Vido Ramassar

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.

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.

Heterogeneity in the Evolution and Mechanisms of the Lesions of Kidney Allograft Rejection in Mice

The natural history and pathogenesis of the pathologic lesions that define rejection of kidney transplants have not been well characterized. We studied the evolution of the pathology of rejection in mouse kidney allografts, using four strain combinations across full major histocompatibility complex (MHC) plus nonMHC disparities, to permit more general conclusions. Interstitial infiltrate, MHC induction, and venulitis appeared by day 5, peaked at day 7–10, then stabilized or regressed by day 21. In contrast, tubulitis, arteritis, and glomerulitis were absent or mild at days 5 and 7, but progressed through day 21, indicating separate regulation and homeostatic control of these lesions. Edema, hemorrhage, and necrosis also increased through day 21. All lesions were T‐dependent, failing to develop in T‐cell‐deficient hosts. Allografts into immunoglobulin‐deficient hosts manifested typical infiltration, MHC induction, and tubulitis at days 7 and 21, indicating that these lesions are alloantibody‐independent. However at day 21 kidneys rejecting in immunoglobulin‐deficient hosts showed decreased edema, arteritis, venulitis, and necrosis.

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.

The induction of class I and II major histocompatibility complex by allogeneic stimulation is dependent on the transcription factor interferon regulatory factor 1 (IRF-1): Observations in IRF-1 knockout mice

Hosts undergoing allograft rejection show increased MHC expression locally in the graft and systemically in the normal host organs, mediated principally by IFN-gamma. The transcription factor IRF-1 has been implicated in the regulation of MHC expression by IFNs in vitro as well as in the regulation of production of some cytokines. We investigated the role of IRF-1 in vivo in the systemic regulation of MHC expression in hosts undergoing rejection of allogeneic tumors by comparing MHC induction in mice with normal IRF-1 genes (wild type or WT mice) with mice with disrupted IRF-1 genes (IRF-1 knockout or IRF-1 KO mice). We assessed MHC product expression by immunohistology and by radiolabeled antibody binding to tissue homogenates, and MHC mRNA levels by Northern blotting. By immunohistology in mice undergoing allogeneic stimulation by the ascites tumor cells, kidneys of WT mice showed massive class I and II induction, but kidneys from IRF-1 KO mice showed almost no class I and II induction. Allograft rejection also increased class I and II product levels by radiolabeled antibody binding and steady state mRNA levels, but again IRF-1 KO mice showed severe impairment of MHC induction. Similar impaired MHC class I and II induction was seen in heart and spleen, but in liver the IRF-1 mice showed impaired class I induction but unimpaired class II induction. The results indicate that IRF-1 has an essential role in both class I and class II MHC induction in allogeneic responses, but that a component of IRF-1 independent MHC induction is also demonstrable in some tissues. The reduction in MHC induction by allogeneic stimulation probably reflects decreased response to IFN-gamma and other cytokines as well as some reduction in the amount of cytokines produced.

Mycophenolate mofetil reduces production of interferon-dependent major histocompatibility complex induction during allograft rejection, probably by limiting clonal expansion

The immunosuppressive drug mycophenolate mofetil (MMF) acts by releasing mycophenolic acid (MPA), which inhibits the enzyme inosine monophosphate dehydrogenase (IMPDH) and thus inhibits de novo purine synthesis. Unlike cyclosporine (CsA), MMF has no direct effect on cytokine gene expression in vitro. We examined the effect of MMF, in comparison to CsA, on in vivo production of interferon-gamma (IFN-gamma) in mice. Two stimuli for IFN-gamma induction were used: (1) allogeneic P815 mastocytoma ascites tumour cells and (2) bacterial lipopolysaccharide (LPS). The allogeneic response is dependent on clonal expansion of T cells, while the LPS response is polyclonal and T cell independent. Since major histocompatibility complex (MHC) induction in mouse kidney is IFN-gamma dependent, we assessed the in vivo induction of IFN-gamma indirectly by measuring MHC induction in mouse kidneys in three systems: radiolabelled antibody binding assay, immunoperoxidase staining in tissue sections, and Northern blotting for steady-state MHC mRNA levels. IFN-gamma steady-state mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). In the allogeneic response, MMF (40-160 mg/kg/day) reduced the production of IFN-gamma in a dose-dependent fashion. MHC class I and II induction was reduced by 35% to 74% and 30% to 74%, respectively. However, MMF had less effect on the induction of MHC by a nonimmune stimulus, bacterial LPS, whereas CsA reduced the induction of IFN-gamma in both responses. We conclude that MMF reduces the IFN-dependent induction of MHC in vivo during specific immune responses, probably by limiting clonal expansion, while preserving nonspecific cytokine production in response to LPS.

In vivo class II transactivator expression in mice is induced by a non-interferon-gamma mechanism in response to local injury.

BACKGROUND Tissue injury induces MHC class II expression, which could be important in the recognition of that tissue as an allograft. The class II transcriptional activator (CIITA) is the major regulator of basal and induced MHC class II expression and is essential for antigen presentation. The role of CIITA in the induction of class II by tissue injury is unknown. In this study, we examined CIITA induction in the course of acute ischemic or toxic renal injury in mice, including the role of interferon (IFN)-gamma and of the transcription factor, interferon regulatory factor (IRF)-1. METHODS Kidneys were injured by ischemia or by gentamicin toxicity and were then studied for changes in gene expression using Northern blot, reverse transcriptase-polymerase chain reaction, radioimmunoassay, and tissue staining. We compared wild-type (WT) mice to IFN-gamma knockout (GKO) or IRF-1 knockout mice. RESULTS Ischemic injury induced CIITA and class II expression in the kidney, in WT and GKO mice. Gentamicin injury also induced both CIITA and class II expression, independent of IFN-gamma, in WT and GKO mice. After ischemic injury, the induction of class II protein levels and CIITA and class II mRNA levels were induced, to a lesser degree, in IRF-1 knockout mice. CONCLUSIONS These data indicate that CIITA is induced by tissue injury, and probably accounts for class II induction during tissue injury. CIITA induction by injury is largely IFN-gamma independent but requires IRF-1. The similarities of the pattern of CIITA and class II induction in ischemic and toxic injury suggest that this is a stereotyped response of injured tissue and not a consequence of a particular mechanism of injury.