Amalia Molinero

Strongly compromised inflammatory response to brain injury in interleukin‐6‐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.

Metallothionein-1+2 protect the CNS after a focal brain injury

We have evaluated the physiological relevance of metallothionein-1+2 (MT-1+2) in the CNS following damage caused by a focal cryolesion onto the cortex. In comparison to normal mice, transgenic mice overexpressing the MT-1 isoform (TgMTI* mice) showed a significant decrease of the number of activated microglia/macrophage and of CD3+ T lymphocytes in the area surrounding the lesion, while astrocytosis was increased. The TgMTI* mice showed a diminished peripheral macrophage but not CD3 T cell response to the cryolesion. This altered inflammatory response produced a decreased expression of the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha and an increased expression of the growth factors bFGF, TGFbeta1, and VEGF in the TgMTI* mice relative to control mice, which might be related to the increased angiogenesis and regeneration of the parenchyma of the former mice. The overexpression of MT-1 dramatically reduced the cryolesion-induced oxidative stress and neuronal apoptosis. Remarkably, these effects were also obtained by the intraperitoneal administration of MT-2 to both normal and MT-1+2 knock-out mice. These results fully support the notion that MT-1+2 are essential in the CNS for coping with focal brain injury and suggest a potential therapeutic use of these proteins.

Enhanced seizures and hippocampal neurodegeneration following kainic acid‐induced seizures in metallothionein‐I + II‐deficient mice

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‐1β‐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‐κB (NF‐κB), 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.

Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures

The role of interleukin-6 in hippocampal tissue damage after injection with kainic acid, a rigid glutamate analogue inducing epileptic seizures, has been studied by means of interleukin-6 null mice. At 35mg/kg, kainic acid induced convulsions in both control (75%) and interleukin-6 null (100%) mice, and caused a significant mortality (62%) only in the latter mice, indicating that interleukin-6 deficiency increased the susceptibility to kainic acid-induced brain damage. To compare the histopathological damage caused to the brain, control and interleukin-6 null mice were administered 8.75mg/kg kainic acid and were killed six days later. Morphological damage to the hippocampal field CA1-CA3 was seen after kainic acid treatment. Reactive astrogliosis and microgliosis were prominent in kainic acid-injected normal mice hippocampus, and clear signs of increased oxidative stress were evident. Thus, the immunoreactivity for inducible nitric oxide synthase, peroxynitrite-induced nitration of proteins and byproducts of fatty acid peroxidation were dramatically increased, as was that for metallothionein I+II, Mn-superoxide dismutase and Cu/Zn-superoxide dismutase. In accordance, a significant neuronal apoptosis was caused by kainic acid, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and interleukin-1beta converting enzyme/Caspase-1 stainings. In kainic acid-injected interleukin-6 null mice, reactive astrogliosis and microgliosis were reduced, while morphological hippocampal damage, oxidative stress and apoptotic neuronal death were increased. Since metallothionein-I+II levels were lower, and those of inducible nitric oxide synthase higher, these concomitant changes are likely to contribute to the observed increased oxidative stress and neuronal death in the interleukin-6 null mice. The present results demonstrate that interleukin-6 deficiency increases neuronal injury and impairs the inflammatory response after kainic acid-induced seizures.

Astrocyte-targeted expression of IL-6 protects the CNSagainst a focal brain injury

The effect of CNS-targeted IL-6 gene expression has been thoroughly investigated in the otherwise nonperturbed brain but not following brain injury. Here we examined the impact of astrocyte-targeted IL-6 production in a traumatic brain injury (cryolesion) model using GFAP-IL6 transgenic mice. This study demonstrated that transgenic IL-6 production significantly increased wound healing following the cryolesion. Thus, at 20 days postlesion (dpl) the GFAP-IL6 mice showed almost complete wound healing compared to litter mate nontransgenic controls. It seems likely that a reduced inflammatory response in the long term could be responsible for this IL-6-related effect. Thus, while in the acute phase following cryolesion (1-6 dpl) the recruitment of macrophages and T lymphocytes was higher in GFAP-IL6 mice, at 10-20 dpl it was significantly reduced compared to controls. Reactive astrogliosis was also significantly increased up to but not including 20 dpl in the GFAP-IL6 mice. Oxidative stress as well as apoptotic cell death was significantly decreased throughout the time period studied in the GFAP-IL6 mice compared to controls. This could be linked to the altered inflammatory response as well as to the transgenic IL-6-induced increase of the antioxidant, neuroprotective proteins metallothionein-I + II. These results indicate that although in the brain the chronic astrocyte-targeted expression of IL-6 spontaneously induces an inflammatory response causing significant damage, during an acute neuropathological insult such as following traumatic injury, a clear neuroprotective role is evident.

