Methylprednisolone treatment, immune activation, and intrathecal inflammation in multiple sclerosis

This review has been accepted as a thesis together with eight previously published papers, by the University of Copenhagen, September 17, 2003, and defended on February 27, 2004.

Department of Neurology, University of Copenhagen, Glostrup Hospital, Glostrup, Denmark.

Correspondence to: Finn Sellebjerg, Slettevej 22, DK-2870 Dyssegård.

Official opponents: Alastair Compston, UK, and Carsten Geisler, MD.

Dan Med Bull 2004;51:167-83.

3. T CELLS IN MULTIPLE SCLEROSIS

3.1 T cell activation in MS

Autoreactive T cells have a central role in autoimmune models of the pathogenesis of MS. T cells from MS patients have been studied by the analysis of antigen-specific T cell clones or T cell lines using proliferation and cytokine production as a read-out, or by immunospot and in situ hybridization assays for the enumeration of cytokine expressing cells. These studies have provided strong evidence that myelin-reactive Th1 cells are activated in the blood, CSF and brain of patients with MS (Navikas and Link, 1996; Wekerle and Lassmann, 1998; Martino and Hartung, 1999). Furthermore, the potential of a MBP-reactive T cell receptor derived from a patient with MS to initiate autoimmune demyelination has been demonstrated in transgenic mice (Madsen et al, 1999). We used flow cytometry to study the expression of activation-related molecules on the surface of freshly isolated T cells from patients with POSMS or CDMS in order to assess CD4 T cell activation status in MS in general. Flow cytometry is a standard method for analysing T cell activation in immunological research. The method is based on the use of fluorochrome-conjugated antibodies, and modern flow cytometers readily allow the analysis of the expression of several different molecules on the surface of single leukocytes.

3.2 CD26 expression on CD4 T cells

The CD26 molecule, dipeptidyl peptidase IV, is widely expressed on various cell types (Fleischer, 1994). CD26 occurs in multiple isoforms, and isoforms recognized by some monoclonal antibodies, including Ta1 and L272, are preferentially expressed on T cells involved in delayed type hypersensitivity reactions and on Th1 cells (Torimoto et al, 1992; Kahne et al, 1996; Seitzer et al, 1998). In one previous study an increased percentage of peripheral blood lymphocytes from patients with SPMS bound the anti-CD26 antibody Ta1 (Hafler et al, 1985), but this was not confirmed in another study (Crockard et al, 1988).

Increases in the percentage of CD26+ CD4 T cells were associated with clinical and MRI disease activity in MS in a recent study (Khoury et al, 2000), and we observed an increased percentage of blood CD26+ CD4 T cells in POSMS and CDMS when compared to neurological control subjects (IV, V). In both studies the Ta1 antibody was used. We have recently confirmed that there is an increase in CD26 expression on CD4 T cells from patients with active MS using the anti-CD26 antibody L272. These CD26+ CD4 T cells produce IFN- γ and TNF- α , and the percentage of CD26+ CD4 T cells in blood correlates with disease activity both in POSMS and RRMS (Jensen et al, submitted).

CD26 expression on CD4 T cells was not increased in a recent study of peripheral blood T cells from patients with active RRMS (Strunk et al, 2000). The CD26 antibody used in that study did, however, not recognize an isoform of CD26 preferentially expressed on Th1 cells as CD26 positive cells were found to produce more interleukin (IL)-2 but less IFN- γ and TNF- α than did CD26 negative T cells. CD26 expression was not increased in RRMS in other studies, possibly because expression was studied on lymphocytes in general and not specifically on T cells (Hafler et al, 1985; Crockard et al, 1988), or because the patients under study did not have active disease (Svenningsson et al, 1993; Rep et al, 1994).

3.3 CD25 and HLA-DR expression on CD4 T cells

Expression of CD25, the a-chain of the IL-2 receptor complex, is induced by T cell activation but CD25 is also expressed by other activated leukocytes (Minami et al, 1993). In addition there exists, within the subset of CD25+ CD4 T cells from healthy volunteers, a minor subset of in vivo activated, regulatory T cells (Treg) which mediate contact-dependent suppression of T cell activation (Baecher-Allan et al, 2001; Dieckmann et al, 2001; Jonuleit et al, 2001; Levings et al, 2001; Ng et al, 2001; Stephens et al, 2001; Taams et al, 2001).

In some previous studies increased lymphocyte expression of CD25 was detected in active MS lesions and on CSF lymphocytes from patients with active MS (Bellamy et al, 1985; Hofman et al, 1986; Woodroofe et al, 1986; Traugott and Lebon, 1988), but this was not confirmed in other studies (Hafler et al, 1985; Hayashi et al, 1988). Studies of MBP-reactive T cells from patients with MS show them to express functional IL-2 receptors, suggesting in vivo activation (Zhang et al, 1994; Illes et al, 1999). However, in one study MBP-reactive T cells expressing CD25 underwent apoptosis after antigen stimulation in the absence of exogenous IL-2 (Bieganowska et al, 1997). This is consistent with studies showing that at least a subset of CD25+ CD4 T cells are anergic to T cell receptor stimulation and highly susceptible to activation-induced apoptosis (Taams et al, 2001). Indeed, CD25+ CD4 Treg cells co-express high levels of CD25 and cytotoxic T lymphocyte antigen (CTLA)-4, a negative regulator of T cell activation (Baecher-Allan et al, 2001; Dieckmann et al, 2001; Jonuleit et al, 2001; Levings et al, 2001).

