No abstract

Metallothioneins (MTs) are major zinc binding proteins in the CNS that could be involved in the control of zinc metabolism as well as in protection against oxidative stress. Mice lacking MT-I and MT-II (MT-I + II deficient) because of targeted gene inactivation were injected with kainic acid (KA), a potent convulsive agent, to examine the neurobiological importance of these MT isoforms. At 35 mg/kg KA, MT-I + II deficient male mice showed a higher number of convulsions and a longer convulsion time than control mice. Three days later, KA-injected mice showed gliosis and neuronal injury in the hippocampus. MT-I + II deficiency decreased both astrogliosis and microgliosis and potentiated neuronal injury and apoptosis as shown by terminal deoxynucleotidyl transferase-mediated in situ end labelling (TUNEL), detection of single stranded DNA (ssDNA) and by increased interleukin-1beta-converting enzyme (ICE) and caspase-3 levels. Histochemically reactive zinc in the hippocampus was increased by KA to a greater extent in MT-I + II-deficient compared with control mice. KA-induced seizures also caused increased oxidative stress, as suggested by the malondialdehyde (MDA) and protein tyrosine nitration (NITT) levels and by the expression of MT-I + II, nuclear factor-kappaB (NF-kappaB), and Cu/Zn-superoxide dismutase (Cu/Zn-SOD). MT-I + II deficiency potentiated the oxidative stress caused by KA. Both KA and MT-I + II deficiency significantly affected the expression of MT-III, granulocyte-macrophage colony stimulating factor (GM-CSF) and its receptor (GM-CSFr). The present results indicate MT-I + II as important for neuron survival during KA-induced seizures, and suggest that both impaired zinc regulation and compromised antioxidant activity contribute to the observed neuropathology of the MT-I + II-deficient mice.

Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin‐6 (IL‐6) during the inflammatory response in the brain following disruption of the blood–brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto‐parietal cortex in interleukin‐6‐deficient (IL‐6−/−) and normal (IL‐6+/+) mice. In IL‐6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and metallothionein‐I+II (MT‐I+II) were increased in these cells, while the brain‐specific MT‐III was only moderately upregulated. In IL‐6−/− mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage‐induced GM‐CSF and MT‐I+II expression were also markedly depressed compared to IL‐6+/+ mice. In contrast, MT‐III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT‐I+II but not MT‐III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL‐6+/+ and IL‐6−/− mice. The present results demonstrate that IL‐6 is crucial for the recruitment of myelo‐monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL‐6 is important for neuroprotection and the induction of GM‐CSF and MT expression. The opposing effect of IL‐6 on MT‐I+II and MT‐III levels in the damaged brain suggests MT isoform‐specific functions. GLIA 25:343–357, 1999. © 1999 Wiley‐Liss, Inc.

In order to determine the role of the neuropoietic cytokine interleukin‐6 (IL‐6) during the first 3 weeks after a focal brain injury, we examined the inflammatory response, oxidative stress and neuronal survival in normal and interleukin‐6‐deficient (knockout, IL‐6KO) mice subjected to a cortical freeze lesion. In normal mice, the brain injury was followed by reactive astrogliosis and recruitment of macrophages from 1 day postlesion (dpl), peaking at 3–10 dpl, and by 20 dpl the transient immunoreactions were decreased, and a glial scar was present. In IL‐6KO mice, the reactive astrogliosis and recruitment of macrophages were decreased throughout the experimental period. The expression of the antioxidant and anti‐apoptotic factors metallothionein I+II (MT‐I+II) was increased prominently by the freeze lesion, but this response was significantly reduced in the IL‐6 KO mice. By contrast, the expression of the antioxidants Cu/Zn‐superoxide dismutase (Cu/Zn‐SOD), Mn‐SOD, and catalase remained unaffected by the IL‐6 deficiency. The lesioned mice showed increased oxidative stress, as judged by malondialdehyde (MDA) and nitrotyrosine (NITT) levels and by formation of inducible nitric oxide synthase (iNOS). IL‐6KO mice showed higher levels of MDA, NITT, and iNOS than did normal mice. Concomitantly, in IL‐6KO mice the number of apoptotic neurons was significantly increased as judged by TUNEL staining, and regeneration of the tissue was delayed relative to normal mice. The changes in neuronal tissue damage and in brain regeneration observed in IL‐6KO mice are likely caused by the IL‐6‐dependent decrease in MT‐I+II expression, indicating IL‐6 and MT‐I+II as neuroprotective factors during brain injury. GLIA 32:271–285, 2000. © 2000 Wiley‐Liss, Inc.

