Joaquin Hernandez

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.

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.

IL-6 deficiency leads to increased emotionality in mice: evidence in transgenic mice carrying a null mutation for IL-6.

The role of interleukin-6 (IL-6) on emotional behavior was studied in two experiments using transgenic mice carrying a null mutation for IL-6 (IL-6(-/-)). In the first experiment, IL-6(-/-) mice were compared with the two wild-type strains contributing to the genetic background of the transgenic mice, namely C57BL/6J and 129/SvJ, as well as with the F2 offspring of C57BL/6J x 129/SvJ mice. The two parental strains differed substantially in terms of emotional reactivity, suggesting that the F2 offspring were more appropriated for analyzing the effect of the null mutation. IL-6(-/-) mice showed lower levels of ambulation in the holeboard, and lower levels of exploration of the open arms of the plus-maze, than the wild-type F2 C57BL/6J x 129/SvJ mice. In the second experiment, IL-6(-/-) mice were backcrossed for 10 generations to C57BL/6J mice to decrease the uncertainty of the effect of the genetic background, and when compared with wild-type C57BL/6J mice in the holeboard and the plus-maze, the same results were obtained. Therefore, IL-6(-/-) mice seem to be more emotional than their appropriate controls, suggesting that the major cytokine IL-6 is involved in the control of emotionality.

Impaired inflammatory response to glial cell death in genetically metallothionein-I- and -II-deficient mice.

Metallothionein I+II (MT-I+II) are acute-phase proteins which are upregulated during pathological conditions in the brain. To elucidate the neuropathological importance of MT-I+II, we have examined MT-I+II-deficient mice following ip injection with 6-aminonicotinamide (6-AN). 6-AN is antimetabolic and toxic for bone marrow cells and grey matter astrocytes. In MT+/+ mice, injection with 6-AN resulted in breakdown of the blood-brain barrier (BBB) and absence of GFAP-positive astrocytes in specific grey matter areas of the brain stem. Reactive astrocytosis encircled the damaged grey matter areas, which were heavily infiltrated by microglia/macrophages. The recruitment of hematogenous macrophages was accompanied by leakage of the BBB. The immunoreactivity (ir) of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and the receptor for GM-CSF (GM-CSFrec) was significantly upregulated in astrocytes and microglia/macrophages, respectively. MT-I+IIir was also clearly increased in astrocytes surrounding the damaged areas, while that of the CNS-specific MT isoform, MT-III, was mildly increased in both astrocytes and microglia/macrophages. In MT-/- mice injected with 6-AN, the BBB remained almost intact. The damage to specific grey matter areas was similar to that observed in MT+/+ mice, but reactive astrocytosis, microglia/macrophages infiltration, and GM-CSFir and GM-CSFrecir were clearly reduced in MT-/- mice. In contrast, MT-IIIir was dramatically increased in MT-/- mice. Total zinc decreased and histochemically detectable zinc increased in the brain stem after 6-AN similarly in MT+/+ and MT-/- mice. Bone marrow myeloid monocytes and macrophages were increased as a reaction to 6-AN only in MT+/+ mice. The results demonstrate that the capability of MT-/- mice to mount a normal inflammatory response in the brain is severely attenuated, at least in part because of 6-AN-induced bone marrow affectation, involving MT-I+II for the first time as major factors during CNS tissue damage.

Effect of Stress on Mouse and Rat Brain Metallothionein I and III mRNA Levels

The effect of immobilization stress on brain and liver metallothionein (MT) mRNA levels has been studied in mice and rats. Stress increased brain and liver MT-I mRNA levels in mice in a time-dependent manner, in agreement with the MT-I+II protein levels, suggesting an increased gene transcription during stress. In contrast, the brain-specific isoform, MT-III, tended to decrease during stress. In selected brain areas of rats, the overall tendency for both MT-I and MT-III mRNA levels was to be transiently decreased by stress in hippocampus, and increased in hypothalamus, cerebellum and the remaining brain tissues; adrenalectomy significantly affected MT mRNA levels either in basal conditions or during stress, with very different temporal patterns of response depending on the brain area studied. These results suggest that glucocorticoids could be involved in MT-I but also MT-III regulation. In both rats and mice, the subtle response to stress observed in the brain contrasts with the robust response in the liver, suggesting that the factors involved in MT regulation in both tissues differ substantially. In primary cultures enriched in astrocytes or neurons, MT-III mRNA was clearly detected by Northern blotting in both cases, suggesting that it is expressed in both types of cells. Dexamethasone appeared to decrease MT-III mRNA levels in cultured neurons and to increase them in astrocytes, which indicates that glucocorticoids have a different role in MT-III regulation in both cell types.

