3.1.1 Cortical grey matter pathology in MS
The prevalence of GMLs in MS and their contribution to disease progression has been underestimated historically, in part due to the difficulty in detecting them using histological techniques (Peterson et al., 2001) or more recently MRI (Calabrese et al., 2007; Gilmore et al., 2009). Chronic GML pathology is suggested to drive disease progression and result in the accumulation of irreversible disability which characterizes SPMS (Calabrese et al., 2010; Reynolds et al., 2011; Geurts et al., 2012), while a low burden of cortical pathology is associated with a more benign disease course (Calabrese et al., 2013).
Cortical lesions in MS have been broadly classified into 3 types by location within the tissue, namely leukocortical (spanning the border between the grey and white matter), intracortical (GM involvement only), and subpial (adjacent to the pia mater) (Peterson et al., 2001). Subpial lesions are the most extensive and most numerous type, and have recently been shown to be associated with the presence of meningeal inflammation (Bo et al., 2003l; Magliozzi et al., 2007; Magliozzi et al., 2010; Howell et al., 2011).
3.1.2 Meningeal inflammation contributes to cortical GM pathology
The observation of extensive subpial GMLs in SPMS cases with high levels of meningeal inflammation, coupled with the relative lack of inflammation associated with intracortical lesions (Bo et al., 2003a), suggests that soluble cytotoxic factors produced by cells in the meninges may contribute to GML pathogenesis (Kutzelnigg et al., 2005; Magliozzi et al., 2007; Magliozzi et al., 2010). Indeed, the extent of GML pathology, as well as the age of attainment of several clinical milestones, are significantly correlated with the degree of meningeal inflammation, implicating meningeal inflammation as a major driver of cortical GM pathology (Magliozzi et al., 2007; Magliozzi et al., 2010; Howell et al., 2011).The organization of meningeal inflammation into structures resembling ectopic lymphoid follicles in approximately 40% SPMS cases (termed follicle positive SPMS cases; F+SPMS) is associated with a more severe disease course compared to cases in which diffuse meningeal inflammation is observed (follicle negative cases; F-SPMS) (Magliozzi et al., 2007; Howell et al., 2011). These follicle-like structures have been shown to contain Ki67+ proliferating CD20+ Chapter 3 – Expression of lymphoid cytokines and chemokines in SPMS 84
B cells, CD35+ follicular dendritic cells, IgG, A, or M+ plasma cells and CD3+T cells (Serafini et al., 2004; Magliozzi et al., 2007), and resemble tertiary lymphoid organs (TLOs), the formation of which has been described in several other chronic inflammatory diseases (Randen et al., 1995; Schroder et al., 1996; Stott et al., 1998; Houtkamp et al., 2001). The presence of meningeal TLOs is associated with a significant decrease in the age of attainment of several clinical markers of disease progression, including onset of disease, time from onset to progression, first wheelchair use and death (Magliozzi et al., 2007; Magliozzi et al., 2010; Howell et al., 2011). In line with increased disease severity, meningeal TLOs are associated with increased cortical pathology, including increased subpial demyelination and cortical atrophy, a gradient of neuronal loss that is greatest in cortical layers closest to the pial surface, and a gradient of increased microglial activation which parallels the gradient of neuronal loss (Magliozzi et al., 2010). While the diffuse meningeal inflammation observed in F-SPMS cases represents a source of soluble factors that may contribute to cortical damage, gradients in cortical pathology were not observed in F-SPMS cases (Magliozzi et al., 2010), suggesting that TLO formation represents the more extreme end of a spectrum of inflammation, and that organisation of meningeal inflammation into TLOs is important in contributing to damage of the underlying GM.
3.1.3 TLO neogenesis in MS
Given the association of TLOs with increased disease severity in SPMS, further research into TLO neogenesis in the meninges is warranted. The presence of TLOs has only been observed in SPMS cases (Serafini et al., 2004; Magliozzi et al., 2007). Due to a relative lack of autopsy material from RRMS cases it is unknown whether TLOs may form at an earlier disease stage, or represent a shift towards progressive disease (Serafini et al., 2004). However a study of cortical biopsies from early MS cases found meningeal aggregates of inflammatory cells adjacent to subpial GMLs, suggesting that significant meningeal inflammation may be present very early in the disease course (Lucchinetti et al., 2011). It remains to be seen whether these have the characteristics of TLOs.
