RESUMEN
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.
Asunto(s)
Rechazo de Injerto/inmunología , Interferón gamma/farmacología , Trasplante de Riñón/inmunología , Transcripción Genética , Animales , Antígenos H-2/inmunología , Trasplante de Corazón/inmunología , Interferón gamma/deficiencia , Interferón gamma/inmunología , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Modelos Animales , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunología , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Trasplante de Corazón/inmunología , Trasplante de Corazón/patología , Interferón gamma/fisiología , Trasplante de Riñón/inmunología , Trasplante de Riñón/patología , Enfermedad Aguda , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Suero Antilinfocítico/biosíntesis , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/prevención & control , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Sueros Inmunes/administración & dosificación , Inyecciones Intraperitoneales , Interferón gamma/administración & dosificación , Interferón gamma/genética , Interferón gamma/inmunología , Riñón/irrigación sanguínea , Riñón/inmunología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Noqueados , Miocardio/inmunología , Miocardio/patología , Necrosis , Proteínas Recombinantes/administración & dosificaciónRESUMEN
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.
Asunto(s)
Rechazo de Injerto/patología , Interferón gamma/metabolismo , Trasplante de Riñón , Animales , Complejo CD3/análisis , Antígenos CD4/análisis , Antígenos CD8/análisis , Regulación de la Expresión Génica , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Antígenos H-2/análisis , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Isoanticuerpos/inmunología , Antígenos Comunes de Leucocito/análisis , Leucocitos Mononucleares/química , Leucocitos Mononucleares/patología , Ratones , Ratones Endogámicos CBA , Ratones Endogámicos , Ratones Noqueados , Necrosis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Receptores de Interferón/metabolismo , Trasplante Homólogo , Receptor de Interferón gammaRESUMEN
BACKGROUND: Patients exposed to allogeneic human tissue sometimes produce anti-HLA antibody for many years in the absence of further obvious antigen exposure. To investigate the mechanism of sustained sensitization, we identified females awaiting renal transplantation with high panel-reactive antibody but no exposure to allogeneic tissue for at least 1 month. METHODS: We analyzed peripheral blood microchimerism using nested polymerase chain reaction amplification specific for the SRY region of the Y chromosome. RESULTS: Microchimerism was detected in 3 of 10 patients but in none of 8 normal female subjects. In two cases, the amplified DNA polymerase chain reaction product was sequenced and was confirmed to be identical to the SRY gene. The estimated level of chimerism as compared with serial dilutions of DNA from male peripheral blood leukocytes was about 1/50000. CONCLUSION: These results do not establish causality but support the possibility that antigens from microchimeric donor cells may sustain the HLA antibody response in certain patients.
Asunto(s)
Trasplante de Riñón/inmunología , Proteínas Nucleares , Factores de Transcripción , Quimera por Trasplante/inmunología , Adulto , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Proteínas de Unión al ADN/genética , Femenino , Antígenos HLA/inmunología , Humanos , Inmunización , Masculino , Reacción en Cadena de la Polimerasa , Procesos de Determinación del Sexo , Proteína de la Región Y Determinante del SexoRESUMEN
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.
