Corline J.A. De Groot

Expression of MCP-1 by Reactive Astrocytes in Demyelinating Multiple Sclerosis Lesions

The pathology of multiple sclerosis (MS) is characterized by breakdown of the blood-brain barrier (BBB), accompanied by infiltration of macrophages and T lymphocytes into the central nervous system (CNS). The migration of these cells into the CNS parenchyma may be partly regulated by chemokines. The aim of this study was therefore to investigate the cellular localization of the potent monocyte- and T-cell-attracting chemokine monocyte chemoattractant protein (MCP)-1 by immunohistochemistry on postmortem brain tissue from MS and normal control cases. Brain tissue samples of six MS patients and four patients without a history of brain disease were neuropathologically classified according to characteristic (immuno)histochemical staining patterns. Frozen tissue sections of active demyelinating MS lesions, chronic active demyelinating MS lesions, and normal control brain were immunohistochemically stained with a monoclonal antibody directed against MCP-1. In active demyelinating MS lesions as well as in chronic active MS lesions, reactive hypertrophic astrocytes were strongly immunoreactive for MCP-1, whereas perivascular and parenchymal foamy macrophages did not express MCP-1 protein. These results suggest a significant role for the beta-chemokine MCP-1, synthesized in vivo by reactive hypertrophic astrocytes, in the recruitment and activation of myelin-degrading macrophages and thereby contributing to the evolution of MS lesions.

Immunocytochemical Characterization of the Expression of Inducible and Constitutive Isoforms of Nitric Oxide Synthase in Demyelinating Multiple Sclerosis Lesions

The cellular localization and distribution of inducible and constitutive nitric oxide synthase (iNOS/cNOS) was determined in tissue sections from multiple sclerosis (MS) and control brain and spinal cord. Immunocytochemical techniques were applied using specific iNOS- and cNOS-directed antibodies. In addition, NADPH-diaphorase histochemistry was performed. To establish the identity of iNOS-, cNOS- and NADPH-diaphorase-positive cells single and double staining was performed on tissue sections with the macrophage marker KP1 (CD68) and with the astrocyte marker glial fibrillary acidic protein (GFAP). Areas of myelin breakdown and demyelination were determined using a staining for neutral lipids, Oil Red O (ORO). Furthermore, macrophages isolated from active demyelinating MS lesions were stained for iNOS, cNOS, KP1 and ORO. In active MS lesions strong iNOS immunoreactivity was found exclusively in perivascular and parenchymal macrophages distributed within regions of active demyelination. In these active MS lesions immunoreactivity for cNOS was also found in macrophages. Macrophages isolated from active MS lesions also showed immunoreactivity for iNOS and cNOS. Moreover, these isolated macrophages produced nitric oxide (NO; >30 μM) in vitro. NADPH-diaphorase activity was detected in KP1-positive perivascular and parenchymal macrophages and in GFAP-positive reactive astrocytes in active MS lesions and in reactive astrocytes located in the hypercellular rims of chronic active MS lesions. cNOS-positive reactive astrocytes were detected in both active and chronic active MS lesions. Inside chronic active lesions some residual macrophages were weakly iNOS-positive. In control brain and spinal cord no iNOS immunoreactivity could be detected. These results suggests an important role for human macrophages capable of producing the free radical nitric oxide (NO), which may contribute to the cytotoxicity of oligodendrocytes and destruction of myelin in MS brain and spinal cord.

CD163-positive perivascular macrophages in the human CNS express molecules for antigen recognition and presentation.

Perivascular macrophages (PVM) constitute a subpopulation of resident macrophages in the central nervous system (CNS) that by virtue of their strategic location at the blood‐brain barrier potentially lend themselves to a variety of important functions in both health and disease. Functional evidence suggests that PVM play a supportive role during experimental autoimmune encephalomyelitis in rodents. However, the function of PVM in the human CNS remains poorly characterized. We first set out to investigate the validity of the antibody EDhu1, which recognizes human CD163, to specifically identify human PVM. Second, we wanted to gain insight into the function of PVM in antigen recognition and presentation and therefore we studied the expression of DC‐SIGN, mannose receptor, MHC class II, and several costimulatory molecules by PVM in the normal and inflamed human CNS (multiple sclerosis (MS) brain lesions). Conventional immunohistochemistry and double‐labeled immunofluorescence techniques were used. We show that CD163 specifically reveals PVM in the normal human CNS. In MS lesions, CD163 staining reveals expression on foamy macrophages and microglia, besides an upregulation of the amount of PVM stained. In contrast, mannose receptor expression is restricted to PVM in both normal and inflamed brain tissue. Furthermore, we show that a subpopulation of PVM in the human brain express several molecules involved in antigen recognition, presentation, and costimulation. Therefore PVM, which occupy a strategic location at the BBB, are equipped to recognize antigen and present it to T cells, supporting a role in the regulation of perivascular inflammation in the human CNS.