Metallothionein-I overexpression alters brain inflammation and stimulates brain repair in transgenic mice with astrocyte-targeted interleukin-6 expression

Transgenic expression of IL‐6 in the CNS under the control of the GFAP gene promoter, glial fibrillary acidic protein‐interleukin‐6 (GFAP‐IL‐6) mice, raises an inflammatory response and causes significant brain damage. However, the results obtained in the GFAP‐IL‐6 mice after a traumatic brain injury, such as a cryolesion, demonstrate a neuroprotective role of IL‐6. Thus, the GFAP‐IL‐6 mice showed faster tissue repair and decreased oxidative stress and apoptosis compared with control litter‐mate mice. The neuroprotective factors metallothionein‐I+II (MT‐I+II) were upregulated by the cryolesion to a higher extent in the GFAP‐IL‐6 mice, suggesting that they could be related to the neuroprotection afforded by the transgenic expression of IL‐6. To examine this possibility, we have crossed GFAP‐IL‐6 mice with transgenic mice overexpressing MT‐I (TgMT), producing double transgenic GFAP‐IL‐6 TgMT mice. The results obtained after cryolesion in GFAP‐IL‐6 TgMT mice, as well as in TgMT mice, consistently supported the idea that the increased MT‐I+II levels observed in GFAP‐IL‐6 mice are a fundamental and important mechanism for coping with brain damage. Accordingly, MT‐I overexpression regulated the inflammatory response, decreased oxidative stress and apoptosis significantly, and increased brain tissue repair in comparison with either GFAP‐IL‐6 or control litter‐mate mice. Overall, the results demonstrate that brain MT‐I+II proteins are fundamental neuroprotective factors. GLIA 42:287–306, 2003.

Effect of nitric oxide synthesis inhibition on mouse liver and brain metallothionein expression.

The role of nitric oxide (NO) production on metallothionein (MT) regulation in the liver and the brain has been studied in mice by means of the administration of nitric oxide synthase (NOS) inhibitors. Mice injected with either the arginine analog NG-monomethyl-L-arginine (L-NMMA) or the heme binding compound 7-nitro indazole (7-NI) showed consistently increased liver MT-I mRNA and MT-I + II total protein levels, suggesting that NO is involved in the hepatic MT regulation. In agreement with the liver results, in situ hybridization analysis demonstrated a significant upregulation of the brain MT-I isoform in areas such as the cerebrum cortex, neuronal CA1-CA3 layers and dentate gyrus of the hippocampus, and Purkinje cell layer of the cerebellum, in 7-NI treated mice. The same trend was observed for the brain specific isoform, MT-III, but to a much lower extent. The effect of NOS inhibition was also evaluated in a MT-inducing condition, namely during immobilization stress. In both the liver and the brain, stress upregulated the MT-I isoform, and 7-NI significantly reduced or even blunted the MT-I response to stress, suggesting a mediating role of NO on MT-I regulation during stress. Stress also increased the MT-III mRNA levels in some brain areas, an effect blunted by the concomitant administration of 7-NI, which in some areas even decreased MT-III mRNA levels below the saline injected mice. Results in primary culture of neurons and astrocytes demonstrate significant effects of the NOS inhibitors in some experimental conditions. The present results suggest that NO may have some role on MT regulation in both the liver and the brain.

Role of Glucocorticoids on Rat Brain Metallothionein-I and-III Response to Stress

The metallothionein (MT) gene family consists of four members (MT-I through -IV) that are tightly regulated during development. Whereas MT-I and MT-II are widely expressed isoforms, MT-III has been found to be mainly expressed in the central nervous system in adult animals, and is the only isoform that inhibits survival and neurite formation of cortical neurons in vitro. A number of models of brain injury have been shown to affect MT-III mRNA levels, which has been suggested to be related to the putative neurotrophic role of this protein. However, a stress response will presumably be associated to the brain injury which could, in turn, drive MT-III regulation. In the present report the effect of a classical stress model, immobilization stress, on brain MT regulation has been studied in rats. MT-I+II protein levels were measured by radioimmunoassay in up to eight brain areas and, as expected, it was found that stress increased selectively MT-I+II levels. Adrenalectomy (ADX) had a general decreasing effect on basal MT-I+II levels; however, ADX blunted the MT-I+II response to stress in cerebellum and presumably in frontal cortex and medulla plus pons but not in the hypothalamus. MT-I mRNA measurements were in accordance with the MT-I+II protein levels in the brain areas studied. In contrast to MT-I mRNA, MT-III mRNA levels of brain cortex tended to decrease during stress, although this effect was not statistically significant. ADX also tended to decrease basal MT-III mRNA levels. Northern blot assays of pooled mRNAs suggested similar differential regulation of these two brain MT isoforms in the cerebellum. These results indicate that glucocorticoids mediate brain MT-I+II response to stress in some but not all brain areas, that a role of these hormones is likely also for MT-III, and that the regulation of MT isoforms differs substantially in the brain.

Effect of stress, adrenalectomy and changes in glutathione metabolism on rat kidney metallothionein content: comparison with liver metallothionein

Eighteen hours of immobilization stress, accompanied by food and water deprivation, increased liver metallothionein (MT) but decreased kidney MT levels. Food and water deprivation alone had a significant effect only on liver MT levels. In contrast, stress and food and water deprivation increased both liver and kidney lipid peroxidation levels, indicating that the relationship between MT and lipid peroxidation levels (an index of free radical production) is unclear. Adrenalectomy increased both liver and kidney MT levels in basal conditions, whereas the administration of corticosterone in the drinking water completely reversed the effect of adrenalectomy, indicating an inhibitory role of glucocorticoids on MT regulation in both tissues. Changes in glutathione (GSH) metabolism produced significant effects on kidney MT levels. Thus, the administration of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased kidney GSH and increased kidney MT content, suggesting that increased cysteine pools because of decreased GSH synthesis might increase kidney MT levels through an undetermined mechanism as it appears to be the case in the liver. However, attempts to increase kidney MT levels by the administration of cysteine or GSH were unsuccesful, in contrast to what is known for the liver. The present results suggest that there are similarities but also substantial differences between liver and kidney MT regulation in these experimental conditions.