Steven M. LeVine

The Role of Iron in the Pathogenesis of Experimental Allergic Encephalomyelitis and Multiple Sclerosis

Abstract: Multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE), are autoimmune disorders resulting in demyelination in the central nervous system (CNS). Pathologically, the blood‐brain barrier becomes damaged, macrophages and T cells enter into the CNS, oligodendrocytes and myelin are destroyed, astrocytes and microglia undergo gliosis, and axons become transected. Data from several biochemical and pharmacological studies indicate that free radicals participate in the pathogenesis of EAE, and iron has been implicated as the catalyst leading to their formation. The primary focus of this article is the examination of the role of iron in the pathogenesis of MS and EAE. Particular attention will be paid to the role and distribution of iron and proteins involved with iron metabolism (e.g., transferrin, ferritin, heme oxygenase‐1, etc.) in normal and disease states of myelin. Furthermore, therapeutic interventions aimed at iron, iron‐binding proteins, and substrates or products of iron‐catalyzed reactions leading to free radical production will be discussed.

Cutting Edge: Resistance to Bacillus anthracis Infection Mediated by a Lethal Toxin Sensitive Allele of Nalp1b/Nlrp1b

Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase-1–dependent pyroptotic death in macrophages and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports, macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to infection by B. anthracis. Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.

Pathogenic implications of iron accumulation in multiple sclerosis

J. Neurochem. (2012) 120, 7–25.

Ferritin, transferrin and iron concentrations in the cerebrospinal fluid of multiple sclerosis patients

The concentrations of ferritin, transferrin and iron were measured in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) and control patients. Ferritin levels were significantly elevated in the CSF of chronic progressive active MS patients (4.71+/-0.54 ng/ml) compared to levels in normal individuals (3.07+/-0.17 ng/ml). MS patients with active or stable relapsing-remitting disease had ferritin levels that were comparable to those found in normal individuals. There were no significant differences in transferrin or iron levels in the CSF between MS and normal individuals. Both ferritin and transferrin levels were elevated in patients that had high CSF IgG values but not in patients with a high IgG index. Since ferritin binds iron, the increase of CSF ferritin levels in chronic progressive MS patients could be a defense mechanism to protect against iron induced oxidative injury. Ferritin levels could be a laboratory measure that helps to distinguish between chronic progressive and relapsing-remitting MS.

Statistical analysis of data from studies on experimental autoimmune encephalomyelitis

Research in multiple sclerosis often employs animal models of the disease, especially experimental autoimmune encephalomyelitis (EAE) in rodents. The statistical analysis procedures chosen for these studies are often suboptimal, either because of violations of the assumptions of the procedure or because the analysis selected is inappropriate for the research question. In this paper, we discuss the types of research questions frequently asked in EAE studies and suggest appropriate and useful research designs and statistical methods that will optimize the information contained within the data. We also discuss other troublesome issues such as missing data, atypical disease profiles, and power analysis.

IL-6 and TNFα expression in brains of twitcher, quaking and normal mice

Abstract Cytokines have been postulated to play a pathogenic role in twitcher mice, which are an animal model of globoid cell leukodystrophy. In particular, TNFα promotes oligodendrocyte and myelin pathology, and IL-6 expression is induced in astrocyte and microglial cultures that have been incubated with TNFα or myelin debris, respectively. It is unknown whether these cytokines are expressed in twitcher mice. The objectives of the present study were to develop an immunohistochemical method to detect TNFα and IL-6 in the mouse CNS, and then utilize this method to identify the cell types expressing these cytokines, and their spatial distribution, in the brains of normal, twitcher and quaking mice. In normal mice, IL-6 was found in ependymal cells, Bergmann glia, in processes that were adjacent or attached to the ventricles or pial surface, and in lightly stained processes in white matter. These processes were identified to belong to astrocytes and microglia. IL-6 staining was dramatically increased in twitcher mice. Astrocytes, with reactive features, and microglia were labeled in the cerebral cortex, basal ganglia, subcortical white matter, pons, medulla and cerebellar white matter. IL-6-positive reactive astrocytes were less abundant in quaking mice than twitcher mice. Cells expressing TNFα were rare or absent in normal and quaking mice. In twitcher mice, TNFα-positive macrophages were present at a lower concentration in cerebral white matter than in the pons and medulla, which have more advanced demyelination. These data demonstrate that pathological events induce the expressions of TNFα and IL-6 in the CNS of twitcher mice.

Chemical analysis of multiple sclerosis lesions by FT-IR microspectroscopy.

