RESUMO
The extracellular matrix (ECM) in the central nervous system actively orchestrates and modulates changes in neural structure and function in response to experience, after injury, during disease, and with changes in neuronal activity. A component of the multi-protein, ECM aggregate in brain, the chondroitin sulfate (CS)-bearing proteoglycans (PGs) known as lecticans, inhibit neurite outgrowth, alter dendritic spine shape, elicit closure of critical period plasticity, and block target reinnervation and functional recovery after injury as the major component of a glial scar. While removal of the CS chains from lecticans with chondroitinase ABC improves plasticity, proteolytic cleavage of the lectican core protein may change the conformation of the matrix aggregate and also modulate neural plasticity. This review centers on the roles of the lecticans and the endogenous metalloproteinase families that proteolytically cleave lectican core proteins, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), in neural plasticity. These extracellular metalloproteinases modulate structural neural plasticity-including changes in neurite outgrowth and dendritic spine remodeling-and synaptic plasticity. Some of these actions have been demonstrated to occur via cleavage of the PG core protein. Other actions of the proteases include cleavage of non-matrix substrate proteins, whereas still other actions may occur directly at the cell surface without proteolytic cleavage. The data convincingly demonstrate that metalloproteinases modulate physiological and pathophysiological neural plasticity.
Assuntos
Encéfalo/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Metaloproteases/metabolismo , Plasticidade Neuronal/fisiologia , Animais , Neurônios/metabolismoRESUMO
Perinatal hypoxia-ischemia (H-I) often manifests as cognitive and/or motor disturbances that appear early in development. Growing evidence indicates that neuroinflammation may exacerbate H-I injury. Resident microglia release proinflammatory cytokines and proteases in response to ischemia. Matrix metalloproteinases (MMPs), in particular, activate cytokines and degrade basement membrane proteins. These actions ultimately permit entry of peripheral leukocytes into the CNS neuropil, enhancing neuroinflammation and cell death. Currently, the relative contributions of resident and peripheral immune cells to ischemic brain injury are unclear. The present study employed an ex vivo model of H-I through oxygen glucose deprivation (OGD) to identify the cellular localization of MMP-9 in organotypic hippocampal slices from rat, and to determine whether inhibiting gelatin-degrading MMPs affords neuroprotection in the absence of peripheral immune cells. Immunohistochemistry revealed ubiquitous neuronal MMP-9 expression in both normoxic and hypoxic slices. Increased MMP-9 expression was detected in CD11b-positive microglia after 48 h exposure to OGD relative to normoxic controls. Consistent with these data, in situ zymography showed increased gelatinolytic activity after OGD. Gelatin-cleaved fluorescence localized to astrocytic processes and somata of various cellular morphologies. Treatment with either the MMP inhibitor AG3340 (prinomastat) or minocycline dampened OGD-induced gelatinolytic activity and neural injury, as measured by Fluoro-Jade staining, relative to vehicle controls. These results show that resident microglia, in the absence of peripheral immune cells, were sufficient to enhance neural injury after OGD in the organotypic hippocampal slice. Additionally, these effects were associated with upregulation or secretion of MMP-9, and were blocked after treatment with either the gelatinase-selective compound AG3340 or the anti-inflammatory compound minocycline. These data, coupled with the effectiveness of these compounds previously shown in vivo, support the selective targeting of gelatin-degrading MMPs and activated microglia as potential therapeutic approaches to combat neonatal H-I injury.
