Suppression of established experimental autoimmune encephalomyelitis and formation of meningeal lymphoid follicles by lymphotoxin β receptor-Ig fusion protein

https://doi.org/10.1016/j.jneuroim.2006.06.015Get rights and content

Abstract

We have recently shown that de novo formation of lymphoid structures resembling B-cell follicles occurs in the inflamed central nervous system (CNS) meninges in a subset of patients with secondary progressive multiple sclerosis and in SJL mice with relapsing–remitting experimental autoimmune encephalomyelitis (EAE). Because lymphotoxin (LT) α1β2 is essential for lymphoid tissue organization, we used real-time PCR to examine LTβ and LTβ receptor (LTβR) gene expression in the CNS of SJL mice immunized with PLP 139–151 peptide. Moreover, we used the decoy receptor LTβR-immunoglobulin fusion protein to block the interaction of lymphotoxin (LT) α1β2 with the LTβ receptor (LTβR) in mice with established EAE and evaluate the effect of systemic and local treatments with the fusion protein on disease progression, CNS lymphocytic infiltration and formation of meningeal B-cell follicles. The present findings indicate that both LTβ and LTβR are upregulated at EAE onset and during subsequent relapses and that systemic and local blockade of the LT pathway with LTβR-Ig results in protracted and transient inhibition of EAE clinical signs, respectively. LTβR-Ig treatment also reduces T- and B-cell infiltration and prevents the induction of the chemokines CXCL10 and CXCL13 and the formation of organized ectopic follicles in the EAE-affected CNS. Targeting of molecules involved in lymphoid organogenesis could represent a valid strategy to inhibit CNS inflammation and formation of ectopic follicles, which may play a role in maintaining an abnormal, intrathecal humoral immune response in CNS autoimmune disease.

Introduction

Lymphotoxin (LT) α1β2, a membrane heterotrimer belonging to the tumor necrosis factor (TNF) family, binds to the LTβ receptor (LTβR) and plays a key role in the development and maintenance of the lymphoid microenvironment (Fu et al., 1995, Gonzales et al., 1998, Ngo et al., 1999). In the adult immune system, LTα1β2 is expressed on the surface of B and T cells (Browning et al., 1997, Gommerman and Browning, 2003) and its binding to the LTβR on reticular stromal cells induces expression of lymphoid chemokines, like CCL19, CCL21 and CXCL13, that regulate the homeostatic trafficking of lymphocytes in lymphoid organs and their compartmentalization in B-cell areas (follicles) and interfollicular T-cell areas (Ngo et al., 1999, Ansel and Cyster, 2001, Cupedo and Mebius, 2003). Signalling through the LTβR is also required for the differentiation and function of high endothelial venules (Browning et al., 2005) and of follicular dendritic cells (FDCs) (Fu et al., 1995, Gonzales et al., 1998, Endres et al., 1999, Tumanov et al., 2002), which provide the microenvironment for the colonisation of primary follicles and the establishment of germinal centres (Cyster et al., 2000, Park and Choi, 2005).

Studies in transgenic mice and observations in pathological tissue specimens indicate that the LTα1β2/LTβR pathway is involved in the formation of ectopic (or tertiary) lymphoid structures in tissues targeted by chronic inflammatory processes, such as infection and autoimmunity (Takemura et al., 2001, Armengol et al., 2003, Drayton et al., 2003). Ectopic lymphoid tissue contains distinct B- and T-cell areas and is thought to play an important role in sustaining local immune reactivity against microbial or self antigens (Hjelmström, 2001, Weyand et al., 2001, Aloisi and Pujol-Borrell, 2006). Recent studies indicate that abnormal lymphoid microenvironments can also form in an immune privileged site like the central nervous system (CNS). Increased levels of LT and of the lymphoid chemokines CCL19, CCL21, CXCL12 and CXCL13 have been detected in the cerebrospinal fluid (CSF) or in the demyelinated lesions of patients with multiple sclerosis (MS), a putatively autoimmune disease of the CNS (Pashenkov et al., 2003, Corcione et al., 2004, Ambrosini et al., 2005, Krumbholz et al., 2006). We have shown that lymphoid structures that resemble secondary follicles and comprise FDC networks, CXCL13-expressing stromal cells, proliferating B cells and plasma cells develop in the inflamed meninges of patients with secondary progressive MS (Serafini et al., 2004) and of mice with relapsing experimental autoimmune encephalomyelitis (EAE) (Magliozzi et al., 2004). Evidence for the presence of functional germinal centres supporting B-cell expansion and maturation in the inflamed CNS also derives from the molecular and phenotypic analysis of B cells isolated from the cerebrospinal fluid and white matter lesions of MS patients (reviewed in Uccelli et al., 2005). Such abnormal lymphoid microenvironments could contribute to the sustained intrathecal production of immunoglobulins which is a hallmark of MS, and is thought to play a role in the development of demyelinated lesions (Cross et al., 2001).

