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Organ allografts deficient in interferon-gamma (Ifng) or major histocompatibility complex (MHC) class I products develop accelerated necrosis when rejection develops, depending on perforin and granzymes. Thus Ifng-induced donor class I products deliver inhibitory signals to host inflammatory cells. We used microarrays to investigate whether Ifng-induced donor class I products also control inflammation patterns in mouse kidney allografts. Compared to wild-type (WT) allografts, many transcripts were increased in both Ifng-deficient allografts (Ifng-suppressed transcripts [GSTs]) and class I-deficient allografts (class I-suppressed transcripts [CISTs]), with 73% overlap between GSTs and CISTs. Some GSTs and CISTs reflected increased necrosis, including known injury-induced transcripts. However, many GSTs and CISTs were independent of perforin, granzymes and necrosis, and were associated with alternative macrophage activation (AMA) (e.g. arginase I [Arg1], macrophage elastase [Mmp12] and macrophage mannose receptor 1 [Mrc1]). AMA transcripts were induced despite absence of host interleukin (IL)4 and IL13 receptors. The AMA inducer may be activins, whose genes (inhibin A [InhbA] and inhibin B [InhbB]) were increased in all allografts with AMA. We conclude that in allograft rejection, Ifng acts via donor Ifng receptors (Ifngr) to induce donor class Ia and Ib products, which engage host inflammatory cells to limit perforin-granzyme-mediated damage and prevent AMA associated with inhibition of activin expression. Thus, Ifng may control T helper type 2 (Th2) cell inflammation by induction of class I products.

Interferon-gamma (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 beta2 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.

We used Affymetrix Microarrays to define interferon-gamma (IFN-gamma)-dependent, rejection-induced transcripts (GRITs) in mouse kidney allografts. The algorithm included inducibility by recombinant IFN-gamma in kidneys of three normal mouse strains, increase in kidney allografts in three strain combinations and less induction in IFN-gamma-deficient allografts. We identified 40 transcripts, which were highly IFN-gamma inducible (e.g. Cxcl9, ubiquitin D, MHC), and 168 less sensitive to IFN-gamma 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-gamma receptors (IFN-gammars): 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-gamma injections. Many GRITs showed transient IFN-gamma-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-gamma produced by the alloresponse locally and systemically, acting via host and donor IFN-gammar, as well as local IFN-gamma production induced by post-operative stress.

IFN-gamma is produced by cytotoxic T lymphocytes (CTL) but can also decrease CTL generation. We used IFN-gamma-R1-deficient (GRKO) and IFN-gamma-deficient (GKO) mice to study the effects of IFN-gamma in MLC on the generation of CTL activity and CTL number, IL-2 production and cell proliferation. CTL activity was increased in MLC when GRKO responders or GKO stimulators and responders were used, compared to wild-type (WT) MLC. The number of cells displaying the CTL phenotype (CD3+, CD8+, CD25+) was also increased, accompanied by increased IL-2 production and proliferation. Combinations of WT or GRKO CD4+ T cells with WT or GRKO CD8+ T cells as responders showed that IFN-gamma mostly affects CD4+ T cells to limit CTL generation. Intracellular staining indicated that IL-2 production was largely by CD4+ T cells. Moreover, addition of IL-2 to WT responders mimicked GKO CTL generation and activity, whereas neutralizing IL-2 decreased CTL activity in GRKO and WT responders. Thus IFN-gamma reduces CTL generation in alloimmune responses largely by limiting proliferation of IL-2 producing CD4+ T cells. This creates a feedback loop in which effectors produce IFN-gamma that limits IL-2 production which in turn limits CTL generation.

Expression of class II transactivator (CIITA), the transcriptional regulator that controls all class II expression, is controlled in cell lines in vitro by three promoters: the dendritic cell promoter PI, the B cell promoter PIII, and the interferon-gamma (IFN-gamma)-inducible promoter, PIV. The authors examined the promoter usage in vivo in mouse kidney in the basal state and in response to IFN-gamma, endotoxin, allostimulation, and renal injury. Genetically modified mice were used to examine the dependency of each promoter on IFN-gamma and on the transcription factor interferon regulatory factor 1 (IRF-1). Usage of distinct CIITA promoters was monitored by real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the unique sequences in the 5' end of the transcript from each promoter. Kidneys in both control mice and IFN-gamma knockouts expressed chiefly PI- and PIV-related products. Administration of recombinant IFN-gamma activated only promoter PIV. Endotoxin or allogeneic stimulation elevated the PIV-related mRNA, dependent on IFN-gamma and on IRF-1. Ischemic renal injury, however, increased the PI- and PIV-driven mRNA expression in wild-type but also in IFN-gamma-deficient mice. Thus the in vivo control of CIITA promoters in kidney is similar to that observed in vitro (i.e., basal-state usage of PI and IFN-gamma-dependent usage of PIV during inflammation), but it also shows additional levels of control: IFN-gamma-independent basal activity of PIV and IFN-gamma-independent induction of PIV during tissue injury.