Assuntos
Infarto Encefálico/tratamento farmacológico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz , Degeneração Neural/tratamento farmacológico , Animais , Animais Recém-Nascidos , Anti-Inflamatórios/farmacologia , Astrócitos/citologia , Astrócitos/enzimologia , Infarto Encefálico/enzimologia , Infarto Encefálico/fisiopatologia , Inibidores Enzimáticos/farmacologia , Gliose/tratamento farmacológico , Gliose/etiologia , Gliose/fisiopatologia , Hipóxia-Isquemia Encefálica/enzimologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Imuno-Histoquímica , Metaloproteinase 9 da Matriz/metabolismo , Microglia/enzimologia , Minociclina/farmacologia , Degeneração Neural/enzimologia , Degeneração Neural/fisiopatologia , Neurônios/enzimologia , Técnicas de Cultura de Órgãos , Compostos Orgânicos/farmacologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Proteolytic fragments generated by ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs)-mediated cleavage of the aggregating chondroitin sulfate proteoglycan, brevican, have been identified, but not localized in the CNS. The purpose of this study, using kainate-induced CNS lesion, was to examine the spatial and quantitative relationship between ADAMTS1 and 4 mRNA expression and ADAMTS-mediated cleavage of brevican (as determined by the abundance of the neo-epitope QEAVESE at the C-terminal of the cleaved brevican G1 domain). In untreated rats, in situ hybridization and reverse transcriptase polymerase chain reaction indicated that ADAMTS4 expression was higher than ADAMTS1 and was localized to hippocampus, temporal lobe and other areas of cortex, striatum and hypothalamus. ADAMTS4 mRNA expression in these regions correlated with the presence of the QEAVESE neo-epitope, which was concentrated in perineuronal nets and in neuropil. In rats that seized after kainate, there was a dramatic elevation in ADAMTS1 and ADAMTS4 transcript that correlated and co-localized with a robust elevation in an extractable, 55-kDa fragment of brevican in temporal lobe and hippocampus. This fragment consisted, at least in part, of the ADAMTS-cleaved epitope G1-QEAVESE. The kainate-induced elevation in this ADAMTS-cleaved fragment was localized to amygdaloid and thalamic nuclei, hippocampus, caudate-putamen, cingulate cortex, and the outer molecular layer of the dentate gyrus where it was accompanied by a robust elevation in ADAMTS1 and 4 mRNA and a 28% decline in synaptic density 5 days after kainate.Thus, complexes of extracellular matrix proteins that exist in perineuronal nets and in the neuropil are cleaved by specific matrix-degrading proteases at early time points during excitotoxic neurodegeneration. The observed ADAMTS-induced cleavage of brevican in the dentate outer molecular layer is closely associated with diminished synaptic density, and may, therefore, contribute to synaptic loss and/or reorganization in this region.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Giro Denteado/enzimologia , Giro Denteado/patologia , Desintegrinas/metabolismo , Metaloendopeptidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/patologia , Proteínas ADAM , Proteína ADAMTS1 , Proteína ADAMTS4 , Animais , Anticorpos , Brevicam , Denervação , Desintegrinas/genética , Desintegrinas/imunologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ácido Caínico/farmacologia , Lectinas Tipo C , Masculino , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Neurotoxinas/farmacologia , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno N-Endopeptidase , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Sinapses/enzimologiaRESUMO
There have been several reports on the use of beta-amyloid (Abeta ) vaccination in different mouse models of Alzheimer's disease (AD) and its effects on pathology and cognitive function. In this report, the histopathologic findings in the APP+PS1 doubly transgenic mouse were compared after three, five, or nine Abeta inoculations. The number of inoculations influenced the effects of vaccination on Congo red levels, microglia activation, and anti-Abeta antibody titers. After three inoculations, the antibody titer of transgenic mice was substantially lower than that found in nontransgenic animals. However, after nine inoculations, the levels were considerably higher in both genotypes and no longer distinguishable statistically. The number of inoculations influenced CD45 expression, an indicator of microglial activation. There was an initial upregulation, which was significant after five inoculations, but by nine inoculations, the extent of microglial activation was equivalent to that in mice given control vaccinations. Along with this increased CD45 expression, there was a correlative reduction in staining by Congo red, which stains compact plaques. When data from the mice from all groups were combined, there was a significant correlation between activation of microglia and Congo red levels, suggesting that microglia play a role in the clearance of compact plaque.
Assuntos
Peptídeos beta-Amiloides/administração & dosagem , Formação de Anticorpos , Microglia/imunologia , Emaranhados Neurofibrilares/imunologia , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Precursor de Proteína beta-Amiloide/genética , Animais , Vermelho Congo , Antígenos Comuns de Leucócito/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Emaranhados Neurofibrilares/metabolismo , Presenilina-1RESUMO
Due to the possible detrimental impact of local inflammatory responses in neurodegenerative disease, it was of interest to measure the expression of extracellular matrix-degrading enzymes, a group of proteases that are induced during an inflammatory response, in the brains of old and young animals in a model of neuronal death. Doses of kainic acid were administered that resulted in comparable hippocampal pyramidal neuron loss in young and old F344/BN hybrid rats, even though each age group received widely differing doses. Two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were differentially induced with respect to time after kainic acid in sensitive brain regions in both young and old rats. However, the elevation of MMP-9 in the temporal lobe 12 h after injection in old rats was significantly greater than that observed in young animals. These results suggest that early and late induction of MMPs may play a role in neuronal death and repair mechanisms, respectively, and that inflammatory mechanisms in the central nervous system (CNS) of old rats are exaggerated compared to young rats.
