Restricted immune responses lead to CNS demyelination and axonal damage

Journal of Neuroimmunology 107 (2000) 178–183 www.elsevier.com / locate / jneuroin

Restricted immune responses lead to CNS demyelination and axonal damage a, b a b Gianluigi Mancardi *, Bert A. ‘t Hart , Elisabetta Capello , Herbert P.M. Brok , c a a a Avraham Ben-Nun , Luca Roccatagliata , Debora Giunti , Paola Gazzola , Mariella Dono d , Nicole Kerlero de Rosbo c , Monica Colombo d , Antonio Uccelli a a

Department of Neurological Sciences and Vision, University of Genova, Via De Toni 5, 16132 Genoa, Italy b Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands c The Weizmann Institute of Science, Rehovot, Israel d Clinical Immunology, National Institute for Cancer Research, Genova, Italy

Abstract Although autoreactive T-cells have a pivotal role in initiating the inflammatory process in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), recent evidence suggests a relevant role for autoantibodies specific for myelin proteins as well. To examine the role of B-cells in the cerebrospinal fluid of patients with MS, we analyzed the VH gene usage in ten MS patients by PCR technologies. Analysis of HCDR3 length revealed an oligoclonal accumulation of B-cells. Sequence analysis of the VH 3 and VH 4 g transcripts of two MS individuals demonstrated that this accumulation was related to the expansion and somatic diversification of a limited groups of B-cell clones. These findings are indicative of a chronic and intense antigenic stimulation occurring in the CNS. Animal models, such as EAE, are of particular importance in order to elucidate the pathogenetic effector mechanisms in autoimmune demyelination. In a non-human primate model of EAE, we describe that the immunodominant T-cell epitope is presented exclusively by a monomorphic DRB1 allele, suggesting that susceptibility to EAE may be linked to this unique restriction and, therefore, providing a possible mechanism for MHC linkage to diseases. Moreover, we report on the presence of inflammation, sharp demyelination and axonal damage in EAE induced with whole myelin as well as with recombinant myelin oligodendrocyte glycoprotein (MOG), but not with myelin basic protein alone. The presence of axonal pathology was supported by immunohistochemistry with anti-amyloid precursor protein and anti-non phosphorilated neurofilaments monoclonal antibodies within early active demyelinated plaques. These findings suggest that axonal damage may be an early event in the pathogenesis of autoimmune demyelinating diseases of the CNS and highlights the importance of animal models in which therapies targeting repair and axonal survival may be exploited.  2000 Elsevier Science B.V. All rights reserved. Keywords: Multiple sclerosis; Autoantibody; Callitrix jacchus; EAE; MOG; Axonal damage

1. Introduction Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) possibly initiated, in a susceptible host, by an infection caused by a virus or another pathogen, which results in a recruitment of inflammatory cells which move within the CNS and recognize, at low affinity, myelin proteins whose molecular

*Corresponding author. Tel.: 139-10-353-7057; fax: 139-10-3538639. E-mail address: [email protected] (G. Mancardi).

structure mimicks microbial motifs (Gran et al., 1999). The inflammatory infiltrates are composed of T-cells, macrophages and, in a lesser amount, B-cells. Autoreactive T-cells have a pivotal role in initiating the inflammatory process, as indicated by studies on experimental autoimmune encephalomyelitis (EAE) (Zamvil and Steinman, 1990). In rodents as well as in primates, autoreactive T-cells are part of the repertoire of healthy individuals but, under antigenic stimulation, may undergo clonal expansion and acquire encephalitogenicity (Schlusener and Wekerle, 1985; Genain et al., 1994). Production of antibodies has relevance as well in the pathogenesis of disease, as it is now clear that they contribute to the process of demyelination (Genain et al., 1995).