Metallothionein reduces central nervous system inflammation, neurodegeneration, and cell death following kainic acid-induced epileptic seizures

We examined metallothionein (MT)‐induced neuroprotection during kainic acid (KA)‐induced excitotoxicity by studying transgenic mice with MT‐I overexpression (TgMT mice). KA induces epileptic seizures and hippocampal excitotoxicity, followed by inflammation and delayed brain damage. We show for the first time that even though TgMT mice were more susceptible to KA, the cerebral MT‐I overexpression decreases the hippocampal inflammation and delayed neuronal degeneration and cell death as measured 3 days after KA administration. Hence, the proinflammatory responses of microglia/macrophages and lymphocytes and their expression of interleukin (IL)‐1, IL‐6, IL‐12, tumor necrosis factor‐α and matrix metalloproteinases (MMP‐3, MMP‐9) were significantly reduced in hippocampi of TgMT mice relative to wild‐type mice. Also by 3 days after KA, the TgMT mice showed significantly less delayed damage, such as oxidative stress (formation of nitrotyrosine, malondialdehyde, and 8‐oxoguanine), neurodegeneration (neuronal accumulation of abnormal proteins), and apoptotic cell death (judged by TUNEL and activated caspase‐3). This reduced bystander damage in TgMT mice could be due to antiinflammatory and antioxidant actions of MT‐I but also to direct MT‐I effects on the neurons, in that significant extracellular MT presence was detected. Furthermore, MT‐I overexpression stimulated astroglia and increased immunostaining of antiinflammatory IL‐10, growth factors, and neurotrophins (basic fibroblastic growth factor, transforming growth factor‐β, nerve growth factor, brain‐derived neurotrophic factor, glial‐derived neurotrophic factor) in hippocampus. Accordingly, MT‐I has different functions that likely contribute to the increased neuron survival and improved CNS condition of TgMT mice. The data presented here add new insight into MT‐induced neuroprotection and indicate that MT‐I therapy could be used against neurological disorders.

Altered central nervous system cytokine-growth factor expression profiles and angiogenesis in metallothionein-I+II deficient mice.

To study the importance of metallothionein-I and -II (MT-I+II) for brain inflammation and regeneration, the authors examined normal and MT-I+II knock-out (MT-KO) mice subjected to a cortical freeze injury. Normal mice showed profound neurodegeneration, inflammation, and gliosis around the injury, which was repaired by 20 days postlesion (dpl). However, in MT-KO mice the lesion-associated inflammation was still present as late as 90 dpl. Scanning electron microscopy demonstrated that the number of capillaries was lower, and ultrastructural preservation of the lesioned parenchyma was poorer in MT-KO mice, suggesting an altered angiogenesis. To gain insight into the mechanisms involved, a number of cytokines and growth factors were evaluated. The number of cells expressing the proinflammatory cytokines IL-1β, IL-6, and TNF-α was higher in MT-KO mice than in normal mice, which was confirmed by RNase protection analysis, whereas the number of cells expressing the growth factors bFGF, TGFβ1, VEGF, and NT-3 was lower. Increased expression of proinflammatory cytokines could be involved in the sustained recruitment of CD-14+ and CD-34+ inflammatory cells and their altered functions observed in MT-KO mice. Decreases in trophic factors bFGF, TGFβ1, and VEGF could mediate the decreased angiogenesis and regeneration observed in MT-KO mice after the freeze lesion. A role for MT-I+II in angiogenesis was also observed in transgenic mice expressing IL-6 under the control of the promoter of glial fibrillary acidic protein gene (GFAP-IL6 mice) because MT-I+II deficiency dramatically decreased the IL-6-induced angiogenesis of the GFAP-IL6 mice. In situ hybridization analysis indicated that the MT-III expression was not altered by MT-I+II deficiency. These results suggest that the MT-I+II isoforms have major regulatory functions in the brain inflammatory response to injury, especially in the angiogenesis process.