Assuntos
Encéfalo/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Fatores Etários , Animais , Encéfalo/enzimologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Células Piramidais/enzimologia , Ratos , Ratos Endogâmicos F344 , Convulsões/induzido quimicamenteRESUMO
Vaccinations with amyloid-beta peptide (A beta) can dramatically reduce amyloid deposition in a transgenic mouse model of Alzheimer's disease. To determine if the vaccinations had deleterious or beneficial functional consequences, we tested eight months of A beta vaccination in a different transgenic model for Alzheimer's disease in which mice develop learning deficits as amyloid accumulates. Here we show that vaccination with A beta protects transgenic mice from the learning and age-related memory deficits that normally occur in this mouse model for Alzheimer's disease. During testing for potential deleterious effects of the vaccine, all mice performed superbly on the radial-arm water-maze test of working memory. Later, at an age when untreated transgenic mice show memory deficits, the A beta-vaccinated transgenic mice showed cognitive performance superior to that of the control transgenic mice and, ultimately, performed as well as nontransgenic mice. The A beta-vaccinated mice also had a partial reduction in amyloid burden at the end of the study. This therapeutic approach may thus prevent and, possibly, treat Alzheimer's dementia.
Assuntos
Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/administração & dosagem , Transtornos da Memória/prevenção & controle , Fragmentos de Peptídeos/administração & dosagem , Vacinação , Envelhecimento , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Anticorpos/análise , Anticorpos/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Lobo Frontal/patologia , Humanos , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto , Transtornos da Memória/etiologia , Camundongos , Camundongos Transgênicos , Placa Amiloide/patologiaRESUMO
We have previously reported that invasiveness of mouse glioma G-26, which expresses CD44 adhesion molecule, was inhibited in vitro following treatment with anti-CD44 antibody or mouse interferon alpha/beta (MuIFN alpha/beta). Here, we evaluated whether the expression of transmembrane CD44 adhesion molecule and/or secretion of extracellular matrix metalloproteinases (MMPs) were affected when glioma cell invasion was inhibited. Flow cytometric evaluation of CD44 adhesion molecule expression in G-26 glioma using anti-CD44 antibody, confirmed that G-26 cells were CD44+. Following 3-day treatment with MuIFN alpha/beta at 8 x 10(2) or 8 x 10(3) IU/ml of glioma cells, the expression of CD44 was not significantly affected as reflected by CD44+ cell number and fluorescence intensity. The pretreatment of glioma cells for 1 day with anti-CD44 antibody resulted in a 30-60% decrease of CD44 expression. This coincided with significantly (p < 0.05) lower cell activity as judged by MTT assay for mitochondrial activity. The zymographic evaluation of MMP activity in the G-26 glioma cell culture showed a high level of the active form of MMP-2. This level of MMP-2 was decreased following 3 day treatment of G-26 glioma cells with either 8 x 10(2) or 8 x 10(3) IU/ml of MuIFN alpha/beta but only the latter concentration produced statistically significant 55% decrease. However, following a 1 day treatment of G-26 glioma cells with anti-CD44 antibody, the level of active MMP-2 form was not significantly affected. These findings indicate that while the inhibitory effect of IFN on glioma invasion was accompanied by a decreased level of the active form of MMP-2 released extracellularly, the expression of the transmembrane CD44 adhesion molecule was not affected. Conversely, anti-CD44 antibody pretreatment of G-26 glioma, which led to the inhibition of glioma invasion, resulted in decreased CD44 expression and lower cell activity but had no effect on the MMP-2.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Glioma/metabolismo , Receptores de Hialuronatos/metabolismo , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Citometria de Fluxo , Glioma/patologia , Receptores de Hialuronatos/imunologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Camundongos , Invasividade Neoplásica , Proteínas de Neoplasias/imunologiaRESUMO
Prior studies using rat primary hippocampal cultures indicated induction of matrix metalloproteinases (MMPs) in response to beta-amyloid (A beta). Hence, it was of interest to determine whether MMP activity in a human cell line is influenced by A beta. A beta, but not interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS), stimulated an active form of MMP-2 in human U87 glioblastoma cells, as well as increased the expression of the well-known activator of MMP-2, membrane-type (MT)-MMP. Activation experiments carried out with amino phenyl mercuric acetate (APMA), immunoprecipitation, as well as immunoblotting, suggest that the lower molecular weight, gelatin-degrading activity was an activated form of MMP-2. Furthermore, it was demonstrated that a synthetic furin convertase inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, decreased the production of A beta-induced active MMP-2 in U87 cells. The induction of MMP-3 by cytokines, but not by A beta, suggests that the effect of A beta on MMP-2 is selective. Although A beta stimulated tissue inhibitor of metalloproteinase-1 (TIMP-1), there was no obvious effect of A beta on TIMP-2 production in U87 cells. These results demonstrate that A beta induces an active form of MMP-2 likely by increasing the expression of MT-MMP in a human glioblastoma cell line. Active MMP-2 may degrade A beta or act on ECM components critical in neuronal survival mechanisms and possibly play a role in Alzheimer's disease (AD) neuropathology.