Chronic Inactive Multiple Sclerosis Lesions Research Articles

Natalizumab-Associated Progressive Multifocal Leukoencephalopathy in a Patient With Multiple Sclerosis: A Postmortem Study

Natalizumab, a monoclonal antibody directed against α4 integrins, has, to date, been associated with 399 cases of progressive multifocal leukoencephalopathy (PML) worldwide in patients receiving treatment for multiple sclerosis (MS). Because of the limited number of histologic studies, the possible interplay between MS and PML lesions has not been investigated. We report the clinical, radiologic, and histologic findings of an MS patient who developed PML after 32 months of natalizumab monotherapy. After withdrawal of natalizumab, she received plasma exchange, mefloquine, and mirtazapine but died soon thereafter. Postmortem examination was restricted to examination of the brain and spinal cord. Extensive PML lesions, characterized by the presence of JC virus DNA were found in the cerebral white matter and neocortex. Sharply demarcated areas of active PML lesions contained prominent inflammatory infiltrates composed of approximately equal numbers of CD4-positive and CD8-positive T cells, consistent with an immune reconstitution inflammatory syndrome. Conversely, all MS lesions identified were hypocellular, long-standing inactive plaques characterized by myelin loss, relative axonal preservation, and gliosis and, importantly, were devoid of JC virus DNA and active inflammation. Chronic inactive MS lesions were separate and distinct from nearby PML lesions. This case demonstrates the coexistence and apparent lack of interplay between chronic inactive MS and PML lesions, and that immune reconstitution inflammatory syndrome seems to affect the shape and appearance of PML but not MS lesions.

Adenosine triphosphate-binding cassette transporters mediate chemokine (C-C motif) ligand 2 secretion from reactive astrocytes: relevance to multiple sclerosis pathogenesis

Adenosine triphosphate-binding cassette efflux transporters are highly expressed at the blood-brain barrier and actively hinder passage of harmful compounds, thereby maintaining brain homoeostasis. Since, adenosine triphosphate-binding cassette transporters drive cellular exclusion of potential neurotoxic compounds or inflammatory molecules, alterations in their expression and function at the blood-brain barrier may contribute to the pathogenesis of neuroinflammatory disorders, such as multiple sclerosis. Therefore, we investigated the expression pattern of different adenosine triphosphate-binding cassette efflux transporters, including P-glycoprotein, multidrug resistance-associated proteins-1 and -2 and breast cancer resistance protein in various well-characterized human multiple sclerosis lesions. Cerebrovascular expression of P-glycoprotein was decreased in both active and chronic inactive multiple sclerosis lesions. Interestingly, foamy macrophages in active multiple sclerosis lesions showed enhanced expression of multidrug resistance-associated protein-1 and breast cancer resistance protein, which coincided with their increased function of cultured foamy macrophages. Strikingly, reactive astrocytes display an increased expression of P-glycoprotein and multidrug resistance-associated protein-1 in both active and inactive multiple sclerosis lesions, which correlated with their enhanced in vitro activity on astrocytes derived from multiple sclerosis lesions. To investigate whether adenosine triphosphate-binding cassette transporters on reactive astrocytes can contribute to the inflammatory process, primary cultures of reactive human astrocytes were generated through activation of Toll-like receptor-3 to mimic the astrocytic phenotype as observed in multiple sclerosis lesions. Notably, blocking adenosine triphosphate-binding cassette transporter activity on reactive astrocytes inhibited immune cell migration across a blood-brain barrier model in vitro, which was due to the reduction of astrocytic release of the chemokine (C-C motif) ligand 2. Our data point towards a novel (patho)physiological role for adenosine triphosphate-binding cassette transporters, suggesting that limiting their activity by dampening astrocyte activation may open therapeutic avenues to diminish tissue damage during multiple sclerosis pathogenesis.

Nrf2 and DJ1 are consistently upregulated in inflammatory multiple sclerosis lesions

Oxidative stress plays a major role in multiple sclerosis (MS), a chronic inflammatory central nervous system (CNS) disease. Invading leukocytes contribute to cell damage and demyelination by producing excessive amounts of cytotoxic mediators, including reactive oxygen species (ROS). To counteract the damaging effects of ROS the CNS is endowed with a repertoire of endogenous antioxidant enzymes, which are regulated by the transcription factor NF-E2-related factor 2 (Nrf2). Upon exposure to ROS, Nrf2 translocates to the nucleus allowing transcriptional activation of various antioxidant enzymes. DJ1 is a protein that is involved in the stabilization of Nrf2 and hence acts as a positive regulator of Nrf2-driven antioxidant protection. Here, we investigate the (sub)cellular localization of Nrf2 and DJ1 in various MS lesion stages and show that Nrf2 is strikingly upregulated in active MS lesions, in both the nucleus and the cytoplasm of infiltrating macrophages and to a lesser extent in reactive astrocytes. Simultaneously, DJ1 protein expression is predominantly increased in astrocytes in both active and chronic inactive MS lesions compared to control brain tissue and normal-appearing white matter. Together, our findings suggest that persistent Nrf2-mediated transcription occurs in active MS lesions, but that this endogenous response is insufficient to prevent ROS-induced cellular damage, which is abundant in inflammatory MS lesions.