Expression of Transforming Growth Factor (TGF)-β1, -β2, and -β3 Isoforms and TGF-β Type I and Type II Receptors in Multiple Sclerosis Lesions and Human Adult Astrocyte Cultures

It is known that the pleiotropic cytokine transforming growth factor beta (TGF-beta) has a regulatory role in the process of tissue repair and remodelling following injury. As reports on these molecules in multiple sclerosis (MS) lesion with different lesional activity are rare, we studied the cellular localization of TGF-beta1, -beta2, and -beta3 isoforms, and TGF-beta receptor type I (TGF-betaR-I) and TGF-betaR-II expression by immunohistochemistry on postmortem brain tissue from MS and normal control cases. To validate the TGF-beta staining results we demonstrated that cultured human adult astrocytes that produce biological active TGF-beta2, and to a lesser extent TGF-beta1, were immunoreactive for all 3 TGF-beta isoforms. Moreover, at mRNA level TGF-beta1 was detected in MS and normal control brain tissue. In normal control brain tissue, TGF-beta isoforms were expressed in ramified microglia and TGF-beta2, and -beta3 on neuronal cells in the gray matter TGF-betaR-I and TGF-betaR-II expression was found on endothelial cells, astrocytes, microglia, and neurons. In active demyelinating MS lesions a strong to intense immunoreactivity was detected for all 3 TGF-beta isoforms in perivascular and parenchymal (foamy) macrophages and in hypertrophic astrocytes. Strong immunoreactivity for TGF-betaR-I and TGF-betaR-II was found on macrophages in both parenchymal and perivascular areas and on hypertrophic astrocytes and endothelial cells in active demyelinating MS lesions. In chronic active and inactive MS lesions, all 3 TGF-beta isoforms and their receptors were strongly expressed in hypertrophic astrocytes. Our findings strongly suggest that the expression of the various TGF-beta isoforms and their receptor types found in MS lesions with different cellular activity participate in reactive processes leading to the formation of chronic MS lesions.

Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization

We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3–9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte‐specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP‐positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1–3%) were present in the human adult astrocyte cultures, using a panel of macrophage‐specific markers. However, the monoclonal antibodies (mAbs KP1, EBM11, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non‐lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay). J. Neurosci. Res. 49:342–354, 1997.

Differential expression of stress proteins in human adult astrocytes in response to cytokines

Various lines of evidence suggest a close relationship between heat shock proteins (hsp) and several autoimmune diseases such as arthritis, diabetes and multiple sclerosis. While enhanced expression of hsp in autoimmune diseases is often regarded as a non-specific bystander effect of the inflammatory process, surprisingly little is known on hsp regulation by inflammatory mediators such as cytokines. In this study cytokine-induced expression of hsp60, hsp27 and alphaB-crystallin was studied in cultures of primary human adult astrocytes at the mRNA as well as at the protein level. We show differential hsp expression patterns in response to pro-inflammatory and immunoregulatory cytokines. Hsp60 expression was found to be enhanced in response to cytokines as diverse as IL-1beta, TNF-alpha, IL-4, IL-6 and IL-10. Upregulation of hsp27, however, was primarily induced by immunoregulatory cytokines like IL-4, IL-6 and TGF-beta whereas alphaB-crystallin expression was found to be enhanced by the pro-inflammatory cytokine TNF-alpha only. None of the cytokines studied was able to enhance expression of all three hsp simultaneously. These results show that in human astrocytes induced expression of hsp27 and alphaB-crystallin is dependent on the presence of a defined set of stimuli, while induced expression of hsp60 is a much less selective event. This highly differential pattern of hsp expression in response to inflammatory mediators known to play an important role in the pathogenesis of autoimmune diseases indicates that hsp responses are specific rather than non-specific bystander responses.

Multiple sclerosis as a generalized CNS disease—comparative microarray analysis of normal appearing white matter and lesions in secondary progressive MS

We used microarrays to compare the gene expression profile in active lesions and donor-matched normal appearing white matter (NAWM) from brain autopsy samples of patients with secondary progressive multiple sclerosis (MS) with that from controls who died from non-neurological diseases. The 123 genes in lesions, and 47 genes in NAWM(MS) were differentially expressed. Lesions distinguished from NAWM(MS) by a higher expression of genes related to immunoglobulin synthesis and neuroglial differentiation, while cellular immune response elements were equally dysregulated in both tissue compartments. Current results provide molecular evidence of a continuum of dysfunctional homeostasis and inflammatory changes between lesions and NAWM(MS), and support the concept of MS pathogenesis being a generalised process that involves the entire CNS.

Upregulation of transcription factors controlling MHC expression in multiple sclerosis lesions.