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Neoplasias Encefálicas/enzimologia , Gelatinases/biossíntese , Glioblastoma/enzimologia , Metaloendopeptidases/biossíntese , Fragmentos de Peptídeos/farmacologia , Peptídeos beta-Amiloides/química , Neoplasias Encefálicas/patologia , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Furina , Gelatinases/antagonistas & inibidores , Glioblastoma/patologia , Humanos , Immunoblotting , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Metaloproteinase 2 da Matriz , Metaloendopeptidases/antagonistas & inibidores , Fragmentos de Peptídeos/química , Testes de Precipitina , Subtilisinas/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
Specific recognition of cell-surface molecules with other cells or extracellular matrix (ECM) is fundamental for cellular motility, reorganization, and proliferation. To carry out these actions, cells often displace space previously occupied by cells or the ECM, thus proteolysis may be required. More functionally in different model systems, integrin-mediated interaction of cells with ECM influences or directs cell growth, differentiation and survival via specific intracellular signaling pathways (1-3). Thus, the interplay between binding of integrins (and other surface molecules) with ECM and the proteolysis of ECM must be highly orchestrated. The mechanism of degradation of ECM for these physiological purposes is under stringent control, turning on only when appropriate signals are in place for a subsequent function. The importance of ECM-degrading proteases in such interactions was shown recently in transgenic animals expressing an autoactivated, ECM-degrading metalloprotease targeted to mammary epithelial cells. These epithelial cells underwent early apoptosis near the end of pregnancy. When these transgenic mice were crossed with mice overexpressing an endogenous inhibitor of the protease, early apoptosis was not observed (4). These results emphasize the importance of proteinases and their inhibitors in regulating the functions of cell-ECM interactions.
RESUMO
Indirect evidence from in vitro studies implicates a functional role for matrix metalloproteinases (MMPs) in the central nervous system (CNS), including induction of neuronal migration during development and enhancement of neurite extension. Few reports have documented the expression of these enzymes in the brain, especially after injury in vivo. The objective of this study was to determine whether MMPs are expressed in various regional areas of rat brain after administration of the neurotoxin, kainic acid. Limbic motor seizures and neuronal degeneration were induced in Sprague-Dawley rats by systemic administration of kainate (10 mg/kg). Rats were subsequently divided into convulsive and non-convulsive groups, after observing their behaviour in response to the drug. Animals were killed 6, 12, 24, 72 and 168 h (7 days) after injection of kainate. Gelatinases were extracted from various brain regions and assayed by gelatin-substrate zymography. Levels of glial fibrillary acidic protein (GFAP) in corresponding regions were measured by ELISA. In the absence of treatment, MMP-2 and MMP-9 activities were expressed differentially in various brain regions with the highest levels in the hippocampus and the lowest in the cerebellum. In areas from convulsive rats, MMP-9 activity was markedly elevated at 6 h, and reached a maximum at 12 h after injection of kainate (8.1-fold hippocampus, 7.7-fold diencephalon, 7.2-fold striatum, 5.7-fold frontal cortex, 5.5-fold cerebellum, 2.6-fold midbrain). MMP-2 activity was induced more than two-fold in the hippocampus, diencephalon and striatum, to a lesser extent in the frontal cortex and midbrain, and was unchanged in the cerebellum, 72 h after injection. Neither MMP activity was altered in any brain region derived from non-convulsive rats. Treatment with the GABAA antagonist, bicuculline, resulted in increased levels of MMP-9, 12 h after drug administration, but no change in levels of MMP-2 up to 3 days following treatment. GFAP levels were induced 3 days after kainic acid injection in brain regions where MMP-2 was elevated. Nissl staining displayed the classical, regional neurodegeneration in kainate-treated animals that exhibited seizures. No obvious degeneration was detected in kainate-treated, non-convulsive rats or bicuculline-treated animals. These data demonstrate that MMP-9 and MMP-2 are differentially expressed with respect to time after kainic acid injection, and suggest that they are regulated by convulsion and/or neurodegenerative-associated mechanisms, respectively. Although similar in catalytic activity, MMP-9 and MMP-2 may play different roles in response to kainic acid-induced seizure and neuronal degeneration.