6-aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes mainly in the brainstem. We have examined the role of interleukin-6 (IL-6) in this degenerative process by using transgenic mice with astrocyte-targeted IL-6 expression (GFAP-IL6 mice). This study demonstrates that transgenic IL-6 expression significantly increases the 6-AN-induced inflammatory response of reactive astrocytes, microglia/macrophages, and lymphocytes in the brainstem. Also, IL-6 induced significant increases in proinflammatory cytokines IL-1, IL-12, and tumor necrosis factor-alpha as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor-beta, neurotrophin-3, angiopoietin, vascular endothelial growth factor, and the receptor for bFGF. In accordance, angiogenesis was increased in GFAP-IL6 mice relative to controls after 6-AN. Moreover, oxidative stress and apoptotic cell death were significantly reduced by transgenic IL-6 expression. IL-6 is also a major inducer in the CNS of metallothionein I and II (MT-I+II), which were significantly increased in the GFAP-IL6 mice. MT-I+II are antioxidants and neuroregenerative factors in the CNS, so increased MT-I+II levels in GFAP-IL6 mice could contribute to the reduction of oxidative stress and cell death in these mice.

Intestinal inflammation results in disturbed intestinal motility in humans as well as in animal models. This altered function of smooth muscle cells and/or the enteric nervous system may be caused by activation of macrophages in muscularis externa and a thereby following release of cytokines and chemokines that causes influx of mononuclear cells and neutrophilic granulocytes. We subjected osteopetrotic (op/op) mice that lack certain macrophage subtypes, e.g. macrophages in the muscularis externa and +/+ mice to LPS to induce inflammatory cell influx. The densities of F4/80+, MHCII+, and myeloperoxidase+ cells were quantified using stereological sampling. In +/+ mice we found that MHCII+ cells outnumber F4/80+ cells and that LPS injection increased the density of MHCII+ cells temporarily but not that of F4/80+ cells. This indicates that an upregulation of MHCII antigen takes place and that two or more macrophage subtypes with comparable morphologies exist. Osteopetrotic mice lacked MHCII+, CD169+, and F4/80+ cells after either treatment, which indicate that these cells are CSF-1-dependent. LPS induced VCAM-1 activation of the vessels, modest influx of granulocytes, as well as an iNOS-activation in a cell type different from macrophages in both +/+ and op/op mice.

Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin-6 (IL-6) during the inflammatory response in the brain following disruption of the blood-brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto-parietal cortex in interleukin-6-deficient (IL-6-/-) and normal (IL-6+/+) mice. In IL-6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and metallothionein-I+II (MT-I+II) were increased in these cells, while the brain-specific MT-III was only moderately upregulated. In IL-6-/- mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage-induced GM-CSF and MT-I+II expression were also markedly depressed compared to IL-6+/+ mice. In contrast, MT-III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT-I+II but not MT-III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL-6+/+ and IL-6-/- mice. The present results demonstrate that IL-6 is crucial for the recruitment of myelo-monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL-6 is important for neuroprotection and the induction of GM-CSF and MT expression. The opposing effect of IL-6 on MT-I+II and MT-III levels in the damaged brain suggests MT isoform-specific functions.