Sphingosine 1‐phosphate receptor 1 and 3 are upregulated in multiple sclerosis lesions

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1-phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well-characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S1P(1/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.

Enhanced number and activity of mitochondria in multiple sclerosis lesions

Mitochondrial dysfunction has been implicated in the development and progression of multiple sclerosis (MS) lesions. Mitochondrial alterations might occur as a response to demyelination and inflammation, since demyelination leads to an increased energy demand in axons and could thereby affect the number, distribution and activity of mitochondria. We have studied the expression of mitochondrial proteins and mitochondrial enzyme activity in active demyelinating and chronic inactive MS lesions. Mitochondrial protein expression and enzyme activity in active and chronic inactive MS lesions was investigated using (immuno)histochemical and biochemical techniques. The number of mitochondria and their co-localization with axons and astrocytes within MS lesions and adjacent normal-appearing white matter (NAWM) was quantitatively assessed. In both active and inactive lesions we observed an increase in mitochondrial protein expression as well as a significant increase in the number of mitochondria. Mitochondrial density in axons and astrocytes was significantly enhanced in active lesions compared to adjacent NAWM, whereas a trend was observed in inactive lesions. Complex IV activity was strikingly up-regulated in MS lesions compared to control white matter and, to a lesser extent, NAWM. Finally, we demonstrated increased immunoreactivity of the mitochondrial stress protein mtHSP70 in MS lesions, particularly in astrocytes and axons. Our data indicate the occurrence of severe mitochondrial alterations in MS lesions, which coincides with enhanced mitochondrial oxidative stress. Together, these findings support a mechanism whereby enhanced density of mitochondria in MS lesions might contribute to the formation of free radicals and subsequent tissue damage.

Reduced raft‐association of NF155 in active MS‐lesions is accompanied by the disruption of the paranodal junction

Neurofascin155 (NF155) is required for the establishment of the paranodal axo-glial junction, the predominant interaction site between myelin and axon. It has been shown that the distribution of NF155 is altered in demyelinating diseases such as multiple sclerosis (MS). However, little is known about the biochemical mechanisms underlying these changes. We therefore compared NF155 in postmortem tissue of active and chronic inactive MS lesions with white matter from healthy controls. Although NF155 showed a very similar expression in all control white matter samples, a strong individual variation was observed in MS-lesions with NF155-levels reduced in most samples. At the same time an NF155-fragment was increased in MS-lesions, suggesting that NF155 is subject to protein degradation in lesion sites. Interestingly, the association of NF155 to membrane microdomains (rafts) was reduced in all lesions, irrespective of the amount of NF155, indicating that membrane association of NF155 was generally affected. Therefore, myelin fractionation experiments were performed to analyze the fate of paranodal proteins during demyelination. Although NF155 was enriched in heavy myelin from both control white matter and active MS-lesions, association of Caspr1/paranodin with heavy myelin was abolished in MS-lesions, demonstrating that paranodal junctions are disrupted. In conclusion, the data support the hypothesis that efficient raft-association of NF155 is essential for the assembly of the paranodal junction and demonstrate that reduced association of NF155 to lipid rafts is accompanied by the disassembly of the paranodal junction and thus contributes to the demyelination process in MS.

Extensive extracellular matrix depositions in active multiple sclerosis lesions

In the central nervous system, basement membrane (BM) constituents are predominantly associated with the vasculature. However, under inflammatory conditions, the expression of BM components may alter. Here, we investigated the distribution of several BM components, including laminin, collagen type IV and heparan sulfate proteoglycans in various multiple sclerosis (MS) lesions. We observed irregular and discontinuous BMs in active lesions. Throughout active MS lesions, we found dense networks of BM proteins, which were surprisingly not associated with the cerebrovasculature. These striking parenchymal networks were not observed in chronic inactive MS lesions and brains of non-neurological controls. In addition, we studied the distribution of transforming growth factor-beta1 (TGF-beta1), since it is known as a major modulator of ECM production. Leukocytes, in particular CD68-positive macrophages, expressed high levels of TGF-beta1 and were located in close proximity to parenchymal BM deposits in the MS lesions. We postulate that these BM networks may play a role in the further recruitment of inflammatory cells and form a barrier for axonal regeneration.