Only few studies have specifically addressed CD25 expression on CD4 T cells from patients with MS, and in these studies no major differences were observed between patients with MS and healthy controls (Khoury et al, 2000; Wu et al, 2000). We found a significantly lower expression of CD25 on CD4 T cells from the CSF of patients with POSMS or RRMS than in patients with non-inflammatory neurological diseases (VI). Interestingly, this change was specific for a CD4 T cell subset that coexpressed CD25 and HLA-DR whereas there was no significant difference in the percentage of HLA-DR- CD25+ CD4 T cells between patients with POSMS, RRMS, and controls (VI).

A decrease in the expression of HLA-DR on T cells in peripheral blood was recently found to precede clinical and MRI disease activity in MS whereas there was no concomitant decrease in CD25 expression (Khoury et al, 2000). The decrease in HLA-DR expression in blood does not appear to reflect the migration of activated, HLA-DR positive cells to the brain as we and others found that even in CSF a lower percentage of T cells from patients with active MS express HLA-DR (VI, Fredrikson et al, 1987a; Konttinen et al, 1987; Oksaranta et al, 1995), and T cells in MS plaques do not express HLA-DR (Hayashi et al, 1988).

CD25 and HLA-DR expression is often considered to reflect T cell activation, but there is no simple relationship between CD4 T cell expression of CD25 and HLA-DR. The kinetics of HLA-DR expression on stimulated human T cells in vivo differs from that of CD25 expression, and HLA-DR expression after in vitro T cell activation is a late phenomenon which is temporally associated with decreasing levels of T cell proliferation (Yachie et al, 1983; Ko et al, 1979). As many CD25+ CD4 Treg cells coexpress HLA-DR, we suggest that there could be an insufficient recruitment of CD4 Treg cells to the CNS in MS (Baecher-Allan et al, 2001; Dieckmann et al, 2001; Jonuleit et al, 2001; Levings et al, 2001).

3.4 Genetic control of T cell activation

The HLA haplotype associated with the DRB1*1501 allele confers susceptibility to MS. The DRB1*1501 molecule has a high affinity for myelin peptides, the DRB1*1501 allele is associated with T cell reactivity to myelin proteins, and transgenic mice expressing a human MBP-specific T cell receptor, human CD4, and DRB1*1501 are highly susceptible to EAE (Ota et al, 1990; Valli et al, 1993; Wallström et al, 1998; Madsen et al, 1999). These findings suggest that the peptide binding characteristics of DRB1*1501 could directly influence susceptibility to develop MS. Other studies have, however, suggested that the closely linked DQB1*0602 is a susceptibility allele independent of DRB1*1501 (Serjeantson et al, 1992; Spurkland et al, 1997; Caballero et al, 1999). Furthermore, it has been reported that T cell clones from DRB1*1501 positive subjects have increased production of TNF- α regardless of the HLA restriction element used in peptide recognition (Zipp et al, 1995; Vandevyver et al, 1998).

We found lower expression of HLA-DR molecules on CD4 T cells from patients carrying DRB1*1501 (VI). All patients had a lower percentage of CD4 T cells coexpressing CD25 and HLA-DR than did neurological control subjects. Carriers of DRB1*1501 did, however, have a further loss of HLA-DR+ CD4 T cells not expressing CD25. Although patients carrying DRB1*1501 also had somewhat higher levels of intrathecal IgG synthesis and CSF activity of MMP-9, the latter differences were not statistically significant after correction for multiple comparisons. These findings suggest that alterations in T cell activation, i.e., a lower level of HLA-DR on CD4 T cells is associated with the increased risk of developing MS conferred by the DRB1*1501 allele.

3.5 Methylprednisolone treatment and
T cell activation

Methylprednisolone treatment resulted in an increase in CD25 and HLA-DR expression on CD4 T cells in CSF (IV). As CD4 Treg cells from healthy volunteers express CD25 and HLA-DR, this could reflect a treatment-induced increase in the activity of Treg cells. A glucocorticoid-induced TNF-receptor (GITR) is over-expressed in regulatory T cells, and modulation of GITR activity is associated with changes in suppressive activity, suggesting that this molecule could be involved in glucocorticoid-induced generation of Treg activity (Gavin et al, 2002; McHugh et al, 2002; Shimizu et al, 2002). It is, however, also possible that the increase in CD25 expression after methylprednisolone treatment reflects a general increase in the expression of cytokine receptors, a recognized effect of glucocorticoids (Almawi et al, 1996).