Transgenic expression of interleukin 6 in the central nervous system regulates brain metallothionein-I and -III expression in mice.

The metallothionein (MT) gene family consists of several members (MT-I-IV) that are tightly regulated during development. MT-I and MT-II are expressed in many tissues, including the brain, whereas MT-III is expressed mainly in the central nervous system. However, the physiological roles of these isoforms in the brain and their regulation are poorly characterized. In this report, we have studied the putative role of IL-6 in the regulation of brain MT. The present results demonstrated that transgenic mice expressing IL-6 under the regulatory control of the glial fibrillary acidic protein gene promoter (GFAP-IL6 mice), and which develop chronic progressive neurodegenerative disease, show significantly increased MT-I + II protein levels in specific brain areas. Thus, the MT-I + II levels of 1- and 3-month-old GFAP-IL6 mice (G16 and/or G36 lines) were not altered in hippocampus but they were elevated in the cerebellum (highest induction), medulla plus pons, hypothalamus and remaining brain (lowest induction). The effect of the transgenic expression of IL-6 was more dramatic for MT-I + II protein than for MT-I mRNA levels, with the latter only marginally elevated in the G16 line at 3 months but not at 6 months of age where there was a tendency to decreased levels. Brain MT-I mRNA levels also tended to decrease in the higher expressor G36 line in 3-month-old mice despite the strongly elevated MT-I + II protein levels at this age. Therefore, in addition to increasing MT gene transcription, these results suggest a post-transcriptional effect of IL-6 or of a IL-6-dependent factor, in this chronic situation. The up-regulated brain MT-I + II protein levels in the GFAP-IL6 mice was comparable to the expression of the acute-phase response gene EB22/5, suggesting that these MT isoforms could be considered acute-phase response proteins in the brain. Brain MT-III mRNA levels followed a somewhat similar pattern that those of MT-I mRNA but the decreasing effect of IL-6 transgene production with age was more dramatic for the former, suggesting differential regulation of these MT isoforms by IL-6. The results indicate that these transgenic mice might be a valuable tool for further examining the role of the MT isoforms in brain physiology and pathobiology.

Astrocyte-targeted expression of interleukin-6 protects the central nervous system during neuroglial degeneration induced by 6-aminonicotinamide

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‐α as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor‐β, 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.

Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury

Tumor necrosis factor‐α (TNF‐α) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF‐α can induce both apoptosis and necrosis via intracellular signaling. This cytokine exerts its functions via interaction with two receptors: type‐1 receptor (TNFR1) and type‐2 receptor (TNFR2). In this work, the inflammatory response after a freeze injury (cryolesion) in the cortex was studied in wild‐type (WT) animals and in mice lacking TNFR1 (TNFR1 KO) or TNFR2 (TNFR2 KO). Lack of TNFR1, but not of TNFR2, significantly decreased the inflammatory response and tissue damage elicited by the cryolesion at both 3 and 7 days postlesion, with decreased gliosis, lower IL‐1β immunostaining, and a reduction of apoptosis markers. Cryolesion produced a clear induction of the proinflammatory cytokines interleukin (IL)‐1α, IL‐1β, IL‐6, and TNF‐α; this induction was significantly lower in the TNFR1 KO mice. Host response genes (ICAM‐1, A20, EB22/5, and GFAP) were also induced by the cryolesion, but to a lesser extent in TNFR1 KO mice. Lack of TNFR1 signaling also affected the expression of apoptosis/cell death‐related genes (Fas, Rip, p53), matrix metalloproteinases (MMP3, MMP9, MMP12), and their inhibitors (TIMP1), suggesting a role of TNFR1 in extracellular matrix remodeling after injury. However, GDNF, NGF, and BDNF expression were not affected by TNFR1 deficiency. Overall, these results suggest that TNFR1 is involved in the early establishment of the inflammatory response and that its deficiency causes a decreased inflammatory response and tissue damage following brain injury.