Anne-Marie van Dam

Interleukin-10, interleukin-4, and transforming growth factor-β differentially regulate lipopolysaccharide-induced production of pro-inflammatory cytokines and nitric oxide in co-cultures of rat astroglial and microglial cells

The pro‐inflammatory cytokines interleukin‐1β (IL‐1β), IL‐6, tumor necrosis factor‐α (TNF‐α), and nitric oxide (NO) can be produced by activated glial cells and play a critical role in various neurological diseases. Using primary co‐cultures of rat microglial and astroglial cells, we investigated the effects of the anti‐inflammatory cytokines transforming growth factor‐β1 (TGF‐β1)/β2, IL‐4, and IL‐10 on the production of (pro‐) inflammatory mediators after stimulation of the cells with lipopolysaccharide (LPS; 0.1 μg/ml, 24 h). IL‐10 (10 and 100 ng/ml) and IL‐4 (5 and 50 U/ml) suppressed the LPS‐induced production of NO, IL‐6, and TNF‐α in a dose‐dependent manner, whereas TGF‐β1/β2 (2 and 20 ng/ml) only suppressed NO production. LPS‐induced levels of IL‐1β were suppressed by IL‐10, but not by IL‐4 and TGF‐β1/β2. Conversely, co‐incubation of the glial cells with LPS and antibodies to TGF‐β1/β2 selectively enhanced LPS‐induced NO production, whereas co‐incubation with antibody to IL‐10 enhanced LPS‐induced production of all pro‐inflammatory cytokines and NO. This finding strongly suggests that effective concentrations of TGF‐β1/β2 and IL‐10 are produced by LPS‐stimulated glial cell co‐cultures. Production of IL‐10 in these co‐cultures was confirmed by measurement of rat IL‐10 by radioimmunoassay. We conclude that anti‐inflammatory cytokines affect the production of inflammatory mediators in LPS‐activated co‐cultures of microglial and astroglial cells differentially. GLIA 30:134–142, 2000.

Apolipoprotein E Protects Against Bacterial Lipopolysaccharide-induced Lethality A NEW THERAPEUTIC APPROACH TO TREAT GRAM-NEGATIVE SEPSIS

Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present. Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines. Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo. We now report that intravenously administered LPS strongly increases the serum levels of apoE. In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents. Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice. These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia.

Detoxication enzyme inducers modify cytokine production in rat mixed glial cells

Pro-inflammatory cytokines, e.g. interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFalpha) as well as neurotoxic molecules such as nitric oxide (NO), that are produced and released by activated glial cells, play an important role in inflammation and oxidative stress occurring during Multiple Sclerosis (MS). Reduction of these processes could therefore be of therapeutic interest. Dimethylfumarate (DMF) and sulforaphane (SP) are well known for their detoxicating properties. Furthermore, they have anti-inflammatory effects as shown clinically by the treatment of inflammatory skin diseases. However, their detoxication and anti-inflammatory action on brain-derived cells is unknown. In the present study we have studied, within the same concentration range, the anti-inflammatory and detoxicating effects of DMF and SP on the production and release of mediators of inflammation and detoxication from lipopolysaccharide (LPS) activated primary co-cultures of rat microglial and astroglial cells. DMF and SP attenuated the LPS-induced production and release of TNFalpha, IL-1beta, IL-6 and NO. In addition, DMF and SP increase both mRNA level and activity of NAD(P)H:quinone reductase (NQO-1), a detoxication enzyme, as well as the cellular glutathione content. We conclude that DMF or SP simultaneously can (1) reduce mediators of inflammation and (2) enhance detoxication enzymes in LPS stimulated co-cultures of astroglial and microglial cells. This double-sided effect could potentially be of therapeutic interest.