Chemokines in multiple sclerosis: CXCL12 and CXCL13 up-regulation is differentially linked to CNS immune cell recruitment

Understanding the mechanisms of immune cell migration to multiple sclerosis lesions offers significant therapeutic potential. This study focused on the chemokines CXCL12 (SDF-1) and CXCL13 (BCA-1), both of which regulate B cell migration in lymphoid tissues. We report that immunohistologically CXCL12 was constitutively expressed in CNS parenchyma on blood vessel walls. In both active and chronic inactive multiple sclerosis lesions CXCL12 protein was elevated and detected on astrocytes and blood vessels. Quantitative PCR demonstrated that CXCL13 was produced in actively demyelinating multiple sclerosis lesions, but not in chronic inactive lesions or in the CNS of subjects who had no neurological disease. CXCL13 protein was localized in perivascular infiltrates and scattered infiltrating cells in lesion parenchyma. In the CSF of relapsing-remitting multiple sclerosis patients, both CXCL12 and CXCL13 were elevated. CXCL13, but not CXCL12, levels correlated strongly with intrathecal immunoglobulin production as well as the presence of B cells, plasma blasts and T cells. About 20% of CSF CD4+ cells and almost all B cells expressed the CXCL13 receptor CXCR5. In vitro, CXCL13 was produced by monocytes and at much higher levels by macrophages. CXCL13 mRNA and protein expression was induced by TNFalpha and IL-1beta but inhibited by IL-4 and IFNgamma. Together, CXCL12 and CXCL13 are elevated in active multiple sclerosis lesions and CXCL12 also in inactive lesions. The consequences of CXCL12 up-regulation could be manifold. CXCL12 localization on blood vessels indicates a possible role in leucocyte extravasation, and CXCL12 may contribute to plasma cell persistence since its receptor CXCR4 is retained during plasma cell differentiation. CXCL12 may contribute to axonal damage as it can become a neurotoxic mediator of cleavage by metalloproteases, which are present in multiple sclerosis lesions. The strong linkage of CXCL13 to immune cells and immunoglobulin levels in CSF suggests that this is one of the factors that attract and maintain B and T cells in inflamed CNS lesions. Therefore, both CXCL13 and CXCR5 may be promising therapeutic targets in multiple sclerosis.

VCAM-1-positive microglia target oligodendrocytes at the border of multiple sclerosis lesions.

The distribution and lineage of vascular cell adhesion molecule-1 (VCAM-1)-positive cells was investigated in 43 lesions from the brain tissue of patients with multiple sclerosis (MS). Numerous VCAM-1-positive macrophages/microglia were detected at the edges of MS lesions. Quantitative analysis of 6 active, 7 chronic active, and 4 chronic inactive MS lesions identified most VCAM-1-positive cells at the actively demyelinating borders of active (102/mm3) and chronic active (29/mm3) lesions, but rarely in chronic inactive lesions (4/mm3). Further, approximately 17% of the VCAM-1-positive cells closely apposed or surrounded oligodendrocyte perikarya at the edges of active and chronic active lesions that were sites of ongoing demyelination. Endothelial cells were VCAM-1-negative in both lesion and non-lesion MS brain tissue. This report is the first to document direct microglial interaction with oligodendrocytes in MS.

Cellular localization and expression patterns of interleukin-10, interleukin-4, and their receptors in multiple sclerosis lesions.

Cytokines have been shown to play a crucial role in the pathogenesis of multiple sclerosis (MS). However, still limited data are available on the expression of anti-inflammatory cytokines within the central nervous system (CNS) during MS lesion development. Therefore, we have examined the expression of the anti-inflammatory cytokines, interleukin-10 (IL-10) and IL-4, and their specific receptors, IL-10R and IL-4R, in postmortem human brain tissue obtained from MS patients. Specific patterns of protein localization and expression for both proteins could be observed within active and chronic MS lesions. Strongest IL-10 immunoreactivity was observed in reactive astrocytes within active demyelinating lesions and the hypercellular rim of chronic active MS lesions. Moreover, perivascular macrophages were immunoreactive for IL-10 in (chronic) active MS lesions. Most intense IL-4 immunoreactivity was detected in reactive fibrillary astrocytes within the hypocellular regions of chronic active and chronic inactive MS lesions. Strong immunoreactivity for IL-10R and IL-4R was detected on macrophages in both parenchymal and perivascular areas and on reactive astrocytes in active and chronic MS lesions. Our results indicate that IL-10 and IL-4 have an active role in CNS immune responses. The specific patterns of protein localization and protein expression for both IL-10 and IL-4 in MS lesions at different stages of development suggest that these anti-inflammatory cytokines and their receptors participate in processes leading to the formation of chronic MS lesions.