Intracerebral expression of CXCL13 and BAFF is accompanied by formation of lymphoid follicle-like structures in the meninges of mice with relapsing experimental autoimmune encephalomyelitis

Abstract

Given the abnormalities in B-cell activity occurring in the central nervous system (CNS) of patients with multiple sclerosis (MS), we have explored the possibility that CNS inflammation induced in mouse models of experimental autoimmune encephalomyelitis (EAE) triggers expression of molecules that control the development and functional organization of lymphoid follicles, the sites where B-cell responses are initiated. By reverse transcription–polymerase chain reaction (RT-PCR), we find that gene expression of CXCL13, a chemokine involved in B-cell recruitment into lymphoid follicles, and BAFF, a key regulator of B-cell survival, is markedly and persistently upregulated in the CNS of mice with relapsing-remitting and chronic-relapsing EAE. Using immunohistochemical techniques, we also show the presence of lymphoid follicle-like structures containing B cells and a reticulum of CXCL13⁺ and FDC-M1⁺ follicular dendritic cells within the meninges of several mice undergoing progressive relapsing EAE. These observations indicate that, under chronic inflammatory conditions, the less immunoprivileged meningeal compartment is the site where ectopic lymphoid follicles preferentially develop and where pathogenic B-cell responses could be sustained in autoimmune disorders of the CNS.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by focal areas of demyelination, reactive gliosis and a variable extent of axonal damage. Increasing evidence supports an autoimmune pathogenesis, with myelin antigens among the most plausible targets. Although MS is thought to be primarily mediated by autoreactive T cells, humoral immune responses also appear to be involved (reviewed by Archelos et al., 2000, Cross et al., 2001). Abnormal B-cell activity is a predominant feature of MS, which is manifested by the accumulation of B cells and plasma cells in chronic MS lesions Esiri, 1977, Prineas and Wright, 1978, by the presence of antibodies directed against myelin proteins in serum and cerebrospinal fluid (CSF) (Archelos et al., 2000) and in areas of active myelin breakdown Genain et al., 1999, Raine et al., 1999, and by a stable intrathecal production of oligoclonal IgG in more than 90% of MS patients (Tourtellotte et al., 1984). The recent demonstration of hypersomatically mutated, clonally related B cells in brain lesions and CSF of MS patients strongly argues for an antigen-driven immune response, suggesting that the microenvironment of the inflamed CNS may support B-cell expansion and maturation Qin et al., 1998, Baranzini et al., 1999, Colombo et al., 2000.

Several studies performed in experimental autoimmune encephalomyelitis (EAE), the animal model for MS, strongly support a pathogenic role for antibodies in autoimmune demyelination. Both in rodent and primate models of EAE, myelin-oligodendrocyte glycoprotein (MOG)-specific antibodies were shown to exacerbate or to be directly implicated in demyelination Linington et al., 1988, Genain et al., 1995, Litzenburger et al., 1998. Recent experiments in B-cell-deficient mice clearly indicate that, following immunization with recombinant MOG, B cells are critical for EAE induction or contribute to disease severity, depending on the mouse strain used Lyons et al., 2002, Svensson et al., 2002. Similarly to MS, B cells are recruited to the EAE-affected CNS (Traugott et al., 1981), most likely under the influence of chemokines produced in the inflamed tissue (Alter et al., 2003).

Lymphoid follicles with germinal centers are crucial sites in the development of B-cell responses because they are the sites where B cells undergo somatic hypermutation and affinity maturation. Tissues affected by autoimmune diseases, such as rheumatoid arthritis, Sjogren's syndrome and Hashimoto's thyroiditis, often contain follicle-like structures with germinal centers that are thought to be relevant to pathogenesis, not only for the production of autoreactive antibodies but also for the maintenance of the autoimmune response (Hjelmström, 2001). Expression of relevant homing lymphoid chemokines, such as CCL19, CCL21 and CXCL13, in ectopic lymphoid structures suggests that the process of lymphoid neogenesis during chronic inflammation involves the same mechanisms underlying lymphoid organ development Ansel and Cyster, 2001, Luther et al., 2002.

Despite its immune privileged status, the CNS is emerging as a site where chronic neuroinflammation is likely sustained by an abnormal, local lymphoid milieu. In earlier morphological studies, immune infiltrates reminiscent of lymphoid structures were described in chronic MS lesions (Prineas, 1979) and in the CNS of mice with passively induced EAE (Raine et al., 1984). More recently, the lymphoid chemokines CCL19 and CCL21 were shown to be upregulated intracerebrally during EAE Alt et al., 2002, Columba-Cabezas et al., 2003 and to be elevated in the CSF of MS patients Pashenkov et al., 2003, Giunti et al., 2003. Despite this evidence, it is not yet clear whether lymphoid-like structures containing distinct T- and B-cell zones develop in the chronically inflamed CNS, and in which CNS compartment(s) lymphoid neogenesis might occur preferentially.

With respect to the mechanisms underlying abnormal B-cell activity during autoimmune disease, evidence is emerging that molecules regulating B-cell survival, proliferation, maturation and tolerance could play an important role in altering peripheral B-cell homeostasis. Among these, B-cell-activating factor of the tumor necrosis factor family (BAFF), also termed BlyS, TALL-1, THANK, TNFSF13B and zTNF4, is a potent regulator of B-cell survival and differentiation, and is thought to participate in pathogenic mechanisms leading to autoimmune diseases (Mackay and Browning, 2002). It has been shown recently that BAFF overexpression leads to an expansion of the B-cell compartment and autoimmunity in mice (Mackay et al., 1999), and that elevated amounts of BAFF are present in the serum and target tissues of autoimmune patients Cheema et al., 2001, Groom et al., 2002. So far, nothing is known about BAFF production in the CNS and its possible role in sustaining abnormal B-cell responses in neuroinflammatory diseases.

In this study, we have used different mouse models of progressive EAE to begin to explore whether the inflamed CNS may become the site for dysregulated or inappropriate synthesis of molecules that normally regulate B-cell recruitment, survival and maturation in lymphoid tissue. To this purpose, we have assessed CNS gene expression of the lymphoid homing chemokine CXCL13, also called B-lymphocyte chemoattractant, and of BAFF. CXCL13 is produced by follicular dendritic cells of secondary lymphoid organs and is essential for the recruitment of B cells into B-cell follicles allowing their interaction with T cells and follicular dendritic cells (Cyster et al., 2000). We have also used immunohistochemical techniques to localize and identify the cell source(s) of CXCL13 protein in the CNS and its relationship to tissue-infiltrating B and T cells. Our study demonstrates that both CXCL13 and BAFF mRNAs are induced intracerebrally during EAE and, most importantly, that B-cell follicle-like structures containing CXCL13-producing cells and follicular dendritic cells can form in the meninges of some mice developing relapsing-remitting and chronic relapsing EAE. This less immune privileged CNS compartment could thus represent the preferential site where pathogenic B-cell responses are stimulated locally during autoimmune disease.