CD26 expression decreased significantly on CD4 T cells in peripheral blood and CSF after treatment with oral high-dose methylprednisolone (IV). This finding suggests that a decrease in Th1 cytokine production following glucocorticoid treatment may reflect a direct effect on CD4 T cell activation. Indeed, a decrease in CD4 T cell production of IFN- γ and decreased expression of the Th1-associated chemokine receptors CCR5 and CXCR3 was reported after methylprednisolone treatment of MS attacks in a recent study (Marinez-Caceres et al, 2002). This effect was observed one month after a three days treatment course but was not maintained after six months.

3.6 Conclusions

Several studies have shown that there is systemic and intrathecal T cell activation in MS. We extend the results of previous studies by showing that not only RRMS patients but also POSMS patients have systemic T cell activation as evidenced by increased expression of the CD26 molecule. The expression of other measures of T cell activation such as CD25 and HLA-DR was lower in patients with POSMS and RRMS than in neurological control subjects. As discussed above, the functional roles of CD25+ and HLA-DR+ CD4 T cells in the pathogenesis of MS remain to be established but in healthy humans CD4 Treg cells are characterized by coexpression of these molecules, and we hypothesize that the lower levels of CD25 and HLA-DR expression on CD4 T cells in POSMS and CDMS may reflect insufficient generation or insufficient recruitment of regulatory T cells to the CNS in MS. Conversely, increased expression of CD26 on CD4 T cells may be directly linked to the activation of pathogenetic T cells. An association of lower levels of HLA-DR expression on T cells with the MS-associated DRB1*1501 allele, and the observed increase in CD25 and HLA-DR expression and decrease in CD26 expression on CD4 T cells after methylprednisolone treatment are consistent with this hypothesis.

5. INTRATHECAL INFLAMMATION IN MS

5.1 Intrathecal inflammation in the
pathogenesis of MS

As discussed in section 1.2 CNS infiltration by T cells and mononuclear phagocytes is the hallmark of active MS lesions. Only few leukocytes, mainly activated memory T cells, cross the intact blood-brain barrier where they can be detected in low numbers in normal CSF, perivascular spaces and even in the brain parenchyma (Booss et al, 1983). However, in MS and EAE the expression of proinflammatory cytokines results in the expression of molecules which direct the migration of additional leukocytes across the blood-brain barrier (Ransohoff, 1999). In EAE induced by the adoptive transfer of myelin-reactive CD4 T cells these are first detected in the subarachnoid space and the perivascular space, presumably reflecting the ability of activated T cells to cross the intact blood-brain barrier (Skundric et al, 1993; Shin et al, 1995). In the perivascular space the autoreactive T cells undergo a secondary activation step by cognate recognition of myelin antigen presented by perivascular macrophages. This is followed by the recruitment of a broader range of T cells and monocytes from the blood and their migration into the brain parenchyma (Offner et al, 1993; Flügel et al, 2001). The recruitment of mononuclear phagocytes is essential as monocyte-depleted animals do not develop classical EAE in spite of persisting perivascular T cell infiltration (Brosnan et al, 1981; Huitinga et al, 1990, 1995; Tran et al, 1998).

Leukocyte migration is a complex, multi step process which is initiated by a weak adhesion between circulating leukocytes and endothelial cells, mediated mainly by selectins and their glycolipid ligands (Ransohoff, 1999). This allows rolling of the leukocyte along the vessel wall. If the leukocyte is activated by chemotactic factors, e.g., chemokines immobilized on the endothelium, tight adhesion between activated integrins on the leukocyte and immunoglobulin superfamily adhesion molecules expressed on the endothelium ensues. Chemokines are small chemotactic cytokines which are divided in four different families (CC, CXC, CX3C, and C chemokines) based on conserved amino acid motifs. They bind to G protein-coupled 7-transmembrane spanning receptors expressed on the surface of leukocytes (Olson and Ley, 2002). The chemokine-induced adhesion of leukocytes is followed by transmigration across the endothelium and migration into the parenchyma. The migration is directed by chemotactic cues and is facilitated by proteolytic enzymes, e.g., MMPs which degrade basement membrane and extracellular matrix proteins (Cuzner and Opdenakker, 1999; Ransohoff, 1999; Hartung and Kieseier, 2000).

5.2 Magnetic resonance imaging
of inflammation in MS

Gd-enhanced MRI is generally used as a measure of inflammatory activity in RRMS patients. This is based partly on histopathology studies showing that Gd-enhancing lesions are inflammatory with macrophage infiltration of the parenchyma, and partly on studies showing that the development of new lesions on T2-weighted MRI is generally preceded by the occurrence of Gd-enhancing lesions on T1-weighted MRI (Estes et al, 1990; Nesbit et al, 1991; Katz et al, 1993; Brück et al, 1997; Rovaris and Filippi, 1999). In patients with RRMS Gd-enhancing lesions on MRI are much more common than clinical attacks, but the presence of enhancing lesions is associated with clinical disease activity (Kappos et al, 1999).