0165-5728 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00223-X

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2. Antibody response in MS The importance of the antibody response is well known to clinicians who utilize the presence of IgG molecules with a restricted isoelectric focusing mobility within the cerebrospinal fluid (CSF) for the diagnosis of MS. The role of the humoral response, as a possible pathogenetic effector mechanism of demyelinating lesions in MS and EAE, has recently been highlighted by the demonstration that autoantibodies specific for myelin oligodendrocyte glycoprotein (MOG) are bound to disintegrating myelin membranes surrounding axons in acute MS and in the marmoset model of EAE (Genain et al., 1999). Many studies in the past, especially in EAE and organotypic cultures, have demonstrated the possible role of antibodies in the process of demyelination (Appel and Borstein, 1964), although the role of B-cells in MS pathogenesis has not been properly addressed, likely due to insufficient technical methods. The advent of PCR methodologies now make it possible to have information on the development and maturational history of B-cells, studying the genes of the variable region of the heavy or light chain (VH and VL ). During a T-cell dependent response, B-cells expand in the germinal centers, where they undergo somatic mutation and affinity maturation. During these processes, B-cells accumulate point mutations in the VH and VL genes to increase the affinity of the Ab for the stimulating antigen. B-cell clones carrying V gene variants more favorable for antigen binding are selected. Among these stimulated and selected B-cells, there may be a predominance of B-cell clones that are the progeny of a single precursor. Using PCR technologies and HCDR3 fingerprinting, we recently demonstrated that in the CSF of ten out of ten MS subjects there was an oligoclonal B-cell accumulation in the m, g and a cDNA amplified with VH specific primers. VH 3 and VH 4 genes from the CSF of two MS patients were amplified from g cDNA, cloned and sequenced. DNA sequence analysis demonstrated that both VH 3 and VH 4 genes were extensively mutated compared to germline counterparts. Moreover, a substantial proportion of analyzed molecular clones shared the same HCDR3 and the same VH genes, albeit with different numbers and locations of point mutations. The presence of clonally-related V gene sequences in the CSF of both patients, indicated an ongoing process of intraclonal diversification. In connection with this, it is of note that clonally related VH 4 sequences were found in different areas of an acute MS brain (Owens et al., 1998). These data corroborate our findings of the accumulation of oligoclonal B-cells in the CSF of MS subjects. Accumulation of clonally related B-cells has been previously described in the target tissue of other autoimmune diseases, such rheumatoid arthritis (Randen et al., 1992). Collectively, these findings suggest that in MS the accumulation of B-cells in the brain or in the CSF is not a

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random process but, on the contrary, arises from the compartmentalization of few clones selectively targeting the CNS.

3. Axonal damage in marmoset EAE Although the identification of the antigen involved in MS pathogenesis is still lacking, MOG is now considered a possible and very attractive candidate. The importance of the cellular and humoral response against MOG has been recently demonstrated in the marmoset model of EAE (Genain et al., 1995; Brok et al., unpublished observations). EAE in the non-human primate Callitrix jacchus (C. jacchus) is highly reminiscent of human MS (Massacesi et al., 1995) and is of particular interest because marmoset bone marrow chimerism allows adoptive transfer of pathogenetic T-cells between genetically distinct siblings (Genain et al., 1994). Following immunization either with whole myelin homogenate (WMH) or with recombinant MOG, but not with purified human myelin basic protein (MBP), we induced EAE in three sets of marmoset twins. Perivascular mononuclear cell infiltration, demyelination (Fig. 1a) and reactive gliosis characterized EAE induced both with WMH or MOG. No pathological changes were observed when the animals were challenged with MBP alone. The pathologic picture of EAE induced in the marmoset is very similar to the pathology of MS (Raine et al., 1999) and the lesions can be detected and studied with magnetic resonance images (Hart et al., 1998). Although much attention has been recently focused on axonal pathology in MS (Ferguson et al., 1997; Trapp et al., 1998), this issue has not yet been fully addressed in EAE. We performed a neuropathological study in marmosets immunized with WMH or MOG using immunohistochemistry and electron microscopy (EM) techniques, primarily focusing our attention on the degree, topography and timing of axonal damage. In areas of demyelination stained with Bodian to visualize axons, they appeared well preserved. In contrast, axons were sharply stained by antiamyloid precursor protein (APP) and anti-non-phosphorylated neurofilaments (SMI32) mabs within the same plaques. SMI32 or APP positive axons typically had a smooth round or linear appearance (Fig. 1b); occasionally, constrictions, dilatation or swellings, morphological features suggestive of more severe axonal damage or axonal degeneration, were observed (Fig. 1c). Definite axonal ovoids, indicative of axonal transections, were not detected. APP positive elements were counted in demyelinated areas under a 3100 objective. Early active lesions were characterized by the presence of inflammation and demyelination, associated with LFB-and / or MAG positive products within macrophages stained by MRP14 antibodies; late active lesions were defined by the presence of inflammation and demyelination associated with PAS