Assuntos
Bicuculina/toxicidade , Encéfalo/enzimologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Antagonistas GABAérgicos/toxicidade , Gelatinases/biossíntese , Ácido Caínico/toxicidade , Animais , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Colagenases/biossíntese , Ensaio de Imunoadsorção Enzimática , Proteína Glial Fibrilar Ácida/biossíntese , Immunoblotting , Masculino , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Metaloendopeptidases/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Several methods have been developed for the measurement of gelatinase activity from various tissues using detergent extraction. Gelatin-affinity chromatography has been employed for the large-scale purification of gelatinases from conditioned medium obtained from cultured cells. The objective of this paper was to develop a rapid method whereby gelatinase activity could be extracted from regional brain tissues without tedious, intervening purification steps. After Triton X-100 extraction and gelatin-Sepharose 4B purification of rat brain tissue extracts, two major activities were observed on gelatin zymograms. These were identified as gelatinase A and B using co-migration with astrocyte-derived enzymes and inhibition of activity by tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). The non-ionic detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) were equally effective in extracting activities from brain tissue. Little difference in recovery was observed among 0.1, 1 and 10% concentrations of Triton X-100. The method developed here was capable of recovering gelatinase activities from rat brain tissue over a 4-10-fold range using gelatin zymography for the measurement of activity. It is possible that this method may be modified for the measurement of gelatinases in tissues such as biopsy samples of gliomas or astrocytomas or other cancers where gelatinases are thought to play a role in tumor invasion and/or metastasis.
Assuntos
Encéfalo/enzimologia , Gelatinases/análise , Animais , Astrócitos/enzimologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Cromatografia de Afinidade , Colagenases/análise , Colagenases/isolamento & purificação , Colagenases/metabolismo , Meios de Cultivo Condicionados , Detergentes , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Gelatinases/isolamento & purificação , Gelatinases/metabolismo , Ácido Caínico/farmacologia , Linfócitos/enzimologia , Masculino , Metaloproteinase 8 da Matriz , Metaloproteinase 9 da Matriz , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/isolamento & purificação , Ratos , Ratos Sprague-DawleyRESUMO
Since administration of anti-inflammatory drugs has been reported to possibly slow the progression of Alzheimer's disease, an effort was made to define effector functions induced by beta-amyloid (A beta) which may be inhibited by these drugs. Rat microglia in culture were treated with various A beta fragments and gelatinase (GLase) activity was determined using gelatin-substrate zymography; the ability of indomethacin (INDO) and dexamethasone (DEX) to inhibit A beta-stimulated GLase, specifically matrix metalloproteinase-9 (MMP-9), activity was also evaluated. A beta (1-38) and A beta (1-40) were effective in stimulating MMP-9 activity in a dose-dependent fashion. Co-treatment with INDO (50 microM) or DEX (100 nM) resulted in a 54% and 66% inhibition, respectively, of A beta (1-40)-stimulated MMP-9 production in microglia.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios/farmacologia , Colagenases/biossíntese , Dexametasona/farmacologia , Indometacina/farmacologia , Microglia/enzimologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Meios de Cultivo Condicionados , Densitometria , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Metaloproteinase 9 da Matriz , Microglia/efeitos dos fármacos , RatosRESUMO
Growing evidence supports the notion of a functional relationship between the presence of the beta-amyloid (A beta) peptide and the production of inflammatory mediators in and around neuritic plaques of Alzheimer's disease. Tissue remodeling enzymes that are critical in peripheral inflammatory responses are the matrix metalloproteinases (MMPs), enzymes produced by neurons and glia. Thus, it was of interest to determine whether A beta may alter the expression of MMPs in glial and neuronal cultures. It was demonstrated that A beta (1-40) is a potent stimulator of MMP-9 and MMP-2 activity in addition to inducing the expression of a lower molecular weight, unidentified gelatinase activity in mixed hippocampal and astrocyte cultures. Shorter fragments of A beta were less effective in stimulating the production of these enzymes. The lower molecular weight activity was observed only in response to A beta, and not after treatment with various cytokines. In addition, both cultures express MMP-3 (stromelysin-1) in response to A beta peptides. These results suggest that MMPs may play a role in the development or progression of neuritic plaques, i.e., abnormal neurite outgrowth.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Astrócitos/enzimologia , Colagenases/metabolismo , Gelatinases/metabolismo , Hipocampo/citologia , Metaloendopeptidases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Meios de Cultivo Condicionados , Feminino , Immunoblotting , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Metaloproteinase 1 da Matriz , Metaloproteinase 2 da Matriz , Metaloproteinase 3 da Matriz , Metaloproteinase 9 da Matriz , Peso Molecular , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Gravidez , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In neurodegenerative disease or after brain injury, parenchymal cells in the central nervous system are activated to produce inflammatory mediators, mainly consisting of cytokine-induced factors, in a manner similar to, but clearly different from a peripheral inflammatory response. The upregulated expression of several extracellular matrix proteins in astrocytes located surrounding a neuritic plaque in Alzheimer's disease is a good example of such a response. A family of mediators which is cytokine-induced during an inflammatory response in the periphery are the matrix metalloproteinases. Matrix metalloproteinases are calcium-requiring, zinc-containing endopeptidases that