Mercedes Giralt

Interleukin-6 Regulation of AMP-Activated Protein Kinase: Potential Role in the Systemic Response to Exercise and Prevention of the Metabolic Syndrome

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined.

Impaired inflammatory response and increased oxidative stress and neurodegeneration after brain injury in interleukin-6-deficient mice.

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.

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.

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.

Interleukin-6 receptor expression in contracting human skeletal muscle: regulating role of IL-6

Contracting muscle fibers produce and release IL‐6, and plasma levels of this cytokine are markedly elevated in response to physical exercise. We recently showed autocrine regulation of IL‐6 in human skeletal muscle in vivo and hypothesized that this may involve up‐regulation of the IL‐6 receptor. Therefore, we investigated IL‐6 receptor regulation in response to exercise and IL‐6 infusion in humans. Furthermore, using IL‐6‐deficient mice, we investigated the role of IL‐6 in the IL‐6 receptor response to exercise. Human skeletal muscle biopsies were obtained in relation to: 3 h of bicycle exercise and rest (n=6+5), or recombinant human IL‐6 infusion (rhIL‐6) or saline infusion (n=6+6). We further obtained skeletal muscle samples from IL‐6 knockout (KO) mice and wild‐type C57/BL‐6 mice in response to a 1‐h bout of exercise. In exercising human skeletal muscle, IL‐6 receptor mRNA increased sixfold with a peak at 6 h postexercise. Detection of the IL‐6 receptor protein by immunohistochemistry revealed a pronounced staining following exercise that was primarily located at the cell membrane of the muscle fibers, whereas muscle gp130 expression and plasma levels of soluble IL‐6 receptor were unaffected. Infusion of rhIL‐6 to humans had no effect on the mRNA level of the IL‐6 receptor, whereas there was an increase at the protein level. IL‐6 receptor mRNA increased similarly in muscle of both IL‐6 KO mice and wild‐type mice in response to exercise. In conclusion, exercise increases IL‐6 receptor production in human skeletal muscle. This effect is most prominent 6 h after the end of the exercise bout, suggesting a postexercise‐sensitizing mechanism to IL‐6 when plasma IL‐6 is concomitantly low. Exercise‐induced increases in IL‐6 receptor mRNA most likely occurs via an IL‐6 independent mechanism as shown in IL‐6 KO mice and the human rhIL‐6 infusion study, whereas IL‐6 receptor protein levels are responsive to elevated plasma IL‐6 levels.

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.

Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain

The knowledge of brain metallothionein (MT) regulation and especially of MT presence in specific cell types is scarce. Therefore, the effect of several well-known MT inducers, measured by radioimmunoassays using antibodies that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been studied in primary cultures of neurons or astrocytes obtained from rat cerebrum. MT-I levels in glial cells were about ten times higher than those in neuronal cells (538 +/- 194 vs. 49 +/- 16 pg MT-I/micrograms protein, mean +/- S.D. from three separate cell preparations). Increasing the concentration of Zn in the bovine serum albumin (BSA)-containing culture medium up to 50 microM significantly increased MT-I levels by up to 3.5-fold in neurons and 2.5-fold in astrocytes. In contrast, Cu up to 50 microM increased MT-I levels in a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1.5-fold), the maximum effect occurring at 5 microM Cu. In general, the combination of Zn and Cu further increased MT-I levels. The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml. Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration-dependent manner. Endotoxin, IL-1 and IL-6 did not have a significant effect on glial MT levels at the concentrations studied. The administration of dexamethasone to rats increased MT-I levels in non-frontal cortex, cerebellum, pons+medulla, midbrain and hippocampus, but not in hypothalamus, frontal cortex and striatum. Endotoxin increased liver but not brain MT-I levels. Immunocytochemical studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostaining was always nuclear in glial cells, whereas in neurons it was nuclear in the cerebral cortex, hippocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purkinje cells in the cerebellum, hypothalamic nuclei and gigantocellular reticular nucleus in the brain stem. Meninges, choroidal plexus, ependymal and endothelial cells were also MT-immunoreactive.

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.