Sigrid R. Ruuls

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.

Interferon-β directly influences monocyte infiltration into the central nervous system

Interferon-beta (IFN-beta) has beneficial effects on the clinical symptoms of multiple sclerosis (MS) patients, but its exact mechanism of action is yet unknown. We here suggest that IFN-beta directly modulates inflammatory events at the level of cerebral endothelium. IFN-beta treatment resulted in a marked reduction of perivascular infiltrates in acute experimental allergic encephalomyelitis (EAE), the rat model for MS, which was coupled to a major decrease in the expression of the adhesion molecules ICAM-1 and VCAM-1 on brain capillaries. In vitro, IFN-beta reduced the mRNA levels and protein expression of adhesion molecules of brain endothelial cell cultures and diminished monocyte transendothelial migration. Monocyte adhesion and subsequent migration was found to be predominantly regulated by VCAM-1. These data indicate that IFN-beta exerts direct antiinflammatory effects on brain endothelial cells thereby contributing to reduced lesion formation as observed in MS patients.

Macrophage phagocytosis of myelin in vitro determined by flow cytometry: phagocytosis is mediated by CR3 and induces production of tumor necrosis factor-α and nitric oxide

Demyelination of axons in the central nervous system (CNS) during multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE) is a result of phagocytosis and digestion by macrophages (M phi) and the local release of inflammatory mediators like tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). We have investigated the process of myelin phagocytosis by M phi in vitro using flow cytometric analysis. The binding and uptake of CNS-derived myelin was dose dependent, was abolished in the presence of EDTA and was enhanced after opsonization with complement. The phagocytosis of opsonized myelin could be inhibited by antibodies directed against complement receptor type 3 (CR3). Furthermore, CR3 also contributes to phagocytosis of non-opsonized myelin, e.g. under serum-free conditions. The phagocytosis of CNS-derived myelin induced the production of substantial amounts of TNF-alpha and NO by the M phi. Our results indicate an important role for CR3 in myelin phagocytosis. The induction of TNF-alpha and NO which accompanies this phagocytosis may further contribute to the overall process of demyelination during MS or EAE.

Discontinuation of treatment with IFN-β leads to exacerbation of experimental autoimmune encephalomyelitis in Lewis rats. Rapid reversal of the antiproliferative activity of IFN-β and excessive expansion of autoreactive T cells as disease promoting mechanisms

IFN-beta has recently been shown to exert remarkable beneficial effects on disease development in patients with early stage relapsing-remitting MS. The specific immune mechanism(s) by which IFN-beta ameliorates this human demyelinating disease is at present undefined. One potential mechanism may reside in the antiproliferative activity of IFN-beta which may inhibit the expansion of autoaggressive T cells thereby limiting disease progression. In the present study we investigated whether the administration of recombinant rat IFN-beta (rrIFN-beta) to Lewis rats with actively induced experimental autoimmune encephalomyelitis (EAE) inhibits the expansion of encephalitogenic T cells in lymphoid organs and as such may contribute to suppression of disease activity in this widely used animal model for MS. Our data show that daily administrations of > or = 3 x 10(5) u rrIFN-beta to EAE rats, starting two days before MBP sensitization and continued for 10 days led to a dramatic and dose-dependent reduction in encephalitogenic T cells in both spleen and inguinal lymph nodes at day 8 post-immunization (p.i.). However, the rrIFN-beta-mediated reduction in effector T cells did not ameliorate paralytic disease as expected but significantly enhanced the severity of EAE. Analyses of lymphoid organs in the remission phase of EAE revealed strongly elevated numbers of encephalitogenic T cells in rrIFN-beta-treated versus control rats suggesting a rapid reversal of the antiproliferative action of rrIFN-beta followed by an overshoot in the subsequent expansion of these effector T cells. In conformity with higher numbers of encephalitogenic T cells and worsening of disease, animals also showed significantly greater perivascular inflammation in the CNS. The relevance of our findings in relation to the beneficial effects of IFN-beta in MS is discussed.

The role of macrophage subpopulations in autoimmune disease of the central nervous system

SummaryIn this review the role of various subpopulations of macrophages in the pathogenesis of experimental autoimmune encephalomyetitis is discussed. Immunohistochemistry with macrophage markers shows that in this disease different populations of macrophages (i.e. perivascular cells, microglia and infiltrating blood-borne macrophages) are present in the central nervous system. These subpopulations partially overlap in some functional activity while other activities seem to be restricted to a distinct subpopulation, indicating that these subpopulations have different roles in the pathogenesis of encephalomyelitis. The studies discussed in this review reveal that immunocytochemical and morphological studies, combined with new techniques such asin situ nick translation and experimental approaches like the use of bone marrow chimeras and macrophage depletion techniques, give valuable information about the types and functions of cells involved in central nervous system inflammation. The review is divided in three parts. In the first part the experimental autoimmune encephalomyelitis model is introduced. The second part gives an overview of the origin, morphology and functions of the various subpopulations. In the third part the role of these subpopulations is discussed in relation to the various stages (i.e. preclinical, clinical and recovery) of the experimental disease.

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.