constitute a major component of the enzyme cascade responsible for degradation of extracellular matrix proteins such as collagen, proteoglycan and laminin. Little is known about the cellular source or the function of matrix metalloproteinases in the central nervous system or how their expression is regulated in brain. Thus, it was of interest to determine which factors of the so-called 'brain inflammatory response' regulate the expression of these proteases in the nervous system. To this end, we measured the expression of matrix metalloproteinases in cultured rat astrocytes and microglia after treatment with various cytokines. Interleukin-1 beta, tumor necrosis factor-alpha and lipopolysaccharide were potent stimulators of matrix metalloproteinase-2 (gelatinase A) and matrix metalloproteinase-9 (gelatinase B) in cultured rat astrocytes; the effect of each secretagogue was inhibited in the presence of glucocorticoid. Interleukin-1 beta and lipopolysaccharide also stimulated the production of matrix metalloproteinase-3 (stromelysin-1) in astrocytes. In addition, activated microglia release matrix metalloproteinase-9. The 'coactivator' of monocytic phagocytes, interferon-gamma, rather than augmenting the response to lipopolysaccharide, inhibited it. Thus, cytokines appear to be potent regulators of matrix metalloproteinase production in astrocytes and microglia. The presence of these enzymes in 'inflamed' central nervous system may suggest their involvement in the pathogenesis or progression of neurodegenerative diseases which are associated with an inflammatory component. Much remains to be learned about the potential substrates for these enzymes and the mechanism of their activation in the central nervous system.
Assuntos
Astrócitos/enzimologia , Proteínas da Matriz Extracelular/biossíntese , Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases/biossíntese , Microglia/enzimologia , Doenças do Sistema Nervoso/enzimologia , Neurônios/enzimologia , Animais , Citocinas/fisiologia , Encefalite/enzimologia , Encefalite/imunologia , Proteínas da Matriz Extracelular/genética , Humanos , Metaloendopeptidases/genética , Doenças do Sistema Nervoso/imunologia , RatosRESUMO
The objectives of this study were to characterize further the effects of phenobarbital (PB) on cytochrome P4502B1 and 2B2 (P4502B1/2) enzyme activity and immunoreactivity in rat hepatocytes and to investigate the mechanism(s) mediating the ability of interleukin-6 (IL-6) to inhibit this induction. PB caused a concentration-dependent increase in benzyloxyresorufin O-deethylase (BROD) activity with maximal effects (a 25-fold increase) at concentrations of 0.3 to 1 mM. The induction of BROD activity was linear over 24 hr of exposure. Immunoblot profiles of P4502B1/2 agreed with measurements of enzyme activity. In addition to inducing P4502B1/2, PB (0.75 mM) also increased the levels of P450 reductase by approximately 2-fold following a 24-hr exposure to PB. When IL-6 was added concomitantly with or up to 12 hr after the addition of PB, the PB induction of BROD activity and immunoreactivity was inhibited significantly. When 18 hr elapsed between the time of addition of PB and IL-6, the inhibitory effects of IL-6 were no longer apparent, suggesting that the actions of IL-6 were mediated by early events in the induction process. IL-6 did not affect the PB induction of P450 reductase. To determine whether IL-6 altered the degradation of P4502B1/2, hepatocytes were exposed to PB for 24 hr, then washed, and the loss of BROD activity and immunoreactivity following incubation with a protein synthesis inhibitor was measured. IL-6 did not alter the rate of loss of either enzyme activity or immunoreactivity, indicating that the effects of IL-6 could not be attributed to the enhanced degradation of P4502B1/2. Results suggest that the inhibition of PB-induced BROD activity by IL-6 is due to an action on early cellular and molecular events in the induction process.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Interleucina-6/farmacologia , Fenobarbital/farmacologia , Esteroide Hidroxilases/efeitos dos fármacos , Animais , Células Cultivadas , Sistema Enzimático do Citocromo P-450/biossíntese , Indução Enzimática/efeitos dos fármacos , Humanos , Masculino , NADPH-Ferri-Hemoproteína Redutase/biossíntese , NADPH-Ferri-Hemoproteína Redutase/efeitos dos fármacos , Fenobarbital/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Esteroide Hidroxilases/biossínteseRESUMO
Activated macrophages produce several matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM)-degrading enzymes, during wound healing and in other inflammatory states. In response to brain injury, brain microglia become "activated," in a way similar to peripheral tissue macrophages, a process which includes differentiation and probably invasion and proliferation. Little is known about the ECM-degrading MMPs that are secreted by microglia upon activation. Thus, it was of interest to determine whether activated microglia secrete MMPs. Conditioned media samples obtained from cultured microglia that were stimulated with various activating agents were subjected to gelatin-substrate zymography. Microglia constitutively express low levels of a 94-kDa gelatinase (GLase) activity. Treatment with LPS, zymosan, and fixed Staphylococcus aureus for 24 hr stimulated the activity of the 94-kDa GLase, 4-20-fold, in a dose-dependent manner. Addition of INF gamma inhibited the LPS-stimulated activity of MMP-9. LPS, zymosan, and fixed Staphylococcus aureus also stimulated the secretion of IL-6 from microglia in a dose-dependent manner. The 94-kDa GLase activity was Ca++ dependent, it was inhibited by 1,10-phenanthroline, and it was activated by organomercurial compounds. When immunoblots were performed using specific antisera against the 94-kDa gelatinase B (MMP-9) with untreated and LPS-stimulated conditioned medium samples, a 94-kDa immunopositive band was observed. Thus, it appears that the 94-kDa GLase is gelatinase B (MMP-9). These results indicate that activators of peripheral macrophages are potent secretagogues for the MMPs in cultured microglia. The ability of activated microglia to secrete MMPs suggests that these enzymes may play an important function in the brain parenchyma during inflammatory states.