Experimental liver allografts undergo spontaneous acceptance despite undergoing rejection during the first few weeks post transplant. We explored the role of interferon-gamma (IFN-gamma) 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-gamma-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-gamma-deficient hosts, but livers in interferon-gamma-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-gamma on the liver, we transplanted livers from IFN-gamma-receptor-deficient mice into normal hosts. Liver allografts lacking IFN-gamma receptors also developed hemorrhage and necrosis with minimal induction of MHC expression. Thus IFN-gamma mediates a direct effect on rejecting liver allografts that reduces hemorrhage and necrosis, induces MHC expression, and is absolutely required for spontaneous acceptance.

Interferon (IFN)-gamma is a cytokine produced mostly by activated T cells and NK cells that has complex effects on immune and nonimmune cells. IFN-gamma plays important roles in inflammation, usually in synergy with other cytokines, such as IL-1beta and TNF-alpha. The uniqueness of IFN-gamma lies in its ability to induce major histocompatibility complex (MHC) expression in many tissues, making it particularly relevant to transplantation. The results of graft rejection in the absence of IFN-gamma show that IFN-gamma modulates but is not essential for the allogeneic responses, suppressing generation of CTL. In vivo IFN-gamma has a protective role early in the response to vascularized organ allografts: transplants in mice have a tendency to develop necrosis when IFN-gamma is not available, apparently by failure of the microcirculation. The lack of IFN-gamma greatly reduces the induction of MHC in organ allografts, and it is possible that this is indirectly related to the protective effect of IFN-gamma. Nevertheless IFN-gamma also promotes graft vessel disease later in the course ofthe transplant. Thus IFN-gamma has diverse and potentially contradictory effects on organ allograft survival, acting both on the immune system and on the graft itself, the net effect depending on the graft type and the time post-transplant.

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.

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 pathogenetic role of interferon-gamma (IFN-gamma) in acute graft-versus-host disease (GVHD) was examined in a murine model. IFN-gamma gene expression was evaluated by northern blotting and mRNA in situ hybridization. The temporal and tissue specific patterns of IFN-gamma gene expression were related to the patterns of major histocompatibility complex (MHC) antigen induction and of tissue injury. Markedly increased levels of IFN-gamma transcripts were seen in the spleen during the early lymphoproliferative phase and coincided with widespread MHC induction in non-lymphoid tissues. Increased IFN-gamma transcripts were also found in the non-lymphoid target tissues during the phase of subsequent tissue injury. These findings support a role for IFN-gamma in leading to widespread MHC induction during acute GVHD and suggest that IFN-gamma may also contribute to target tissue injury during acute GVHD., (Copyright 1999 John Wiley & Sons, Ltd.)

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.

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 class II transactivator (CIITA) is a protein that induces the transcription of MHC class II genes. We studied the expression of CIITA in vivo, comparing steady state levels of CIITA and class II mRNA in various mouse tissues. Many tissues in normal mice contained mRNA for CIITA, correlating with class II mRNA. The basal expression of CIITA and class II mRNA in mice with disrupted IFN-gamma genes (GKO mice) was similar to that in wild-type mice. Injection of rIFN-gamma strongly induced CIITA and class II mRNA: CIITA mRNA increased at 2 hr and declined to baseline by 48 hr, whereas class II mRNA increased at 24 hr and returned to baseline at 7 days. Proinflammatory stimuli that induce IFN-gamma production (allogeneic cells and LPS) induce CIITA and class II expression in wild-type mice, but not in GKO mice. CIITA induction by IFN-gamma was partially sensitive to cycloheximide, suggesting that another protein is required for CIITA induction. The data suggest that CIITA is a major regulator of basal and induced class II expression in vivo.

Cyclosporine (CsA) inhibits cytokine transcription by preventing the activation of key promoter sites, in particular the binding of nuclear factor of activated T cells (NFAT) to the IL-2 NFAT site and the "P" site in IL-4. To identify potential NFAT-like sites in the IFN-gamma promoter, we sought areas of homology with the known sites in other promoters. In the promoter region of the mouse and human IFN-gamma gene, we identified two repeats of a consensus sequence ATTTCCnnT, designated P1 and P2 because of their homology to the calcium-inducible and CsA-sensitive "P" sequences in the IL-4 promoter. In electrophoretic mobility shift assay (EMSA), a probe containing the second P sequence "P2" in the human IFN-gamma gene bound nuclear proteins from stimulated, but not unstimulated, humans T cells. The cytosol of unstimulated cells contained similar binding activity that decreased after stimulation, indicating that this binding activity translocated to the nucleus after stimulation. CsA inhibited nuclear translocation. Competition studies demonstrated that oligomers containing the sequences P1 and P2 in IFN-gamma gene, the NFAT site in the IL-2 gene, and the IL-4 P site competed with the P2 probe for protein binding, whereas an oligomer containing mutations in the P2 site did not. Addition of anti-NFAT antiserum altered protein binding to P2, indicating that the proteins were either identical or related to NFAT. Stimulation of T cells transfected with constructs containing three copies of the P2 sequence enhanced CAT activity in response to ionomycin, and this effect was blocked by CsA. These results suggest that the P2 sequence, and probably the P1 sequence, in the IFN-gamma promoter are NFAT binding sites and contribute to the calcium inducibility and CsA sensitivity of IFN-gamma production.