Asunto(s)
Rechazo de Injerto/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Inmunosupresores/administración & dosificación , Interferón gamma/biosíntesis , Ácido Micofenólico/análogos & derivados , Inmunología del Trasplante/inmunología , Animales , Ciclosporina/farmacología , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II/genética , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ácido Micofenólico/administración & dosificación , Trasplante de Neoplasias , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Genes MHC Clase II , Genes MHC Clase I , Proteínas Nucleares , Fosfoproteínas/fisiología , Animales , Cicloheximida/farmacología , Regulación de la Expresión Génica , Inflamación/fisiopatología , Factor 1 Regulador del Interferón , Interferón gamma/farmacología , Interferón gamma/fisiología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Oxazolona , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/genética , Proteínas Recombinantes , Factores de Tiempo , Distribución Tisular , Transactivadores/genéticaAsunto(s)
Rechazo de Injerto/epidemiología , Rechazo de Injerto/fisiopatología , Supervivencia de Injerto , Trasplante de Riñón/inmunología , Necrosis Tubular Aguda/patología , Enfermedad Aguda , Citocinas/biosíntesis , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Inflamación , Trasplante de Riñón/mortalidad , Trasplante de Riñón/patología , Necrosis Tubular Aguda/inmunología , Complejo Mayor de Histocompatibilidad , Donantes de Tejidos , Resultado del TratamientoAsunto(s)
Trasplante de Corazón/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Trasplante de Riñón/inmunología , Riñón/inmunología , Complejo Mayor de Histocompatibilidad , Linfocitos T/inmunología , Transcripción Genética , Animales , Genes MHC Clase I , Genes MHC Clase II , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Microglobulina beta-2/biosíntesisAsunto(s)
Moléculas de Adhesión Celular/biosíntesis , Interferón gamma/biosíntesis , Isquemia/inmunología , Riñón/irrigación sanguínea , Riñón/inmunología , Complejo Mayor de Histocompatibilidad , Animales , Gentamicinas/toxicidad , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Interferón gamma/deficiencia , Interferón gamma/genética , Riñón/patología , Cloruro de Mercurio/toxicidad , Ratones , Ratones Noqueados , Ratones DesnudosRESUMEN
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.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/genética , Enfermedades Renales/inmunología , Proteínas Nucleares , Transactivadores/genética , Animales , Proteínas de Unión al ADN/fisiología , Expresión Génica , Genes MHC Clase II/genética , Factor 1 Regulador del Interferón , Interferón gamma/fisiología , Riñón/irrigación sanguínea , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosfoproteínas/fisiología , ARN Mensajero/genética , Factores de TiempoRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/farmacología , Genes MHC Clase II/genética , Genes MHC Clase I/genética , Fosfoproteínas/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Factor 1 Regulador del Interferón , Interferón gamma/genética , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , ARN Mensajero/análisisRESUMEN
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.
Asunto(s)
Interferón gamma/farmacología , Proteínas Nucleares , Transactivadores/fisiología , Animales , Anticuerpos/farmacología , Presentación de Antígeno/inmunología , Unión Competitiva , Cicloheximida/farmacología , Amplificación de Genes , Expresión Génica/efectos de los fármacos , Genes MHC Clase II/genética , Interferón gamma/inmunología , Riñón/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARN , Factores de Tiempo , Distribución Tisular , Transactivadores/genética , Transactivadores/farmacocinéticaRESUMEN
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.
Asunto(s)
Factor de Crecimiento Epidérmico/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos H-2/biosíntesis , Factor I del Crecimiento Similar a la Insulina/uso terapéutico , Isquemia/complicaciones , Riñón/irrigación sanguínea , Nefritis/prevención & control , Precursores de Proteínas/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Secuencia de Bases , Northern Blotting , Factor de Crecimiento Epidérmico/genética , Rechazo de Injerto , Antígenos H-2/genética , Humanos , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/farmacología , Isquemia/metabolismo , Trasplante de Riñón , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Nefritis/etiología , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/genética , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
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.
Asunto(s)
Calcio/fisiología , Ciclosporina/farmacología , Proteínas de Unión al ADN/metabolismo , Interferón gamma/genética , Regiones Promotoras Genéticas/fisiología , Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Secuencia de Consenso , Citoplasma/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Humanos , Interferón gamma/biosíntesis , Interleucina-4/genética , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/fisiología , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Proteínas Nucleares/metabolismo , Unión Proteica , Sensibilidad y Especificidad , Homología de Secuencia de Ácido Nucleico , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Interferón gamma/deficiencia , Animales , Secuencia de Bases , Regulación de la Expresión Génica , Isquemia/inmunología , Riñón/inmunología , Riñón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Citocinas/biosíntesis , Antígenos de Histocompatibilidad/biosíntesis , Necrosis Tubular Aguda/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Citocinas/efectos de los fármacos , ADN Complementario , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/análisis , Factor de Crecimiento Epidérmico/farmacología , Antígenos de Histocompatibilidad/efectos de los fármacos , Interferón gamma/análisis , Interferón gamma/inmunología , Isquemia/inmunología , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta/análisisAsunto(s)
Citocinas/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Interferón gamma/genética , Isquemia , Riñón/patología , Complejo Mayor de Histocompatibilidad , Animales , Secuencia de Bases , Cartilla de ADN , Factor de Crecimiento Epidérmico/biosíntesis , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Inflamación , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Riñón/irrigación sanguínea , Riñón/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Valores de Referencia , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Interferón gamma/biosíntesis , Lipopolisacáridos , ARN Mensajero/análisis , Animales , Secuencia de Bases , Células Cultivadas , Femenino , Interferón gamma/genética , Riñón/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regulación hacia ArribaRESUMEN
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.