We found that patients with POSMS had a lower prevalence of Gd-enhancing lesions in the brain than did patients with RRMS (III). This is consistent with the results of a previous study showing a higher prevalence of Gd-enhancing lesions in patients with ON as a symptom of RRMS than in patients with idiopathic ON (Frederiksen et al, 1997). We also addressed the relationship between Gd-enhancing lesions and spontaneous remission in placebo-treated patients from the oral high-dose methylprednisolone trials (III). An improvement of at least one point on the Kurtzke EDSS score (in patients with attacks of MS) or on the visual function system of the EDSS (in patients with ON) was taken as evidence of a clinically relevant improvement. We found that a higher proportion of patients without Gd-enhancing lesions on T1-weighted MRI improved spontaneously after a follow-up period of up to eight weeks. This does not imply that Gd-enhancement is irrelevant in patients without overt enhancement, but in such patients enhancement might be detectable only for a shorter period or only after the administration of higher doses of Gd-DTPA. Interestingly, the duration of Gd-enhancement correlates with the development of more destructive, hypointense lesions on T1-weighted MRI and more severe changes in magnetization transfer variables, and lesions that enhance only after the administration of triple doses of Gd-DTPA are active for shorter periods and may be less destructive than lesions that enhance after standard doses (Filippi et al, 1998a-b; Rovaris et al, 1998; Ciccarelli et al, 1999).

5.3 Cerebrospinal fluid measures
of intrathecal inflammation

Several studies have addressed the association between inflammation on Gd-enhanced MRI and blood and CSF measures of inflammation. An association between Gd-enhancement and blood T cell production of IL-2 or the expression of T cell activation markers has been observed in several studies (Calabresi et al, 1998a; Khoury et al, 2000; Jensen et al, submitted). A correlation between the CSF leukocyte count and Gd-enhancement was reported in some studies but was not generally confirmed (Trojano et al, 1996; Rieckmann et al, 1997; Calabresi et al, 1998c). There are also several reports of an association between increased serum and CSF concentrations of soluble forms of adhesion molecules with Gd-enhancing lesions on MRI but again the results of different studies are not consistent (Hartung et al, 1993, 1995; Mossner et al, 1996; Trojano et al, 1996; Giovannoni et al, 1997).

The detection of an association between Gd-enhancement on MRI and CSF levels of inflammatory markers may be complicated by the fact that the distance from the active lesion to the CSF influences the possibility to detect molecules produced in the lesion (Rieckmann et al, 1997). Nevertheless, we found an association between inflammation on Gd-enhanced MRI and CSF measures of inflammation as the area of Gd-enhancement correlated with the CSF activity of MMP-9 and the CSF leukocyte count (III). Intrathecal IgG synthesis also correlated with the area of Gd-enhancement on T1-weighted MRI and with the presence of abnormalities on T2-weighted MRI, confirming a previous report from our group (III, VII; Frederiksen et al, 1996).

Neopterin is a molecule produced by human monocytes and macrophages stimulated by IFN- γ and other proinflammatory cytokines, and neopterin has been used as a surrogate measure of proinflammatory cytokine activity (Hamerlinck, 1999). We found that the CSF concentration of neopterin as assessed by a commercially available radioimmunoassay did not correlate with Gd-enhancement, and the CSF concentration of neopterin decreased spontaneously both in methylprednisolone- and placebo-treated patients (IV). This finding is consistent with an earlier study showing a spontaneous decrease in the CSF concentration of neopterin during the course of an MS attack (Fredrikson et al, 1987b). Indeed, increased systemic neopterin production was reported to be associated with the initial development of Gd-enhancing lesions rather than with the maintenance of lesion activity (Giovannoni et al, 2000). We found that patients with high baseline concentrations of neopterin in CSF were somewhat more likely to respond to oral high-dose methylprednisolone treatment after one week but not after eight weeks, consistent with an association between neopterin production and the initiation of an attack but not with the maintenance of disease activity (III).

Transforming growth factor (TGF)- β has been implicated in the pathogenesis of many neurological diseases (Pratt and McPherson, 1998). In MS TGF- β expression has been associated with the control of inflammation (Beck et al, 1991; Link et al, 1993; Mokhtarian et al, 1994; Rieckmann et al, 1994, 1995; Correale et al, 1995; Carrieri et al, 1997; Bertoletto et al, 1999). Bioactive TGF- β can be measured by enzyme-linked immunosorbent assay (ELISA) using recombinant TGF- β receptor for catchment and anti-TGF- β antibody for detection of bound TGF- β . By this method the total TGF- β concentration can be measured after acid activation of latent TGF- β . We found a lower CSF concentration of total TGF- β 1 in untreated patients with attacks of MS than in neurological control subjects whereas active TGF- β 1 was not detected (IV). The CSF concentration of total TGF- β 1 correlated negatively with the area of Gd-enhancement on T1-weighted MRI (unpublished observations: Spearman's ρ = -0.44, p = 0.03, n = 23). This finding is in agreement with a previous study showing an inverse correlation between
TGF- β 1 mRNA expression in blood cells and MRI disease activity in MS (Bertoletto et al, 1999). Furthermore, high levels of TGF- β mRNA expressing blood cells has been associated with low levels of disability in MS (Link et al, 1993).