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Fig. 1. (a) A large demyelinating plaque in the hemispheric white matter in a MOG immunized marmoset (LFB-PAS, 3100). (b) In an area diffusely infiltrated by inflammatory cells (early active lesion) numerous APP positive axons are present (mabs anti APP, 3100). (c) Near the wall of the lateral ventricle, a large number of APP positive axons, some with constrictions, interruptions or swellings, are observed (mabs anti APP, 3100). (d) At EM, enlarged axons with a diffuse increase of neurofilaments are found usually surrounded by numerous macrophages containing products of myelin degeneration (uranyl acetate and lead citrate, 312 000).

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Fig. 1. (continued)

positive products within macrophages containing a scarce amount of LFB positive products and macrophages stained by anti-lysozyme but not by MRP14 mabs. This quantitative study showed that APP positive axons were significantly more numerous in early active lesions as com-

pared to late active lesions. Very rare APP positive axons were also observed in normal appearing white matter. At EM, many lipid-laden macrophages, near demyelinated axons or surrounding vessels, were detected on plaques. Naked axons were observed, often surrounded by pro-

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cesses of fibrous astrocytes containing densely packed glial filaments. Axons devoid of myelin were often enlarged with an increase of neurofilaments diffusely accumulated in the axoplasm (Fig. 1d). Dystrophic axons, characterized by accumulation of abnormal mithocondria and electron dense organelles, were not observed. These data indicate that in marmoset EAE axonal damage is an early event, more severe in areas of acute inflammation and demyelination. These findings match the picture of human MS, axonal pathology seems to be more evident, possibly due to the long period of time during which this phenomenon occurs. Moreover, the characterization of axonal damage in experimental models of MS can be exploited at designing therapies targeting repair and axonal survival.

4. Restriction of the response to MOG in marmoset EAE EAE is a model which also offers the opportunity to study the genetic influence on the development of disease. The identification of the genetic background of EAE, and consequently of MS, is of main relevance in order to improve our knowledge of the physiopathology of the disease and therefore for the development of a rational therapy. Because of the outbred condition of marmosets, due to their polymorphic genetic make-up, the role of MHC gene can be conveniently addressed in different experimental conditions. In marmosets immunized with MOG we dissected the humoral and cellular response against the immunogen. In contrast to the heterogeneous response observed in humans (Kerlero de Rosbo and Ben-Nun, 1998), an immunodominant region of the extracellular domain of MOG, namely aa 14–36, is preferentially recognized by marmosets cells. Immunization with this peptide induces EAE. Interestingly, this region is also one of the dominant epitopes, target of antibodies detected in the serum of animals immunized with MOG. We characterized the response to this epitope and demonstrated that the recognition of the minimal epitope, aa 24–36, is restricted by the monomorphic allele Caja DRB1* W1201, which is shared by all the common marmosets (Brok et al., unpublished). The unique pattern of restriction in the response to MOG supports the high susceptibility of this species to EAE induced with this antigen and provides insights of the role of MHC genes in disease susceptibility. Overall, EAE in C. jacchus is an appealing model to study the immuno-pathogenesis of autoimmunity within the CNS in an experimental setting that closely resembles the human condition. Moreover, the molecular and functional organization of the primate immune system (Uccelli et al., 1997; Antunes et al., 1998), together with the possibility of utilizing sophisticated diagnostic tools, leads to the possibility of evaluating the safety and efficacy of biological molecules as therapy for MS.

Acknowledgements This study was financially supported by the EU-Biomed Program, contract number ERB FMGE CT950024, by the Italian Society for Multiple Sclerosis (AISM), by the Istituto Superiore di Sanita’ (Progetto ‘Sclerosi Multipla’) and by the Netherlands MS Society (grants 96-267MS and 98-373MS)

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