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.

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.

As peritoneal macrophages require Interferon-gamma (IFN-gamma) for bacterial lysis, IFN-gamma levels were measured in peritoneal dialysis effluents. (PDE) by a specific radioimmunoassay. High IFN-gamma levels were found in patients with peritonitis compared to low levels in patients without peritonitis (mean 9.73 +/- 2.63 SE U/ml, N = 39 vs. 0.25 +/- 0.04, N = 32). IFN-gamma levels varied among different bacteria: Staph. aureus (highest: 23.4 +/- 5.7, N = 14), Staph. epidermidis (lower: 3.2 +/- 0.8, N = 13), other gram-positive (1.06 +/- 0.32, N = 6), gram-negative bacteria (lowest: 0.57 +/- 0.30, N = 6). After treatment of peritonitis levels decreased. In corresponding blood and PDE samples, by comparing IFN-gamma levels in 10 peritoneal dialysis patients (5 with peritonitis, 5 without), levels were raised only in PDE of patients with peritonitis, implying local IFN-gamma production. Total lymphocytes, T, B and monocyte subsets in patients' plasma and PDE did not differ, except for a higher number of mononuclear cells in PDE of patients with peritonitis (P < 0.05). Further investigation of in vitro IFN-gamma production in PDE with peritoneal monocytes, syngeneic host lymphocytes, and bacteria showed that Staph. aureus induced the highest levels of IFN-gamma and E. coli the lowest, in experiments with T cell enriched host lymphocytic fractions. We conclude that Staph. aureus peritonitis induces high levels of IFN-gamma in PDE, possibly by a T cell dependent superantigen response.

Immune responses to such stimuli as tissue injury, infection, and allografting result in localized IFN-gamma expression. Autoregulation of cytokine expression has been described for some cytokines in vitro, but whether this occurs in vivo is unknown. We therefore examined the role of murine IFN-gamma in the regulation of its own expression in vivo after stimulation with bacterial LPS. This agent is known to induce IFN-gamma expression in both spleen and kidney in a T cell-independent, cyclosporine-sensitive manner. We found that concomitant administration of a neutralizing mAb to IFN-gamma inhibited not only the MHC expression induced by LPS but also the increased IFN-gamma mRNA expression, suggesting an autoregulatory role for IFN-gamma. Inhibition was dose dependent and observed in both spleen and kidney. The effect was not seen with a neutralizing anti-IL-3 mAb, demonstrating specificity. The inhibition of IFN-gamma mRNA expression by the anti-IFN-gamma mAb occurred in both T cell-deficient athymic nude mice and their normal controls, suggesting that the autoamplification of IFN-gamma mRNA in vivo is T cell independent. Administration of rIFN-gamma to unstimulated mice induced IFN-gamma mRNA expression in spleen and kidney, supporting the conclusion that IFN-gamma up-regulates expression of its mRNA. Exposure of resting murine splenocytes to concentrations of rIFN-gamma as low as 10 U/ml in vitro induced expression of IFN-gamma mRNA. Thus, in vivo IFN-gamma may participate in an autoregulatory loop to amplify the amount of IFN-gamma expressed both at the site of local inflammation and at remote sites. This would have relevance in the mechanism by which the host defends itself against and prevents dissemination of an infectious agent.

Despite accumulating information about cytokine expression in vitro, relatively little is known about the regulation and biologic relevance of these mediators in vivo. In order to study the effects of inhibition of protein synthesis and cyclosporine in vivo, we made use of systemically administered LPS, which induces the expression of a variety of cytokines. The expression of IFN-gamma and TNF-alpha mRNA in normal and LPS-treated mice was examined by Northern blot analysis and amplification using the polymerase chain reaction. IFN-gamma activity was monitored using the biologic end point of MHC induction. TNF-alpha activity in serum was assessed using a L929 cytotoxicity assay. Messenger RNA for IFN-gamma and TNF-alpha could not be reliably detected by Northern analysis in spleens or kidneys of normal mice. After treatment with cycloheximide, a protein synthesis inhibitor, IFN-gamma and TNF-alpha mRNA could be detected in both sites in otherwise normal mice. The level of both IFN-gamma and TNF-alpha mRNA increased after LPS, although the temporal patterns of expression were different. The concurrent administration of cycloheximide led to marked superinduction of both cytokine mRNA levels. Similar effects were seen in T cell-deficient nude mice, suggesting that these responses are T cell independent. Cyclosporin A blocked induction of IFN-gamma in a dose dependent manner, but failed to significantly inhibit TNF-alpha mRNA or protein expression. Thus at least part of the immunosuppressive effect of cyclosporin A in vivo may be caused by its ability to inhibit the expression of certain cytokine genes, as has been found in vitro systems. However, the cellular target for this effect may extend to cell populations other than T cells.