Fourier transform infrared microspectroscopy can be used to collect infrared spectra from microscopic regions of tissue sections. If spectra are collected along a grid pattern, then maps of chemical functional groups can be produced and correlated to tissue histopathology. In the present study, white matter from multiple sclerosis and control brains were examined. Mapping experiments were designed such that 17 spectra were collected at 200 microm intervals along a line that was partially or wholly within a multiple sclerosis lesion site or within a representative white matter region of control tissue. Data analysis was based on earlier in vitro studies which found that the carbonyl at 1740 cm(-1) increases when lipids become oxidized (Free Rad. Biol. Med. 16:591-601, 1994), and the amide I peak at approximately 1660 cm(-1) broadens when proteins become oxidized (FEBS Let. 362:165-170, 1995). The results indicated that the C=O to CH2 ratio (1740 cm(-1):1468 cm(-1)) was elevated at several collection points in lesion sites from multiple sclerosis brains compared to values from white matter of control brains. Inspection of the amide I peak at 1657 cm(-1) revealed that it was broadened towards 1652 cm(-1) in multiple sclerosis tissues but not control tissues. These results suggest that lipids and proteins could be oxidized at active multiple sclerosis lesion sites. The localization of these products to lesion sites supports a role for free radicals in the pathogenesis of multiple sclerosis.

Desferrioxamine suppresses experimental allergic encephalomyelitis induced by MBP in SJL mice.

Data from several studies indicate that free radicals have a pathogenic role in experimental allergic encephalomyelitis (EAE). Iron can contribute to free radical damage by catalyzing the formation of hydroxyl radical, inducing secondary initiation of lipid peroxidation and by promoting the oxidation of proteins. The iron chelator, desferrioxamine, can limit these oxidative reactions and it can scavenge peroxynitrite independent of iron chelation. Two previous studies have examined the therapeutic value of desferrioxamine in EAE. One study observed an effect when disease was induced by spinal cord homogenates (J. Exp. Med. 160, p. 1532, 1984), but a second study found no therapeutic value of desferrioxamine for myelin basic protein (MBP)-induced EAE (J. Neuroimmunol. 17, p. 127, 1988). In the second study, the drug was only administered during the preclinical stages of disease. Since desferrioxamine scavenges free radicals and prevents their formation, we hypothesized that the drug should be given during the active stage of disease to have therapeutic value. We first demonstrated that the drug enters the CNS around inflammatory cells in EAE animals. In animals treated during the active stage of MBP-induced EAE, the clinical signs were significantly reduced compared to vehicle-treated animals. The iron-bound form of this drug, ferrioxamine, was without therapeutic value. A derivative of desferrioxamine, hydroxylethyl starch (HES)-desferrioxamine, has a greater plasma half-life than desferrioxamine and it was also tested. Although there was a suggestion of improvement in these animals, the effects were less than that observed for desferrioxamine which may be related to the greater molecular size of HES-desferrioxamine. In summary, these data suggest that chelation of iron is an effective therapeutic target for EAE.

Experimental allergic encephalomyelitis is exacerbated in mice deficient for 12/15-lipoxygenase or 5-lipoxygenase

12/15-Lipoxygenase (12/15-LO) produces 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) which are agonists for peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma agonists reduce clinical severity of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. In contrast, 5-lipoxygenase (5-LO) produces the generally proinflammatory leukotrienes (LTs) which would be expected to worsen EAE. We tested the hypotheses that EAE severity would be exacerbated in 12/15-LO-deficient mice and attenuated in 5-LO-deficient mice. 12/15-LO deficiency conferred a significantly worse disease course, and surprisingly, 5-LO deficiency also caused significantly more severe EAE compared to control mice. These data suggest that PPARgamma-regulated gene expression and that 5-LO production of certain LTs have the ability to diminish EAE. Continued analysis will provide insight into the endogenous LO-generated effectors that assist in tempering EAE.

Hemeoxygenase-1 in SJL mice with experimental allergic encephalomyelitis

The expression of heme oxygenase-1 (HO -1) is increased in the C NS of mice and rats with experimental allergic encephalo myelitis (EAE), an animal model of multiple sclerosis (MS). To investigate the role of HO -1 in EAE, a putative inhibitor [tin-pro toporphyrin IX (Sn-PP IX)] of HO -1 was administered to SJL mice during active disease. Sn-PP IX (200 mmol/kg) attenuated clinical scores, weight loss, and some signs of patho logy in comparison to vehicle treatment. G lutathione levels were greater in treated EAE mice than in those receiving vehicle, indicating lower oxidative stress in the former group. These data suggest that inhibition of HO -1 attenuated disease and suppressed free radical production. In the SJL model of EAE, extravasated blood is present in the C NS, and iron released by HO -1 from this heme source may not be adequately sequestered by ferritin, allowing for iron-mediated tissue damage. Thus, HO -1 may act to amplify the disease process in this model.