(peri)vascular production and action of pro-inflammatory cytokines in brain pathology

In response to tissue injury or infection, the peripheral tissue macrophage induces an inflammatory response through the release of IL-1beta (interleukin-1beta) and TNFalpha (tumour necrosis factor alpha). These cytokines stimulate macrophages and endothelial cells to express chemokines and adhesion molecules that attract leucocytes into the peripheral site of injury or infection. The aims of the present review are to (i) discuss the relevance of brain (peri)vascular cells and compartments to bacterial meningitis, HIV-1-associated dementia, multiple sclerosis, ischaemic and traumatic brain injury, and Alzheimers disease, and (ii) to provide an overview of the production and action of pro-inflammatory cytokines by (peri)vascular cells in these pathologies of the CNS (central nervous system). The brain (peri)vascular compartments are highly relevant to pathologies affecting the CNS, as infections are almost exclusively blood-borne. Insults disrupt blood and energy flow to neurons, and active brain-to-blood transport mechanisms, which are the bottleneck in the clearance of unwanted molecules from the brain. Perivascular macrophages are the most reactive cell type and produce IL-1beta and TNFalpha after infection or injury to the CNS. The main cellular target for IL-1beta and TNFalpha produced in the brain (peri)vascular compartment is the endothelium, where these cytokines induce the expression of adhesion molecules and promote leucocyte infiltration. Whether this and other effects of IL-1 and TNF in the brain (peri)vascular compartments are detrimental or beneficial in neuropathology remains to be shown and requires a clear understanding of the role of these cytokines in both damaging and repair processes in the CNS.

Emerging roles of microglial activation and non-motor symptoms in Parkinson's disease

Recent data has indicated that the traditional view of Parkinsons disease (PD) as an isolated disorder of the nigrostriatal dopaminergic system alone is an oversimplification of its complex symptomatology. Aside from classical motor deficits, various non-motor symptoms including autonomic dysfunction, sensory and cognitive impairments as well as neuropsychiatric alterations and sleep disturbances are common in PD. Some of these non-motor symptoms can even antedate the motor problems. Many of them are associated with extranigral neuropathological changes, such as extensive α-synuclein pathology and also neuroinflammatory responses in specific brain regions, i.e. microglial activation, which has been implicated in several aspects of PD pathogenesis and progression. However, microglia do not represent a uniform population, but comprise a diverse group of cells with brain region-specific phenotypes that can exert beneficial or detrimental effects, depending on the local phenotype and context. Understanding how microglia can be neuroprotective in one brain region, while promoting neurotoxicity in another, will improve our understanding of the role of microglia in neurodegeneration in general, and of their role in PD pathology in particular. Since neuroinflammatory responses are in principle modifiable, such approaches could help to identify new targets or adjunctive therapies for the full spectrum of PD-related symptoms.

Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4+ T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8+ T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4+ T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.

Transglutaminases and Transglutaminase-Catalyzed Cross-Links Colocalize with the Pathological Lesions in Alzheimer's Disease Brain

Alzheimers disease (AD) is characterized by pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self‐aggregated proteins amyloid beta (Aβ) and tau, respectively. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular covalent cross‐links. Both Aβ and tau are substrates for TG cross‐linking activity, which links TGs to the aggregation process of both proteins in AD brain. The aim of this study was to investigate the association of transglutaminase 1 (TG1), transglutaminase 2 (TG2) and TG‐catalyzed cross‐links with the pathological lesions of AD using immunohistochemistry. We observed immunoreactivity for TG1, TG2 and TG‐catalyzed cross‐links in NFTs. In addition, both TG2 and TG‐catalyzed cross‐links colocalized with Aβ in SPs. Furthermore, both TG2 and TG‐catalyzed cross‐links were associated with CAA. We conclude that these TGs demonstrate cross‐linking activity in AD lesions, which suggests that both TG1 and TG2 are likely involved in the protein aggregation processes underlying the formation of SPs, CAA and/or NFTs in AD brain.