The LTβR-immunoglobulin fusion protein (LTβR-Ig) acts as a decoy receptor and represents a useful pharmacological tool to interfere with the activity of LTα1β2 and LIGHT, a second ligand for LTβR (Gommerman and Browning, 2003). LTβR-Ig has been used in various animal models to investigate the involvement of the LT/LIGHT system in autoimmunity and to test its therapeutic potential in preventing disease development or progression. LTβR-Ig was shown to be effective in inhibiting diabetes in nonobese diabetic (NOD) mice (Wu et al., 2001), collagen-induced arthritis and adjuvant arthritis (Fava et al., 2003), experimental autoimmune uveitis (Shao et al., 2003) and EAE (Gommerman et al., 2003). Gommerman and colleagues (2003) showed that systemic administration of LTβR-Ig prior to immunization with myelin peptides inhibited disease development in a rat monophasic EAE model and prevented late-stage disease relapses in a mouse model of relapsing EAE. These authors also showed that disease prevention by LTβR-Ig was due to specific blockade of LTα1β2 binding, rather than LIGHT binding to the LTβR, resulting in impaired T-cell recall responses and intracerebral recruitment.

The aim of this study has been to investigate whether the LTα1β2/LTβR system is involved in disease progression and formation of ectopic lymphoid tissue in SJL mice with PLP 139–151 peptide-induced relapsing–remitting EAE. To this purpose, we examined CNS gene expression of LTβ and LTβR at EAE onset and during relapses and evaluated the effects of LTβR-Ig treatment on clinical score, CNS lymphocytic infiltration, chemokine gene expression and organization of meningeal follicles. Although there is no evidence that B cells and antibodies have a pathogenic role in PLP 139–151-induced EAE in SJL mice, this is so far the only EAE model in which CNS lymphoid neogenesis has been consistently observed, thus allowing modulation of this process to be examined (Magliozzi et al., 2004).

Section snippets

EAE induction

Female adult SJL mice were obtained from Harlan Italy and housed in a controlled environment in accordance with the guidelines of the European Community Council of the Welfare of Experimental Animals (86/609/EEC). All experimental procedures were approved by the Italian Ministry of Health. For the induction of EAE, female SJL mice (6–8 weeks of age) were injected subcutaneously in the flank with 0.2 mg proteolipid protein (PLP) 139–151 peptide, (Primm, Milano, Italy) in complete Freund's

LTβ and LTβR mRNAs are up-regulated in the CNS of EAE-affected SJL mice

To know more about expression of LTα1β2 and its receptor during EAE development and progression, we first evaluated the levels of LTβ and LTβR mRNA in the CNS of control and EAE-affected SJL mice at different times after immunization with PLP 139–151 peptide. Using real time PCR, we found that LTβ mRNA is undetectable in the normal CNS, is markedly up-regulated at the first peak of EAE and during relapses, and returns to nearly baseline levels during the remission phase (Fig. 1A). LTβR mRNA,

Discussion

We show here that LTβ and LTβR gene expression is up-regulated in the CNS of SJL mice developing relapsing–remitting EAE and that treatment of diseased mice with the decoy receptor LTβR-Ig prevents EAE relapses and inhibits T-cell infiltration, formation of organized ectopic B-cell follicles and synthesis of inflammatory (CXCL10) and lymphoid (CXCL13) chemokines in the inflamed CNS. These findings confirm a role for the LT system in EAE development and highlight its involvement in the

Acknowledgements

We thank Dr. J.L. Browning (Biogen Idec, Cambridge, MA, USA) for providing LTβR-Ig fusion proteins and control IgG. This work was supported by the Italian Multiple Sclerosis Foundation (fellowship to R.M.), Program of Collaboration between Istituto Superiore di Sanità and National Institutes of Health and 6th Framework Program of the European Union, NeuroproMiSe, LSHM-CT-2005-01863 (to F.A.).

References (41)

  • E. Ambrosini et al.

    Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions

    J. Neuropathol. Exp. Neurol.

    (2005)
  • K.M. Ansel et al.

    A chemokine-driven positive feedback loop organizes lymphoid follicles

    Nature

    (2000)
  • M.P. Armengol et al.

    Chemokines determine local lymphoneogenesis and a reduction of circulating CXCR4+ T and CCR7+ B and T lymphocytes in thyroid autoimmune diseases

    J. Immunol.

    (2003)
  • M. Bofill et al.

    Follicular dendritic cells share a membrane-bound protein with fibroblasts

    J. Pathol.

    (2000)
  • J.L. Browning et al.

    Visualization of lymphotoxin-β and lymphotoxin-β receptor expression in mouse embryos

    J. Immunol.

    (2002)
  • J.L. Browning et al.

    Characterization of lymphotoxin-alpha beta complexes on the surface of mouse lymphocytes

    J. Immunol.

    (1997)
  • Y.H. Chang et al.

    Proinflammatory effects of LIGHT through HVEM and LTβR interactions in cultured human umbilical vein endothelial cells

    J. Biomed. Sci.

    (2005)
  • S. Columba-Cabezas et al.

    Lymphoid chemokines CCL19 and CCL21 are expressed in the central nervous system during experimental autoimmune encephalomyelitis: implications for the maintenance of chronic neuroinflammation

    Brain Pathol.

    (2003)
  • A. Corcione et al.

    Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis

    Proc. Natl. Acad. Sci. U. S. A.

    (2004)
  • J.G. Cyster et al.

    Follicular stromal cells and lymphocyte homing to follicles

    Immunol. Rev.

    (2000)
  • Cited by (0)

    View full text