RESUMEN
An imbalance between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) may contribute to tissue destruction that is found in various inflammatory disorders. To determine in an in vivo experimental setting whether the inflammatory reaction in the course of lipopolysaccharide (LPS)-induced endotoxemia causes an altered balance in the MMP/TIMP system, we analyzed the expression of a number of MMP and TIMP genes as well as MMP enzymatic activity in the liver, kidney, spleen, and brain at various time points after systemic injection of different doses of LPS in mice. Injection of sublethal doses of LPS led to an organ- and time-specific pattern of up-regulation of several MMP genes and the TIMP-1 gene in the liver, spleen, and kidney, whereas in the brain only TIMP-1 was induced. Injection of a lethal dose of LPS caused similar but more prolonged expression of these MMP genes as well as the induction of additional MMP genes in all organs. In LPS-treated mice in situ hybridization revealed collagenase 3 gene induction in cells resembling macrophages whereas TIMP-1 RNA was detected predominantly in parenchymal cells. Finally, gelatin zymography revealed increased gelatinolytic activity in all organs after LPS treatment. These observations highlight a dramatic shift in favor of increased expression of the MMP genes over the TIMP genes during LPS-induced endotoxemia, and suggest that MMPs may contribute to the development of organ damage in endotoxemia.
Asunto(s)
Endotoxemia/genética , Lipopolisacáridos/farmacología , Metaloproteinasas de la Matriz/genética , Inhibidores Tisulares de Metaloproteinasas/genética , Animales , Encéfalo/metabolismo , Resistencia a Medicamentos , Electroforesis en Gel de Poliacrilamida , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Hibridación in Situ , Riñón/metabolismo , Hígado/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Activación TranscripcionalRESUMEN
The role of IL-12 in the evolution of immunoinflammatory responses at a localized tissue level was investigated. Transgenic mice were developed with expression of either both the IL-12 subunits (p35 and p40) or only the IL-12 p40 subunit genes targeted to astrocytes in the mouse CNS. Glial fibrillary acidic protein (GF)-IL-12 mice, bigenic for the p35 and p40 genes, developed neurologic disease which correlated with the levels and sites of transgene-encoded IL-12 expression. In these mice, the brain contained numerous perivascular and parenchymal inflammatory lesions consisting of predominantly CD4+ and CD8+ T cells as well as NK cells. The majority of the infiltrating T cells had an activated phenotype (CD44high, CD45Rblow, CD62Llow, CD69high, VLA-4 high, and CD25+). Functional activation of the cellular immune response was also evident with marked cerebral expression of the IFN-gamma, TNF, and IL-1alphabeta genes. Concomitant with leukocyte infiltration, the CNS expression of immune accessory molecules was induced or up-regulated, including ICAM-1, VCAM-1, and MHC class II and B7-2. Glial fibrillary acidic protein-p40 mice with expression of IL-12 p40 alone remained asymptomatic, with no inflammation evident at any age studied. The effect of local CNS production of IL-12 in the development of experimental autoimmune encephalomyelitis was studied. After immunization with myelin oligodendrocyte glycoprotein-peptides, GF-IL-12 mice had an earlier onset and higher incidence but not more severe disease. We conclude that localized expression of IL-12 by astrocytes can 1) promote the spontaneous development of activated type 1 T cell and NK cellular immunity and cytokine responses in the CNS, and 2) promote more effective Ag-specific T cell dynamics but not activity in experimental autoimmune encephalomyelitis.
Asunto(s)
Astrocitos/inmunología , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Marcación de Gen , Inmunidad Celular/genética , Interleucina-12/biosíntesis , Interleucina-12/genética , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Astrocitos/metabolismo , Antígeno B7-2 , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Movimiento Celular/genética , Movimiento Celular/inmunología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Relación Dosis-Respuesta Inmunológica , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica/inmunología , Proteína Ácida Fibrilar de la Glía/genética , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/genética , Interferón gamma/biosíntesis , Interferón gamma/genética , Células Asesinas Naturales/inmunología , Activación de Linfocitos/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/biosíntesis , Linfocitos T/inmunología , Transgenes/inmunologíaRESUMEN
RNase protection assay (RPA) is becoming an increasingly popular method for the detection and quantitation of RNA levels in cells and tissues (1-3). Hybridization is conducted in solution using an excess of a labeled antisense single-stranded RNA as probe. Thus, hybridization of the probe with target RNA results in the formation of stable, double-stranded RNA-RNA hybrids. After hybridization, the excess probe is removed by digestion with single-strand specific RNase, leaving behind only those probe molecules that were "protected" from digestion by virtue of having formed a duplex with their complementary mRNA target. These protected hybrids are denatured and separated from remaining labeled probe using standard sequencing polyacrylamide gel electrophoresis. The separated protected probe can then be visualized using routine autoradiography. In comparison with other RNA detection methods, such as Northern blot analysis or RT-PCR, the RPA has a number of advantages. These include: 1. High sensitivity and specificity. 2. Small sample requirement. 3. Tolerant of RNA degradation. 4. Easy quantitation. 5. Rapid and simultaneous analysis of multiple target transcripts. 6. High throughput analysis. 7. Construction and use of "designer" probe sets.
RESUMEN
Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of inflammatory disorders of the central nervous system (CNS) whereas the contribution of the major endogenous counter-regulators of MMPs, the tissue inhibitors of the matrix metalloproteinases (TIMPs), is unclear. We investigated the temporal and spatial expression patterns in the CNS of nine MMP genes and three TIMP genes in normal mice, in mice with EAE, and in transgenic mice with astrocyte (glial fibrillary acidic protein)-targeted expression of the cytokines interleukin-3 (macrophage/microglial demyelinating disease), interleukin-6 (neurodegenerative disease), or tumor necrosis factor-alpha (lymphocytic encephalomyelitis). In normal mice, the MMPs MT1-MMP, stromelysin 3, and gelatinase B were expressed at low levels, whereas high expression of TIMP-2 and TIMP-3 was observed predominantly in neurons and in the choroid plexus, respectively. In EAE and the transgenic mice, significant induction or up-regulation of various MMP genes was observed, the pattern of which was somewhat specific for each of the models, and there was significant induction of TIMP-1. In situ localization experiments revealed a dichotomy between MMP expression that was restricted to leukocytes and possibly microglia within inflammatory lesions and TIMP-1 expression that was observed in activated astrocytes circumscribing the lesions. These findings demonstrate specific spatial and temporal regulation in the expression of individual MMP and TIMP genes in the CNS in normal and inflammatory states. The distinct localization of TIMP-1 and MMP expression during CNS inflammation suggests a dynamic state in which the interplay between these gene products may determine both the size and resolution of the destructive inflammatory focus.