Alteration of the extracellular matrix interferes with raft association of neurofascin in oligodendrocytes. Potential significance for multiple sclerosis

Remyelination, as potential treatment for demyelinating diseases like multiple sclerosis (MS), requires the formation of new axoglial interactions by differentiating oligodendrocyte progenitor cells. Since the oligodendrocyte-specific isoform of neurofascin, NF155 (neurofascin isoform of 155 kDa), may be important for establishing axoglial interactions, we analyzed whether its expression is changed in chronic relapsing experimental allergic encephalomyelinitis (EAE). Although overall expression of NF155 was not changed, immunoreactivity of NF155 was dramatically increased in EAE lesion sites indicating an enhanced accessibility of NF155 epitopes. As this may be due to infiltrating plasma components, for example, fibronectin, we analyzed whether fibronectin affects the intracellular distribution and membrane association of NF155 in primary oligodendrocytes. In oligodendrocytes cultivated on polylysine, NF155 was recruited to membrane microdomains (rafts) during development and became enriched in secondary and tertiary processes. Fibronectin perturbed localization and raft association of NF155 and inhibited the morphological differentiation of oligodendrocytes. Consistent with the in vitro data, raft association of NF155 was reduced in spinal cord of EAE rats. The results suggest that the association of NF155 to microdomains in the oligodendrocyte membrane is required for its participation in intermolecular interactions, which are important for myelination and/or myelin integrity.

Tissue transglutaminase: A novel pharmacological target in preventing toxic protein aggregation in neurodegenerative diseases

Alzheimers disease, Parkinsons disease and Huntingtons disease are neurodegenerative diseases, characterized by the accumulation and deposition of neurotoxic protein aggregates. The capacity of specific proteins to self-interact and form neurotoxic aggregates seems to be a common underlying mechanism leading to pathology in these neurodegenerative diseases. This process might be initiated and/or accelerated by proteins that interact with these aggregating proteins. The transglutaminase (TG) family of proteins are calcium-dependent enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine isopeptide bonds, which can result in both intra- and intermolecular cross-links. Intramolecular cross-links might modify self-interacting proteins, and make them more prone to aggregate. In addition, intermolecular cross-links could link self-aggregating proteins and thereby initiate and/or stimulate the aggregation process. So far, increased levels and activity of tissue transglutaminase (tTG), the best characterized member of the TG family, have been observed in many neurodegenerative diseases, and the self-interacting proteins, characteristic of Alzheimers disease, Parkinsons disease and Huntingtons disease, are known substrates of tTG. Here, we focus on the role of tTG in the initiation of the aggregation process of self-interacting proteins in these diseases, and promote the notion that tTG might be an attractive novel target for treatment of neurodegenerative diseases.

IL-1β immunoreactive neurons in the human hypothalamus: reduced numbers in multiple sclerosis

Corticotropin-releasing hormone (CRH)-containing neurons in the paraventricular nucleus (PVN) in the hypothalamus of multiple sclerosis (MS) patients are hyperactivated. Since interleukin-1 (IL-1)beta is a powerful activator of CRH neurons, its immunohistochemical expression was studied in the postmortem hypothalamus of MS patients (n=11) and matched controls (n=11). Hypothalamic tissue of 10/11 MS patients showed demyelinating lesions that in many cases contained IL-1beta-immunoreactive (ir) macrophages and glial cells. In control subjects IL-1beta-ir was only sporadically found in glial cells. Interestingly, abundant IL-1beta-ir was also present in hypothalamic neurons. Neuronal IL-1beta co-localised with oxytocin and not with vasopressin or CRH. IL-1beta clearly yielded a less intense staining in neurons and numbers of IL-1-ir neurons in the PVN were 4.5-fold reduced in MS. We suggest that IL-1beta produced by activated glial cells in the hypothalamus of MS patients may contribute to the activation of the hypothalamic CRH neurons, while reduced expression of neuronal IL-1beta in MS patients may have consequences for neuroendocrine, behavioural or autonomic functioning.