No evidence of TLO formation has been found in PPMS cases, although increased meningeal inflammation, including formation of dense infiltrates, has been shown to be associated with increased GML burden in PPMS, and GMLs were often situated in close proximity to foci of meningeal inflammation (Serafini et al., 2004; Magliozzi et al., 2007; Choi et al., 2012). The pathology described in PPMS cases was similar but less extensive than that described in Chapter 3 – Expression of lymphoid cytokines and chemokines in SPMS 85
F+SPMS, suggesting that TLO formation may exacerbate the damage mediated by diffuse meningeal inflammation (Choi et al., 2012).
3.1.4 LTα, CXCL13 and CCL21 in TLO neogenesis and MS
TLO neogenesis is postulated to involve the same chemokines and cytokines as those required for secondary lymphoid organ (SLO) formation, and LTα, CXCL13 and CCL21 are implicated in both processes (Weyand et al., 2001; Aloisi and Pujol-Borrell, 2006; Van De Pavert and Mebius, 2010). TLOs develop at sites of ectopic LTα expression in transgenic mice which express the LTα gene under the rat insulin promoter (RIPLT), suggesting a key role of LTα in TLO neogenesis (Kratz et al., 1996; Sacca et al., 1998). LTα ectopic expression in vivo also induces expression of the B cell chemoattractant CXCL13 and the T cell chemoattractant CCL21 (Hjelmstrom et al., 2000). In vitro and in vivo, LTα induces upregulation of the adhesion molecules ICAM, VCAM and P- and E-selectin on endothelial cells, providing a mechanism by which LTα can induce inflammation (Cuff et al., 1998; Cuff et al., 1999). In MS, LTα is upregulated in demyelinated lesions suggesting a role in demyelination (Selmaj et al., 1991b; Lock et al., 1999), and has been shown to play a role in EAE (Powell et al., 1990; Ruddle et al., 1990; Issazadeh et al., 1996).
CXCL13 plays a key role in the organisation of B cell follicles in vivo, as mice lacking the CXCL13 receptor CXCR5 show significantly impaired migration of mature B cells to lymphoid follicles and lack functional germinal centres and splenic follicles (Forster et al., 1996). Ectopic expression of CXCL13 in transgenic mice results in aggregates of B and T cells, organised into separate zones, but lacking follicular dendritic cells or functional GCs (Luther et al., 2000a). The observation of intrathecal Ig synthesis and the accumulation of clonal expansions of B cells in MS have led to increasing interest in CXCL13 as a B cell chemoattractant within the CNS. CXCL13 expression has been detected in B cell follicles in EAE (Columba-Cabezas et al., 2004; Bagaeva et al., 2006) and MS (Serafini et al., 2004; Magliozzi et al., 2007), and CXCL13 concentration in the CSF correlates with the presence of B cells, plasmablasts, T cells, and intrathecal Ig synthesis (Krumbholz et al., 2006).
CCL21 is expressed by high endothelial venules (HEVs) and stromal and dendritic cells within T cell zones of lymphoid organs (Cyster, 1999). Mice deficient in CCL21 or its receptor CCR7 show impaired trafficking of naïve T cells into lymph nodes across HEVs and disorganized T cell zones (Forster et al., 1999; Luther et al., 2000b). CCL21 has been implicated in the Chapter 3 – Expression of lymphoid cytokines and chemokines in SPMS 86
migration of encephalitogenic T cells into the CNS in EAE, as its expression has been described in inflamed CNS venules (Alt et al., 2002) and lesions (Columba-Cabezas et al., 2003; Bagaeva et al., 2006) in EAE.
3.1.5 Aims
These experiments aimed to determine the expression of LTα, CXCL13 and CCL21 in the meninges and CSF of control cases and SPMS cases that had been previously characterized as either having TLOs (F+SPMS) or lacking them (F-SPMS) using snap-frozen cortical tissue (8-10 cases per group). The specific aims of the study were: