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 p16(INK4a). Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16(INK4a). 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 p16(INK4a) 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 p16(INK4a). The measurements of the alloimmune response-infiltrate, cytology, expression of perforin, granzyme B, IFN-gamma 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 p16(INK4a), 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.

IFN-gamma alters the pathology of graft rejection: protection from early necrosis.

We studied the effect of host IFN-gamma 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-gamma genes) IFN-gamma 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-gamma (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-gamma to GKO hosts reduced but did not fully prevent the effects of IFN-gamma deficiency: MHC was induced, but the prevention of necrosis and induction of MIG were incomplete compared with WT hosts. Thus, IFN-gamma 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-gamma produced in large quantities in the allograft.

Interferon-gamma acts directly on rejecting renal allografts to prevent graft necrosis.

In transplant rejection interferon (IFN)-gamma regulates the recipient immune response but also acts directly on IFN-gamma receptors in the graft. We investigated these direct actions by comparing rejecting kidneys from donors lacking IFN-gamma 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-gamma but increased heme oxygenase-1 mRNA. Thus IFN-gamma acting on IFN-gamma receptors in allografts promotes infiltration and MHC induction but prevents early thrombosis, congestion, and necrosis.

The role of the class II transactivator (CIITA) in MHC class I and II regulation and graft rejection in kidney.

Class II transactivator (CIITA) induces transcription of MHC class II genes, and induces class I in some cell lines. We examined the effect of CIITA deficiency on class I and II expression in mouse kidney. In CIITA knockout (CIITAKO) mice, basal class II was absent, but class I levels were mildly but significantly increased. Allogeneic stimulation or ischemic injury increased class I and II expression in kidneys of control (wild-type, WT) mice but induced only class I in CIITAKO mice. Thus, in kidney, all basal and induced class II expression was CIITA-dependent, but neither basal nor induced class I was CIITA-dependent. Rejecting kidney allografts from CIITAKO mice in CBA hosts manifested intense induction of donor class I but no donor class II expression. Rejecting kidneys from both WT and CIITAKO donors showed predominantly CD8 T-cell infiltration at days 7 and 21, with increasing tubulitis and arteritis at day 21. CIITAKO kidneys showed fewer infiltrating cells than WT kidneys at day 21. Thus CIITA-deficient kidneys have no basal and induced class II expression but display intense induction of class I expression, and evoke typical rejection lesions, although some indices of infiltration are reduced at day 21.

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.

IFN regulatory factor-1 plays a central role in the regulation of the expression of class I and II MHC genes in vivo.

Transcription factor interferon regulatory factor-1 (IRF-1) is implicated in regulating class I MHC expression in vitro. We investigated the in vivo relationship between IRF-1 and MHC expression in kidney and other nonlymphoid organs, assessing MHC expression in mice with disrupted IRF-1 genes (IRF-1 KO) compared with mice with intact IRF-1 genes (WT). In kidneys of IRF-1 KO mice, basal class I expression was decreased, particularly on arterial endothelium, but basal class II expression was unchanged. The induction of both class I and class II expression by injected rIFN-gamma was reduced in IRF-1 KOs, compared with WT mice. Similarly, stimuli that induce endogenous IFN-gamma production (LPS or oxazolone) massively increased MHC expression in kidneys of WT mice, with little increase in IRF-1 KO mice. Impaired class II induction by rIFN-gamma in IRF-1 KO mice probably reflects the role of IRF-1 in regulating class II transactivator (CIITA) expression: rIFN-gamma induced CIITA mRNA less in kidneys of IRF-1 KO mice than in WT mice. In organs of WT mice, IRF-1 mRNA was expressed in the basal state, and rIFN-gamma treatment increased IRF-1 mRNA before the induction of class I or CIITA mRNA. Treatment of WT mice with cycloheximide plus rIFN-gamma superinduced IRF-1 mRNA expression, but partially inhibited CIITA mRNA expression, indicating that IRF-1 mRNA induction is not dependent on new protein synthesis, unlike CIITA. Thus, in vivo, IRF-1 plays a major role in basal and induced class I expression and in induction of class II by IFN-gamma, probably via CIITA induction.