MMPs may be involved in the proteolytic degradation of basement membrane and intercellular matrix proteins during leukocyte migration, in the disruption of blood-brain barrier integrity, the degradation of myelin proteins during demyelination, and the induction of axonal pathology in MS (Cuzner and Opdenakker, 1999; Hartung and Kieseier, 2000; Newman et al, 2001). We used zymography to detect MMP-9 activity as 92 kD gelatinase activity, and we found that MMP-9 activity as assessed by zymography correlated with MMP-9 concentrations measured by ELISA (IV). We found increased CSF activity of MMP-9 in CSF from patients with POSMS and CDMS. Patients with MMP-9 activity in CSF had higher levels of intrathecal IgG synthesis, higher CSF leukocyte counts, and higher CSF concentrations of MBP and neopterin (V). A correlation between MMP-9 activity and the CSF leukocyte count was not found in one previous study (Paemen et al, 1994), but in two other studies such correlations were observed (Gijbels et al, 1992; Leppert et al, 1998). In the study by Leppert and coworkers a correlation between MMP-9 activity and the IgG index was also observed.

MMP-9 activity in CSF is not directly related to clinical disease activity as patients in remission and patients with relapses all have increased CSF concentrations of MMP-9 (Leppert et al, 1998). MMP-9 in CSF is, however, observed in its inactive 92 kD proform which requires proteolytic cleavage in complex proteolytic cascade reactions which are initiated by the activity of, e.g., membrane-type 1 MMP (MT1-MMP) or plasminogen activator proteins (Cuzner and Opdenakker, 1999; Hartung and Kieseier, 2000). Increased MT1-MMP and plasminogen activator activity has, indeed, been reported in MS (Akenami et al, 1996, 1997; Cuzner et al, 1996; Galboiz et al, 2001). The lack of correlation between MMP-9 activity and clinical disease activity could also reflect subclinical disease activity in some patients without overt clinical disease activity. Indeed, we found that MMP-9 activity in CSF correlated with the area of enhancement on T1-weighted MRI, and in two other studies increased MMP-9 activity in blood correlated with MRI disease activity (III; Lee et al, 1999; Waubant et al, 1999). We also found an association between presence of MMP-9 activity in CSF and increased risk of recurrent disease activity (V).

5.4 Genetic control of
intrathecal inflammation in MS

We found higher CSF activity of MMP-9 and higher levels of intrathecal IgG synthesis in patients carrying the DRB1*1501-associated HLA haplotype (VI). After correction for multiple comparisons these differences were, however, not significant. As discussed in section 3.5 the mechanism underlying HLA-associations in MS is not clear but DRB1*1501 positive patients produce higher levels of TNF- α than DRB1*1501 negative patients, suggesting that increases in proinflammatory cytokine production are involved (Zipp et al, 1995; Vandevyver et al, 1998).

The chemokine receptor CCR5 has been implicated in the pathogenesis of MS as it is expressed on an increased percentage of T cells and monocytes in blood and CSF from patients with MS, and CCR5 positive T cells produce mainly Th1 cytokines upon activation (Balashov et al, 1999; Sørensen et al, 1999b; Strunk et al, 2000; Zang et al, 2000). In addition, macrophages and microglia in MS lesions express CCR5, and MS patients have increased CSF concentrations of CCR5 ligands (Miyagishi et al, 1995; Balashov et al, 1999; Sørensen et al, 1999b; Simpson et al, 2000; Trebst et al, 2001). The CCR5 Δ 32 allele lacks 32 base pairs and encodes a truncated CCR5 protein not expressed at the cell surface. CCR5 Δ 32 homozygotes have no immunodeficiency phenotype, but have a slightly higher blood lymphocyte count and blood pressure (Nguyen et al, 1999). CCR5 is a coreceptor for the human immunodeficiency virus (HIV), and CCR5 Δ 32 homozygotes are resistant to HIV infection (Dean et al, 1996; Liu et al, 1996; Samson et al, 1996; Fischereder et al, 2001). CCR5 Δ 32 also confers some disease protection in rheumatoid arthritis, asthma, and organ transplantation (Garred et al, 1998; Gomez-Reino et al, 1999; Hall et al, 1999). CCR5 Δ 32 homozygotes may develop MS, but we found that CCR5 Δ 32 heterozygotes have prolonged relapse-free intervals (VII; Bennetts et al, 1997). Whether CCR5 D32 influences the age of onset in MS is controversial. We found a lower age of onset in CCR5 Δ 32 heterozygotes, whereas in familial MS onset of disease occurred later in CCR5 Δ 32 heterozygotes (VII; Barcellos et al, 2000).