Assuntos
Colagenases/biossíntese , Interleucina-6/biossíntese , Microglia/metabolismo , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Matriz Extracelular/enzimologia , Immunoblotting , Interferon gama/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Metaloproteinase 9 da Matriz , Microglia/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Staphylococcus aureus , ZimosanRESUMO
Under a tightly regulated expression mechanism, matrix metalloproteinases degrade extracellular matrix proteins and are thought to play a role in injury repair and tumor metastasis in peripheral tissues. Little is known about the function of matrix metalloproteinases or agents that regulate their production in adult brain; however, it has been shown that the activity of a calcium-dependent metalloproteinase is elevated in Alzheimer's hippocampus. The goals of this study were to determine whether cultured rat astrocytes produce matrix metalloproteinases and to identify agents that regulate protease activity. Enriched astrocyte cultures were prepared from brains of 1-day-old rat pups, and experiments were performed 13 days later. Gelatinase activity in astrocyte conditioned medium was determined using zymography with gelatin copolymerized with acrylamide in the gel. Under basal conditions after a 24-h incubation, rat astrocytes produce gelatinases of 58 and 66 kDa. On stimulation of astrocytes with lipopolysaccharide, interleukin-1 alpha or -beta, or tumor necrosis factor-alpha for 24 h, a dose-dependent increase in the activity of the 58- and 66-kDa gelatinases and the induction of a 94-kDa gelatinase occurred. All three astrocyte-derived proteases showed maximal activity in the presence of millimolar levels of Ca2+, their activity was inhibited in the presence of 1,10-phenanthroline, and their proenzymes were cleaved and activated after incubation with p-aminophenylmercuric acetate. Using immunoblotting, immunopositive bands at the respective molecular sizes indicated that the 58-kDa gelatinase was gelatinase A (matrix metalloproteinase 2) and the 94-kDa activity was gelatinase B (matrix metalloproteinase 9).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Astrócitos/enzimologia , Colagenases/metabolismo , Citocinas/farmacologia , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Animais , Células Cultivadas , Dexametasona/farmacologia , Matriz Extracelular/enzimologia , Immunoblotting , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-1/antagonistas & inibidoresRESUMO
Cultured rat hepatocytes have been used to compare the relative activities of cytokines to inhibit the phenobarbital (PB) or 3-methylcholanthrene (MC) induction of cytochrome P4502B1 and 2B2 (P4502B1/2) or P4501A1 and 1A2 (P4501A1/2), respectively. Recombinant cytokines tested were human interleukin-6 (IL-6), interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta, respectively), and rat gamma-interferon (INF gamma). Hepatocytes were cultured in the presence of 2 mM PB or 1 microgram MC/mL culture medium for 24 hr with or without the cytokines. Benzyloxyresorufin and ethoxyresorufin O-dealkylase (BROD and EROD, respectively) activities were determined as indices of P4502B1/2 and P4501A1/2, respectively. All cytokines produced a concentration-dependent inhibition of the PB induction of BROD activity. IL-1 beta and IL-6 were approximately equipotent with IC50 values of 1-2 U/mL, causing greater than 90% inhibition of PB induction of BROD activity at a concentration of 50 U/mL culture medium. IL-1 alpha tended to be less active. PB induction of BROD activity was also inhibited by INF gamma, but higher concentrations (62.5 to 500 U/mL culture medium) were required. All cytokines were less effective in inhibiting the MC induction of EROD activity than the PB induction of BROD activity. IL-1 beta and IL-6, at 50 U/mL culture medium, inhibited EROD induction by only 35% compared with the greater than 90% inhibitory effect on the PB induction of BROD activity. INF gamma was ineffective in inhibiting EROD activity at the concentrations studied. Western immunoblot analysis indicated that the cytokines prevented the ability of the inducers to increase the expression of P4502B1/2 and P4501A1/2 immunoreactive proteins, and this effect correlated with their inhibitory effect on induction of enzyme activity. The results suggest that inducible isoforms of cytochrome P450 differ in their susceptibility to regulation by the cytokines, and that cytokines possess differential activity to inhibit the induction of P450 isoforms, with IL-1 beta and IL-6 being the most effective.