Experimental Allergic Encephalomyelitis

During multiple sclerosis (MS) multifocal inflammation occurs in the central nervous system (CNS). Perivascular areas around small venules are infiltrated by T cells and macrophages. Myelin sheaths are damaged in these lesion areas which is considered to be a result from aberrant immune responses to myelin associated self-antigens. The role of these infiltrating macrophages, which in active lesions of MS outnumber the lymphocytes a 20-fold (1), is not clear. Since macrophages in the lesion areas have been described to be laden with myelin, their function is sometimes considered to be restricted to phagocytosis of damaged myelin which has been teared off the axons. However, since macrophages appear early in the lesion they could also act as effector cells, initiating and mediating tissue damage and myelin destruction. Activated macrophages secrete pro-inflammatory molecules like interleukin (IL)-lβ, IL-6, TNF-a, proteases and reactive oxygen species (ROS). Both TNF α and ROS have been described to degrade myelin in vitro (2,3), and inhibitors of IL-lβ, proteases, TNF α and ROS suppress neurological symptoms of an animal model for MS, experimental allergic encephalomyelitis (EAE)(4–6). Also in the CNS of rats with EAE macrophages are dominantly present (7). To study the role of these macrophages in the pathogenesis of EAE two different experimental approaches were used. Firstly, macro-phages were depleted from the circulation using a liposome-mediated technique during a T cell line-induced acute form of EAE as well as during a chronic relapsing form of EAE (CR-EAE) (8,9). To evaluate the effect of the macrophage depletion technique on microglia cells during EAE, liposomes were used in bone marrow chimeras (10,11). Secondly, migration of macrophages into the CNS during EAE was inhibited by blockade of the aMβ2 integrin, also named complement receptor type 3 (CR3)(12).

Nitric oxide synthase activity of human adult astrocytes and brain macrophages in multiple sclerosis

25 HUMAN ASTROCYTES, NO AND NEUROTOXICITY. C.C. Chao. S. Hu. aud P. K. Peterson. MiuneaImlis Medical Research Foundation and the University of Mbmemta Medical School, Minneapolis, MN, U.S.A. Activated glial cells are a histopatbologic hallmark of numerous neurodegenerative diseases. In muriue cultures, cytokine-activated microglia induce neuronal injury via a NO mechanism. In contrast, cytokine-activated human microglia are not neurotoxic. nor do human microgIia generate appreciable NO. Instead. human astmcytes generate substantial amounts of NO in response to IL1 which can be potentiated by IFN-y or TNF-a. Treatment of mixed astrocyte/neuronaI all cultures with ILlb plus IFN-1 or IL1 6 plus TNFa resulted in marked neuronal loss. Cytokiue-stimulated neuronal loss cau be blocked partially by a NO synthase inhibitor. Treatment of mixed cell c&urea with N-methyl-D aspartate (NMDA) receptor antagonists attenuates cytokine-induced neurotonicity without affecting NO production. Cytokine-mediated neurotoxicity also involvea an apo~totic neuronal cell death pathway. Because microglia are responsive to irdlammatory mediators and are the keyce8typepromudneILl,we~acaacadeofCNSinflammafion initiated by microglial cell IL1 release with astrocytes and NO playing intermediary r&s. In this model, NMDA receptors are the fmal pathway of neuronal injury. Our findings hop&dIy will stimulate development of therapeutic approaches aimed at gtia-mediated neuronaI injury. (Funded by DA-04381. DA-09924, AI-351 10. and Alzheimc

Macrophages: Effectorcells in experimental allergic encephalomyelitis

s Association).

The length of treatment determines whether IFN-beta prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats.

ENCEPHALOMYELITIS (EAE) ITS POSSIBLE IMPLICATIONS M.P. Pender .~, K.B. Nguyen 1, P.A. MeCombe 1, and J.F.R. Kerr 2 tDepartment of Medicine and 2Department of Pathology, University of Queensland, Brisbane, Australia. Apoptos|s ~s an active process of cellular self-destruction that often serves a biologically meaningful homeostatic function. Here we report apoptosis in the spinal cord in 4 forms of EAE in the Lewis rat: (l) acute EAE induced by inoculation with whole spinal cord; (2) acute EAE induced by inoculation with myelin basic protein (MBP); (3) acute EAE passively transferred by MBP-sensitized splenocytes; (4) chronic relapsing EAE. Cells undergoing apoptosis were recognized at light and electron microscopy by the presence of either crescentic masses of condensed chromatin lying against the nuclear envelope or rounded masses of uniformly dense ehromatin. Although it was not possible to identify definitively the types of cells undergoing apoptosis, the size and location of some of the affected cells suggested that they were o!igodendrocytes. As there is now much evidence that T-cell-induced target cell death takes the form of apoptosis, it is attractive to hypothesize that oligodendrocyte apoptosis is occurring in EAE as a result of oligodendrocyte-directed T-cell eytotoxicity. However, other apoptotie cells were located within the myelin sheath, meninges and perivaseular spaces and were clearly not oligodendroeytes but were most likely blood-derived mononuelear cells. The sparsity of their cytoplasm and the absence of phagocytosed material suggested that they were mainly lymphoeytes rather than macrophages. Apoptosis has been shown to be involved in deleting autoreaetive T-cells in the thymus during the normal development of tolerance. Thus apoptotie deletion of myelin/oligodendrocyte-specifie T-cells in the central nervous system in EAE might explain both the subsidence of inflammation and the acquisition of tolerance in this autoimmune disease. As apoptosis is an active process that can be controlled, agents that modulate its occurrence may prove useful in the therapy of inflamma:ory demyelinating disease.