The mechanism of a protective effect of CCR5 Δ 32 in MS is not clear, but this allele does not appear to be associated with major changes in intrathecal inflammation in POSMS and RRMS although we observed minor changes in nitric oxide production in CCR5 Δ 32 carriers (VII; Sellebjerg et al, 2002). Possibly CCR5 may be more important in monocyte recruitment, activation, or migration into the CNS parenchyma than in the recruitment of T cells and other leukocytes to the subarachnoid and perivascular space (Trebst et al, 2001).

5.5 Methylprednisolone treatment and intrathecal inflammation

As discussed earlier high-dose methylprednisolone treatment improves blood-brain barrier function during attacks of MS (III). The CSF concentration of TGF- β 1 also increased significantly after treatment with oral high-dose methylprednisolone (IV). It is possible that TGF- β may be directly involved in the resolution of lesion activity as there is a negative correlation between Gd-enhancement and CSF concentrations of TGF- β during attacks of MS, but the concomitant changes in CSF concentrations of TGF- β 1 and Gd-enhanced MRI activity does not definitely prove this to be the case.

In a previous study suppression of MMP-9 activity was reported immediately after high-dose methylprednisolone treatment (Rosenberg et al, 1996). We did, however, not find a persisting change in the CSF activity of MMP-9 or in the CSF leukocyte count one week after completing treatment with oral high-dose methylprednisolone (IV). This suggests that suppression of leukocyte recruitment and MMP production is not as important as changes in the activation status of the recruited leukocytes or the induction of TGF- β 1 expression after high-dose methylprednisolone treatment. Indeed, in an additional study we found no change in the CSF concentration of the chemokine CXCL10 (inflammatory protein of 10 kD, IP-10) after treatment with oral high-dose methylprednisolone (Sørensen et al, 2001). CXCL10 and its receptor CXCR3 have been implicated in the recruitment of T cells to the CSF in MS, and we found significant correlations between the CSF concentrations of CXCL10, MMP-9, and the CSF leukocyte count both before and after treatment (Balashov et al, 1999; Sørensen et al, 1999b, 2002).

5.6 Conclusions

There is compelling evidence suggesting a major role of parenchymal inflammation, as assessed by the presence of Gd-enhancing lesions on brain MRI, in the pathogenesis of attacks of MS. Gd-enhancement is temporally associated with attacks of MS and with the development of new lesions on T2-weighted MRI; patients with Gd-enhancement have prolonged attack duration; Gd-enhancement correlates with measures of intrathecal inflammation; Gd-enhancement is attenuated by methylprednisolone treatment; and patients with Gd-enhancement appear to benefit more from methylprednisolone treatment. Although Gd-enhancement correlated with several measures of intrathecal inflammation prior to therapy with oral high-dose methylprednisolone, the CSF leukocyte count and MMP-9 activity in CSF remained elevated after treatment when Gd-enhancement was still strongly suppressed. This finding suggests that whereas methylprednisolone treatment results in the resolution of parenchymal inflammation, leukocytes are still actively recruited across the blood-brain barrier. CSF leukocyte counts are higher early in the course of MS, when the attack rate is high, than at later stages where CSF leukocyte counts and attack rates tend to decrease (reviewed in Walker et al, 1985; Allen, 1991). In addition, CSF leukocyte counts, chemokine concentrations, and MMP-9 activity correlates in patients with RRMS, and patients with MMP-9 activity in CSF have shorter relapse-free intervals. These findings are consistent with a major role of leukocytes and MMP activity in disease activity in RRMS. However, as there is no simple relationship between these measures of inflammation and clinical disease activity in MS additional stages must be involved. The migration of monocytes into the CNS parenchyma, which is associated with Gd-enhancement in histopathology studies, could be one such stage. This would explain why CCR5 Δ 32 may confer some disease protection in RRMS as monocytes are the major leukocyte subset expressing CCR5 in CSF from POSMS and RRMS patients.

6. DISCUSSION AND FUTURE DIRECTIONS

6.1 Immune activation and inflammation in MS

Histopathologically MS is characterized by demyelinated plaques in the white matter of the CNS. Demyelination has long been thought to explain the conduction changes and ensuing symptoms and signs observed in MS. An axonopathy and axonal loss is also present in MS, and the latter is now thought to be a major determinant of irreversible impairment and disability (Ferguson et al, 1997; Trapp et al, 1998, 1999; Evangelou et al, 2000). The etiology of MS remains ill defined but is thought to be multifactorial with an interaction between genetic susceptibility and environmental factors. As discussed in section 3 and 5, there is good evidence that myelin-reactive T cells and intrathecal inflammation are critical in the pathogenesis of MS, and inflammation can induce demyelination and axonal pathology comparable to that observed in MS (McDonald, 1998; Trapp et al, 1999; Kornek et al, 2000; Dandekar et al, 2001). In addition inflammation can induce a reversible conduction block, an effect to which demyelinated axons are especially sensitive (Redford et al, 1997; Coles et al, 1999).