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.

The unique role of interferon-gamma in the regulation of MHC expression on arterial endothelium.

We examined the expression of MHC class I and II in the arterial endothelium of interferon-gamma (IFN-gamma, GKO) and IFN-gamma-R (IFN-gamma-R, GRKO) gene knockout mice in comparison with mice with intact IFN-gamma and IFN-gamma-R genes, BALB/c and 129Sv/J wild-type, respectively. The GKO and GRKO were produced by gene targeting. MHC class I and II expression was assessed by mAb binding to frozen tissue (kidney, spleen, heart, liver) sections by immunoperoxidase staining in the basal state and after various stimuli: allogeneic cells, oxazolone skin sensitization, LPS, and rIFN-gamma. As controls, we also examined the expression of two other IFN-gamma inducible genes present in the endothelium, Ly-6 and ICAM-1. We found that basal class I expression was present in the small arteries and arterioles of BALB/c and 129Sv/J wild-type mice but absent from arterial endothelium of GKO and GRKO mice. Class I was induced in the endothelium of BALB/c and 129Sv/J wild-type mice by three in vivo stimuli: allogeneic, LPS, and oxazolone, whereas class II was only induced after allogeneic stimulus. Administration of rIFN-gamma induced class I in the endothelium of GKO and BALB/c wild-type mice. The basal expression of Ly-6 and ICAM-1 was similar in the arteries of GKO and BALB/c wild-type mice, indicating that, the basal expression of these proteins in endothelium is IFN-gamma independent, unlike class I. In summary, basal class I expression in arterial endothelium is not constitutive as previously believed, but is dependent on basal IFN-gamma production. IFN-gamma has an essential role in the induction of class I and II expression in arterial endothelium. The fact that MHC class I is induced in endothelium may be useful therapeutically for reduction of immune recognition in transplantation.

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.

Quantitating immunosuppression. Estimating the 50% inhibitory concentration for in vivo cyclosporine in mice.

Cyclosporine (CsA) blocks T cell responses in vitro by inhibiting the phosphatase activity of calcineurin (CN) and thus preventing the activation of cytokine transcription. In the study presented here, we measured the extent of inhibition of these functions in the tissues of CsA-fed mice. Mice fed increasing doses of CsA were assessed for CsA blood and tissue levels, spleen cell CN activity, ex vivo spleen cell cytokine induction by A23187, and in vivo interferon-gamma induction during an allogeneic response. The CN activity of spleen homogenates and cell suspensions and the ex vivo cytokine responses of spleen cells from CsA-treated mice were inhibited with a 50% inhibitory concentration (IC50) greater than 300 microg/L. The in vivo interferon-gamma response to an allogeneic ascites tumor was also inhibited by CsA treatment, with IC50s between 517 and 886 microg/L. The true IC50 for CsA in vivo may be even higher, as CsA levels in spleen and kidney were 4-fold higher than concomitant blood levels. We conclude that inhibition of CN activity by systemically administered CsA leads to a parallel reduction in cytokine gene induction in response to an allogeneic stimulus. In light of our previous clinical findings that therapeutic levels of CsA in renal transplant patients were associated with only partial inhibition of CN activity, these current results support the concept that partial CN inhibition can account for both the immunosuppression and the immunocompetence of CsA-treated patients.

Effect of recombinant human insulin-like growth factor-1 on the inflammatory response to acute renal injury.