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.

In rodents, mercuric chloride (HgCl2) causes an autoimmune disorder with glomerulonephritis (GN), and represents an animal model for the pathogenesis of GN. We have tested the hypothesis that HgCl2 induces major histocompatibility complex (MHC) expression in renal parenchymal cells, and studied the kinetics of this induction and its temporal relation to the development of immune complex deposition in the glomeruli. Mice treated with doses of HgCl2 between 2 and 3.2 mg/kg three times for one week had increased renal expression of MHC class I and class II (at the mRNA and the product levels). Class I induction was observed in proximal tubule cells, endothelial cells and glomerular cells. Class II induction was seen mainly in interstitial cells and, to a lesser extent, in tubule cells. Renal MHC expression was maximal at one week, decreased progressively after the second week of HgCl2 administration, and reached basal levels by 23 weeks. In contrast, the amount of lymphocyte infiltration in the kidney increased from the first to the fifth week and was followed by the appearance of glomerular immune deposits from the third week on. Glomerular immune complex deposits were maximal at five weeks and, by 23 weeks, immune deposits in HgCl2-treated mice were only slightly increased over those observed in the sham group. Renal MHC induction by HgCl2 was significantly reduced by treatment with monoclonal antibody against interferon gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple low dose streptozotocin (STZ) is believed to induce immunologically mediated islet cell necrosis. We sought to establish whether this agent also increased MHC expression, as has been reported for other diabetes models. STZ (40 mg/kg/day for 5 days) produced increased levels of both class I and II MHC products in kidney, liver, heart, and pancreas. Class I expression was induced in renal tubular cells, Kupffer cells, hepatocytes, occasional cardiac myocytes, and cells within the pancreatic islet. In contrast, class II products were increased in dendritic cells, renal tubules, and cells within the pancreatic islet. Steady state mRNA levels for class I, class II, and beta 2-microglobulin correlate closely with the level of MHC products measured by radiolabeled antibody binding, suggesting that changes in MHC expression reflect changes in gene transcription. The effect is T cell dependent and inhibitable by cyclosporine. IFN-gamma is an essential mediator of the MHC induction; administration of a neutralizing antibody blocks the increase in expression. Furthermore, this antibody attenuates the hyperglycemic response to STZ, demonstrating a pathogenic role for IFN-gamma in mediating beta cell damage. We conclude that the MHC induction observed after low dose STZ is due to immunologic mechanisms, in particular the release of lymphokines such as IFN-gamma from T cells. The release of IFN-gamma and changes in MHC expression may be relevant to the injury seen with this agent.

The ability of lipopolysaccharide to induce major histocompatibility complex hyperexpression in vivo in a variety of mouse tissues--particularly kidney--and the effect of cyclosporine on this process were studied. MHC expression was measured by a radiolabeled antibody-binding assay using tissue homogenates, as well as by assessment of tissue sections by indirect immunoperoxidase staining. LPS administered to mice in two doses, 4 days apart, induced an increase in class I expression in several tissues but also induced an increase in class II expression in kidney. A similar increase in class II expression in kidney was not elicited with polyinosinic acid/polycytidylic acid, an agent that induces release of IFN-alpha/beta and increases class I MHC product expression. Thus we reasoned that LPS in vivo may release IFN-gamma, which then induces increased expression of MHC products. We validated this hypothesis by demonstrating that monoclonal antibody against IFN-gamma inhibited the induction of renal MHC products by LPS. However, the LPS effects did not require the participation of T cells, being demonstrable in nude mice and in mice with severe combined immunodeficiency. Moreover, the effect of LPS on MHC expression in normal and nude mice was inhibited by in vivo administration of monoclonal antibody against IFN-gamma just as it was in normal mice. Thus the class II hyperexpression that follows LPS is apparently mediated by non T cells and is due to the systemic release of IFN-gamma. This mechanism was inhibited by high doses of CsA in vivo, both in normal and in nude mice. The results indicate that there is a non T cell pathway for IFN-gamma release (and MHC induction) in vivo that is sensitive to CsA. This observation raises the possibility that some of the immunosuppressive effects of CsA may be due to inhibition of mediator release from non T cells.