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Citocinas/farmacologia , Isoenzimas/biossíntese , Isoenzimas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Metilcolantreno/farmacologia , Fenobarbital/farmacologia , Animais , Western Blotting , Células Cultivadas , Citocromo P-450 CYP1A1 , Citocromo P-450 CYP2B1 , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Indução Enzimática/efeitos dos fármacos , Fígado/citologia , Masculino , Oxirredutases/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
To study whether hemorrhage stimulates interleukin-6 (IL-6) production in conscious rats, 30% of the total blood was withdrawn over 3 min through an indwelling venous catheter and the shedblood was reinfused 1 h later. Plasma adrenocorticotropic hormone (ACTH), corticosterone and IL-6 concentration rapidly increased. Plasma ACTH levels peaked at 10 min and corticosterone and IL-6 peaked at 60 min; all started to decrease after reinfusion. In adrenalectomized (ADX) rats with or without a corticosterone pellet implant, there was an inverse relationship between IL-6 and corticosterone concentrations, greatest in ADX rats and lowest in ADX rats in which plasma corticosterone was elevated by crushing the implanted pellet. However, the ADX rats in which plasma corticosterone was maintained at normal or slightly elevated levels showed greater IL-6 responses to hemorrhage and elevated basal plasma IL-6 levels compared to sham-operated control rats. Twenty-four hours after hemorrhage/reinfusion, ACTH, corticosterone, and IL-6 responses to i.v. injection of lipopolysaccharide (LPS) were all reduced compared to the non-hemorrhaged animals, indicating that hemorrhage impaired general host defense. Although very high plasma corticosterone concentrations markedly suppressed the IL-6 response to LPS, in ADX rats in which plasma corticosterone was maintained at slightly higher levels than normal, the reduced IL-6 response to LPS in the posthemorrhage period was not reversed, but enhanced. Thus corticosterone has biphasic effects on the IL-6 response to hemorrhage and the response to LPS during the posthemorrhage period, which has important clinical implications with regard to the optimal dose of glucocorticoid for maintaining the host defense response.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Corticosterona/sangue , Hemorragia/sangue , Interleucina-6/sangue , Lipopolissacarídeos/farmacologia , Adrenalectomia , Hormônio Adrenocorticotrópico/sangue , Hormônio Adrenocorticotrópico/metabolismo , Animais , Transfusão de Sangue , Estado de Consciência , Corticosterona/administração & dosagem , Implantes de Medicamento , Interleucina-6/metabolismo , Masculino , Neuroimunomodulação/fisiologia , Ratos , Fatores de TempoRESUMO
Interleukin-6 (IL-6) is a pleiotropic cytokine that is produced by astrocytes and microglia and may act as a trophic factor in the nervous system. These experiments were intended to identify neuroactive agents that regulate IL-6 production in primary cultured rat astrocytes. Addition of either lipopolysaccharide (LPS) or human recombinant interleukin-1 beta (IL-1 beta) to rat astrocytes in culture stimulated IL-6 secretion. However, LPS was significantly more efficacious in eliciting IL-6 production compared to IL-1 beta. Co-addition of the specific IL-1 receptor antagonist (IL-1ra) completely inhibited IL-1 beta-induced IL-6 secretion but did not affect LPS-stimulated IL-6 production during a 6 h incubation period. Two neuroactive peptides, pituitary adenylate cyclase activating polypeptide (PACAP38) and vasoactive intestinal peptide (VIP), stimulated IL-6 production either alone or in combination with IL-1 beta. PACAP38 was significantly more potent in stimulating IL-6 compared to VIP. Results from these experiments indicate that LPS is an effective inducer of IL-6 production in rat astrocytes. This effect of LPS is independent of astrocyte IL-1 production since the IL-1ra was unable to inhibit LPS-stimulated IL-6 secretion. Also, the neuropeptides PACAP38 and VIP are potential secretagogues for IL-6 secretion, and both peptides synergize with IL-1 to stimulate IL-6 secretion in rat astrocytes.