Renal ischemic injury evokes an inflammatory response with increased cytokine and major histocompatibility complex (MHC) expression and a mild interstitial infiltrate. This "injury response" could contribute to the tendency of ischemically injured renal transplants to reject. The studies presented here evaluated the ability of recombinant human insulin-like growth factor-1 (rhlGF-1) given after renal injury to prevent renal inflammation. The left renal pedicle of CBA and BALB/c mice was clamped for 60 min, and rhlGF-1 (25, 50, 100 micrograms) was administered sc at 2, 24, 48, 72, and 96 h after reflow. Cytokine and MHC expression was monitored in the injured kidney, compared with the contralateral kidney. In untreated mice, a single episode of injury induced the expression of MHC mRNA and products and tumor necrosis factor-alpha (TNF-alpha) mRNA, and depressed preproepidermal growth factor (ppEGF) mRNA, for up to 5 wk. With immunohistology, epithelial Class I and II MHC expression was shown to be increased for 2 wk, and Class II positive interstitial cells were shown to be increased for up to 5 wk. The ischemically injured kidneys from mice treated with rhlGF-1 and examined at 5 days showed a dose-dependent normalization of all of the changes of the injury response. This included prevention of the increased expression of MHC and cytokines and the Class II positive interstitial cells, and restoration of ppEGF mRNA. Thus the complex and long-lasting increase in proinflammatory cytokines and MHC expression that follow renal ischemia can be interrupted by treatment with rhlGF-1 beginning 2 h after the injury. This therapy may have applications to the injury response in renal transplants.

Acute renal injury in the interferon-gamma gene knockout mouse: effect on cytokine gene expression.

We studied major histocompatibility complex (MHC) and cytokine mRNA induction after renal injury in the absence of interferon-gamma (IFN-gamma) using IFN-gamma gene knockout (GKO) mice. The left renal pedicle of normal (wild-type) and GKO BALB/c mice was clamped for 60 minutes; cytokine and MHC mRNA expression were monitored in the injured kidney and compared to the contralateral control kidney. After a single episode of ischemic injury, the expression of mRNA for MHC class I and II, interleukin-2, interleukin-10, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and transforming growth factor-beta 1 was increased in wild-type and GKO mice, whereas preproepidermal growth factor (ppEGF) was reduced. IFN-gamma expression was induced in wild-type mice but absent in the GKO mice. Therefore, local injury was equally effective in both wild-type and GKO mice with equivalent cytokine and MHC mRNA induction, proving that local tissue injury can induce MHC expression by non-IFN-gamma factors.

Disturbed MHC regulation in the IFN-gamma knockout mouse. Evidence for three states of MHC expression with distinct roles for IFN-gamma.

We compared the expression of MHC class I and II products in tissues of IFN-gamma knockout (GKO) vs normal (wild-type) BALB/c mice. We studied expression in the basal state, after local tissue injury, after stimuli that induce systemic MHC expression (allogeneic cells, oxazolone skin painting, or LPS), and after rIFN-gamma. Basal class II expression in interstitial cells was not reduced in GKO mice. However, GKO mice had less basal class I expression in kidney, liver, heart, and arterial endothelium than wild-type mice. Local renal ischemic injury increased class I and II expression in kidney tubules of both GKO and wild-type mice, but induction in GKO was less than in wild-type. Potent inflammatory stimuli increased systemic MHC class I and II markedly in kidney, liver, and heart of wild-type mice, but induced no increase in GKO mice. rIFN-gamma induced class I and II equally in GKO and wild-type mice. Thus, three states of MHC expression can be defined that differ in their dependencies on IFN-gamma: basal, locally induced, and systemically induced. Basal class II expression in interstitial cells is IFN-gamma independent, but basal class I expression, particularly in arterial endothelium, is partially dependent on IFN-gamma. The local increase in MHC class I and II in parenchymal cells in response to injury reflects both IFN-gamma and a non-IFN-gamma factor. Systemic MHC class I and II induction is almost exclusively due to IFN-gamma.

Ischemic acute tubular necrosis induces an extensive local cytokine response. Evidence for induction of interferon-gamma, transforming growth factor-beta 1, granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-10.

We noted previously that ischemic acute tubular necrosis (ATN) induces local expression of MHC products in renal epithelium. The present investigations were conducted to establish the role of IFN-gamma in the regulation of MHC antigen expression in ATN and to explore the changes in cytokine and growth factor expression induced by ischemic renal injury. We produced unilateral ischemic ATN in mice by clamping the left renal pedicle. MHC class I and II steady state mRNA induction was assessed by northern blot analysis, and MHC product was quantified by the extent of binding of radiolabeled monoclonals to tissue homogenates. The steady state mRNA levels for IFN-gamma, IL-2, IL-10, and granulocyte-macrophage CSF were assessed by reverse transcriptase polymerase chain reaction and the levels for transforming growth factor-beta 1 and prepro-epidermal growth factor (ppEGF) were assessed by Northern blot analysis. In the injured kidneys, steady state mRNA levels for IFN-gamma, IL-2, IL-10, granulocyte-macrophage CSF, and transforming growth factor beta-1 were increased, whereas ppEGF mRNA was markedly decreased. The MHC expression was inhibited by treatment of mice with an anti-IFN-gamma mAb (R4-6A2). Murine EGF, administered in an attempt to accelerate recovery, did not reduce the cytokine and MHC changes. These data indicate that ischemic injury, and possibly other forms of injury, triggers a complex circuit of proinflammatory cytokines. This "injury response" could be relevant to clinical renal transplants, where ATN is associated with poor graft outcome.