Studies of Gd-enhancement on T1-weighted MRI show that focal disruption of the blood-brain barrier is associated with clinical attacks of disease but also indicate a high level of subclinical disease activity in many patients with MS. Patients with ON as POSMS have less enhancing lesions than do patients with ON in CDMS, and enhancing lesions are less common in patients with benign MS (Thompson et al, 1992; Kidd et al, 1994; Frederiksen et al, 1997; Losseff et al, 2001). As Gd-enhancement correlates with macrophage infiltration, these findings are consistent with a major role of parenchymal inflammation, i.e., the presence of activated macrophages, in the pathogenesis of MS (Estes et al, 1990; Nesbit et al, 1991; Katz et al, 1993; Brück et al, 1997). Parenchymal inflammation appears to represent the end stage of a multi step process which is initiated by systemic T cell activation followed by the migration of activated T cells across the blood-brain barrier; secondary activation of myelin-reactive T cells within the CNS; and recruitment of additional mononuclear cells and migration of these into the CNS parenchyma. Autoantibodies appear to be involved in the pathogenesis in a subgroup of MS patients. Data obtained in patients with RRMS, including the results of the present studies, and results obtained in EAE generally support this model. Immune reactivity within the CNS may, however, also have beneficial effects and there is still much to be learned about the precise pathogenetic mechanisms in MS (Schwartz and Moalem, 2001).

Histopathology studies have shown that inflammation is a constant feature in active MS plaques, but the role of inflammation in chronic progressive disease is controversial. Patients with SPMS have an altered and to some extent more pronounced immune activation than do patients with RRMS (Balashov et al, 1997; Windhagen et al, 1998; van Boxel-Dezaire et al, 1999; Jensen et al, 2001; Sørensen and Sellebjerg, 2001). Indeed, the development of atrophy and SPMS is preceded by inflammatory activity as assessed by Gd-enhanced MRI, and patients with progressive disease have more widespread changes in blood-brain barrier permeability than do patients with RRMS (McLean et al, 1993; Miller et al, 2000a; Silver et al, 2001; Casanova et al, 2002).

Patients with more pronounced attack activity may be at greater risk of converting to an SPMS disease course, but this has not been observed in all studies (Confavreux et al, 1980; Weinshenker et al, 1991; Runmarker and Andersen, 1993; Trojano et al, 1995). Once the transition to SPMS has occurred attacks do not appear to have much impact on the disease course. Patients with PPMS and patients with SPMS without superimposed relapses have little disease activity on Gd-enhanced MRI but progression rates are similar in patients with or without superimposed attacks (Thompson et al, 1991; Kidd et al, 1996; Confavreux et al, 2000). It is possible that axonal loss and irreversible disability in MS may arise by different mechanisms. Immunoinflammatory changes may directly lead to axonal pathology as suggested by the correlation between inflammation and axonal loss observed in histopathology studies (Trapp et al, 1998; Kornek et al, 2000; Bitsch et al, 2000). Axonal loss in the absence of ongoing inflammation might be caused by the long term effects of demyelination and could resemble the axonal degeneration observed in patients with mutations in the gene encoding PLP. These patients develop a length-dependent axonal loss and in some cases a slowly progressive myelopathy resembling PPMS (Garbern et al, 2002). Elucidating the role of intrathecal inflammation in the pathogenesis of SPMS and PPMS is an important area in future MS research since it is in these stages that there is most development of severe disability.

In EAE susceptibility to autoimmune demyelination is under genetic control, and different genetic loci control susceptibility, chronicity and other disease characteristics (Butterfield et al, 2000). Loci involved in EAE are also involved in the pathogenesis of virus-induced demyelination and animal models of rheumatoid arthritis, and loci involved in the pathogenesis of animal models of autoimmune disease overlap with loci involved in human autoimmune diseases (Teuscher et al, 1997; Becker et al, 1998; Bergsteinsdottir et al, 2000). In a recent study it was found that there is an increased prevalence of autoimmune disease among relatives of patients with MS (Broadley et al, 2000). Furthermore, the prevalence of autoimmune diseases was higher in multiplex families than in families with single members affected with MS. These findings are consistent with a multifactorial model where loci that influence the general risk of autoimmune disease interact with loci that are involved in the pathogenesis of specific autoimmune diseases and are modified by environmental factors (Mackay, 2001; Wandstrat and Wakeland, 2001).

