or granzymes A and B. The ability of Ifng to protect the allograft required donor class I products: allografts lacking class I products due to Tap1 or β2 microglobulin deficiency developed a similar necrosis‐congestion phenotype at day 7 despite Ifng receptors being present. Thus when host cytotoxic T cells infiltrate organ allografts, Ifng prevents their perforin‐granzyme mechanism from compromising the microcirculation by a mechanism requiring donor class Ia or Ib products. We propose that donor class Ia or Ib products are needed to trigger inhibitory receptors on effector T cells.

Nutrient-Related Issues Affecting Successful Experimental Orthotopic Small Bowel Transplantation

Background. This study tested the effectiveness of a nutrient-rich preservation solution in a small animal model of orthotopic whole small bowel transplantation. Methods. Lewis rats received syngeneic total orthotopic small bowel graft after cold storage for 6 h. Donor small bowel was flushed vascularly with University of Wisconsin (UW) solution and flushed luminally with UW solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush; Group 2, UW solution; Group 3, AA solution. Biopsies were taken over 14 days posttransplant; energetics, oxidative stress, neutrophil recruitment and histologic injury were assessed. Results. All animals in Groups 1 and 2 failed to survive 12 h posttransplant due to hemorrhagic shock and fluid loss. In contrast, all animals in Group 3 survived the operation; survival after 14 days was 80% (4/5). In Group 3, full recovery of tissue adenylates (ATP and energy charge) to freshly isolated tissue values occurred within 3 days. Oxidative stress as assessed by malondialdehyde (MDA) levels was low in Group 3 throughout 14 d; Groups 1 and 2 exhibited high oxidative stress over the initial 35 min reperfusion (P<0.05). Neutrophil recruitment (myeloperoxidase activity) was significantly reduced in Group 3 tissues, as was histologic injury (P<0.05 compared to Groups 1 and 2). By day 14, Group 3 exhibited complete mucosal restoration. Conclusions. The data presented in this communication supports the use of an intraluminal preservation solution that is tailored to the metabolic requirements of the small bowel.