Local T cell responses induce widespread MHC expression. Evidence that IFN-gamma induces its own expression in remote sites.

Local inflammation induces increased expression of MHC and other genes in the affected tissue because of the paracrine effects of cytokines such as IFN-gamma. We previously reported that one such process--local allograft rejection--was accompanied by increased expression of MHC in a remote tissue, namely kidney. To explore how local inflammation affects gene expression in remote tissues, we studied MHC, beta 2-microglobulin, and IFN-gamma expression in mice undergoing either of two T cell-dependent localized inflammatory processes: rejection of an ascites tumor allograft, and skin sensitization by oxazalone. As assessed by binding of radiolabeled mAb and by immunohistology, each stimulus increased MHC expression in many remote tissues, including liver, heart, pancreas, and kidney. This was associated with increases in steady state mRNA for class I, class II, and beta 2-microglobulin. MHC induction was inhibited by the in vivo administration of cyclosporine or anti-IFN-gamma mAb and did not occur in nude mice, confirming the key role of IFN-gamma released from T cells. When we examined tissues of mice with these localized inflammatory lesions for IFN-gamma mRNA levels by polymerase chain reaction, we found that IFN-gamma steady state mRNA levels were increased in the spleen and, more surprisingly, in the kidney, and in uninvolved skin. Moreover, anti-IFN-gamma inhibited the induction of IFN-gamma mRNA in the kidney, suggesting that IFN-gamma expression was induced by IFN-gamma in an autoregulatory fashion. Thus the systemic MHC induction accompanying local T cell-mediated inflammation reflects the release of IFN-gamma from the site of inflammation, but may be amplified by the ability of IFN-gamma to induce its own expression in remote tissues. This self-amplification of IFN-gamma may contribute to the ability of local inflammation to induce extensive systemic effects.

The regulation of expression of MHC products in mice. Factors determining the level of expression in kidneys of normal mice.

Cytokines, especially IFN, have been shown to increase MHC expression above basal levels in many immunologic processes, but their contribution to normal MHC expression is unknown. Environmental stimuli, such as bacterial LPS, may provoke the secretion of cytokines which could influence MHC expression in the normal host. We therefore studied the factors influencing the normal expression of MHC products in mice, by using kidney as a representative nonlymphoid tissue. Considerable variation in MHC expression was observed between otherwise normal individuals by using indirect immunoperoxidase staining and a radiolabeled antibody binding assay. An intact T cell compartment was not necessary for normal levels of expression; in athymic nude mice and mice with severe combined immunodeficiency disease, MHC expression was similar to or greater than that of controls. Neither a course of broad spectrum antibiotics nor genetically determined resistance to LPS influenced the level of normal expression. Mice raised under germ-free conditions, on a chemically defined diet also had similar levels of MHC expression when compared with mice maintained under conventional conditions. However, when germfree mice were placed in a conventional colony, induction of both class I and II MHC products occurred. Thus, although exposure to bacterial flora or other sources of endotoxin was not required for normal MHC expression, a change in flora can up-regulate expression, probably by inducing the secretion of cytokines from non-T cells. Treatment with cyclosporine or anti-IFN antibodies produced, at most, a small reduction in the level of renal MHC expression. Therefore in some mice, a small component of MHC expression may reflect the secretion of cytokines in response to stimuli such as bacterial LPS. However, most MHC expression in nonlymphoid tissues is constitutive and independent of a physiologic IFN response.