The expression of major histocompatibility complex (MHC) class I and class II in the CNS has received considerable interest because of its importance in neurodegenerative or inflammatory diseases, such as multiple sclerosis (MS). However, at the moment nothing is known about the expression patterns of transcription factors controlling MHC expression in MS lesions. Here, we performed an extensive immunohistochemical analysis on MS affected postmortem brain tissue to determine the cellular localization and distribution of different MHC‐controlling transcription factors. We show that phagocytic macrophages in active demyelinating MS lesions displayed a moderate to strong immunostaining of the MHC‐specific transcription factors RFX and CIITA, as well as the general transcription factors NF‐κB, IRF1, STAT1, USF, and CREB, which was congruent with a strongly enhanced expression of HLA‐DR, HLA‐DQ, HLA‐DP, and HLA class I. In the normal‐appearing white matter (NAWM), clusters of activated microglial cells forming preactive lesions displayed an overall stronger expression level of these transcription factors, combined with a strong to intense level of MHC class I and class II immunostaining. In general, astrocytes and oligodendrocytes either did not express, or weakly expressed, these transcription factors, correlating with a lack of MHC class II and weak MHC class I expression. Together, the elevated expression level of transcription factors governing expression of MHC class I and class II molecules in activated microglial cells and phagocytic macrophages strongly suggests a general state of microglial cell activation in MS lesions. GLIA 36:68–77, 2001.

Beneficial effect of modified peptide inhibitor of α4 integrins on experimental allergic encephalomyelitis in Lewis rats

An important event in the pathogenesis of the autoimmune disease multiple sclerosis (MS) is the recruitment of lymphocytes and inflammatory macrophages to the central nervous system (CNS). Recruitment requires adhesive interactions between the leukocytes and the microvascular endothelium, perivascular cells, and astrocytes in the CNS parenchyma. Previous studies using an animal model of MS, experimental allergic encephalomyelitis (EAE), have shown the involvement of the α4 integrin VLA‐4 (β4β1). In the present study, the effect of a modified peptide inhibitor of α4 integrins on the clinical course and leukocyte infiltration during EAE is investigated. EAE was either induced actively, by immunizing Lewis rats with whole guinea pig MBP, or passively, by transfer of an MBP‐specific T cell line. Treatment with the inhibitor (CS1 ligand mimic) completely prevented both clinical signs and cellular infiltration in passively induced EAE. Peptide treatment of actively induced EAE, which has a more severe disease course than the transfer model, significantly reduced clinical signs although the recruitment of inflammatory cells and induction of MHC class II expression was not prevented. The α4 inhibitor did inhibit the adhesion of lymphocytes to primary astrocytes in vitro suggesting a role for astrocyte‐leukocyte interactions in the pathogenesis of induced EAE. Astrocytes were found to express an extracellular matrix protein distinct from fibronectin, which shows immune cross‐reactivity with the CS1 domain of fibronectin. Our results show that small‐molecule inhibitors of α4 integrins act therapeutically in EAE possibly by interfering with cell adhesion events involved in this autoimmune disease.

Extracellular matrix proteins expressed by human adult astrocytes in vivo and in vitro: an astrocyte surface protein containing the CS1 domain contributes to binding of lymphoblasts.

Primary cultures of human astrocytes, expressing glial fibrillary acidic protein (GFAP), were obtained from postmortem brain tissue samples. These cultured astrocytes produced an extracellular matrix (ECM), containing laminin (Ln) and fibronectin (Fn), as shown with specific antibodies. The perinuclear staining observed in these cells indicated that these proteins were de novo synthesized. Monoclonal antibody (mAb) 90.45, which recognizes the CS1 sequence found in an alternatively spliced form of Fn, also stained cultured astrocytes. Immunohistochemical analysis of normal human brain tissue showed positive staining for the CS1 domain, both on protoplasmic and fibrous astrocytes located in the gray and white matter. In contrast to cultured astrocytes, no immunoreactivity for Ln or Fn was found on astrocytes in normal human brain tissue. These in situ data indicate that the CS1 domain expressed by astrocytes is not part of a splicing variant of Fn. Western blot analysis confirmed that the CS1 domain expressed by cultured human astrocytes is part of an astrocyte protein which is different from human Fn. The CS1 domain is a known ligand for the adhesion receptor α4β1 (VLA‐4). We found that the human lymphoma cell lines Jurkat and Ramos, which express α4β1, bound to cultured human astrocytes, and that this interaction could be partly blocked by mAb 90.45 or a synthetic CS1 peptide. Thus, the novel CS1‐containing surface protein expressed by astrocytes in vitro and in vivo, contributes to binding of lymphoblasts, and therefore may be a relevant adhesion molecule for the recruitment of α4‐integrin expressing leukocytes into the central nervous system (CNS). J. Neurosci. Res. 50:539–548, 1997.