Understanding how distinct genes influence the immunoinflammatory processes in MS is at an early stage, and the association with the DRB1*1501-associated HLA haplotype is incompletely understood at the functional level (discussed in section 3). Similarly, the protection conferred by CCR5 Δ 32 in different immunoinflammatory diseases is not understood in detail (discussed in section 5). Advances in the fields of genetic epidemiology, immunogenetics, neurobiology, and immunology are expected to improve our understanding of the immunopathogenesis of MS but may well be complicated by heterogeneity in disease processes. Indeed, in the United Kingdom genome screening study differences in genetic linkage were observed between families where disease was associated with the MS-associated HLA haplotype and families where this did not appear to be the case (Coraddu et al, 1998).

6.2 Immunoregulation and glucocorticoids in MS

There is good evidence that myelin-reactive T cells are of pathogenetic significance at least in RRMS but myelin-reactive T cells can also be detected in blood from healthy volunteers and patients with non-inflammatory neurological diseases, suggesting that additional factors are pathogenetically important. T cell tolerance to self antigen is maintained at several different levels. Negative selection in the thymus accounts for the deletion of some T cells with high affinity for self antigens but is insufficient to ensure deletion of all myelin-reactive T cells (Wekerle et al, 1996; Klein et al, 2000). Peripheral deletion of self-reactive T cells and the induction of anergy to T cell receptor-mediated activation are additional mechanisms by which harmful self-reactivity is avoided (Walker and Abbas, 2002). Peripheral tolerance mechanisms are defective in patients with SPMS, possibly explaining the changes in systemic immune activation observed in SPMS (Correale et al, 1996; Balashov et al, 2000). The activation of myelin-reactive T cells is also subject to control by regulatory T cells (Furtado et al, 2001; Maloy and Powrie, 2001).

Pharmacological treatment with glucocorticoids is efficacious in MS, but endogenous glucocorticoids also appear to be involved in the natural regulation of disease activity. In a recent study cortisol release induced by a dexamethasone-corticotropin-releasing hormone stimulation test correlated negatively with the presence of Gd-enhancing MRI lesions, and endogenous glucocorticoid production is strongly involved in the regulation of autoimmunity in animal models (Tonelli et al, 2001; Schumann et al, 2002). Oral high-dose methylprednisolone treatment suppressed disease activity on Gd-enhanced MRI, and the clinical response to therapy was more pronounced in patients with enhancing lesions on the baseline MRI. Hypothetically this could be due to an insufficient induction of endogenous glucocorticoids in patients with prolonged attack duration and a more clear response to methylprednisolone treatment.

Glucocorticoids have multiple effects in the immune system. These include short-lasting effects on leukocyte recirculation patterns which, however, are unlikely to account for the efficacy of glucocorticoid treatment in MS as treatment with oral high-dose methylprednisolone has no persistent effect on the CSF leukocyte count, the CSF activity of MMP-9, and concentrations of CXCL10. Instead alterations in T cell activation and IgG and TGF-b1 synthesis may be involved. Glucocorticoids interact negatively with transcription factors involved in T cell activation and may induce apoptosis of activated T cells (Bosscher et al, 2000; Laethem et al, 2001; Leussinkk et al, 2001). These effects may contribute to the observed changes in CD26 expression on CD4 T cells after oral high-dose methylprednisolone. The functional relevance of the increase in CD4 T cells co-expressing CD25 and HLA-DR after oral high-dose methylprednisolone treatment remains to be determined, but we hypothesize that it could reflect an increase in regulatory T cell activity, possibly related to the induction of TGF- β which induces the differentiation of regulatory CD25+ CD4 T cells in humans (Yamagiwa et al, 2001).

Several studies have shown that whereas glucocorticoids suppress the expression of proinflammatory cytokines they induce TGF- β expression, and this may be an important effect of oral high-dose methylprednisolone treatment of MS. TGF- β inhibits the production of pro-inflammatory cytokines by effector T cells but may also promote the differentiation and survival of T cells by inhibiting the induction of apoptosis (Cerwenka et al, 1994; Link et al, 1995; Genestier et al, 1999; Gorelik et al, 2002). We found that patients with high serum concentrations of IgA had somewhat higher IgG indices, CSF leukocyte counts, prolonged attack duration, and a more pronounced response to oral high-dose methylprednisolone treatment (III). As TGF- β is an isotype shift factor for IgA these results suggest that the role of this cytokine in the pathogenesis of MS may extend beyond a presumed protective effect, but further studies of the relationship between TGF- β and IgA production in MS are needed to substantiate this hypothesis.

Systemic TGF- β 2 treatment did not attenuate disease activity in a previous phase 1 clinical trial in SPMS but this could be due to a loss of sensitivity to TGF- β in SPMS (Correale et al, 1996; Calabresi et al, 1998b). However, the induction by oral tolerance of myelin-reactive Th3 cells secreting TGF- β was not protective in a major clinical trial in patients with MS (Fukaura et al, 1996). Interestingly, the effect of TGF- β on T cell development differs in different inbred mouse strains, and in MS a protective effect of TGF- β on T cell activation may be restricted to DRB1*1501 positive patients suggesting that genetic heterogeneity in the response to TGF- β may be operative in MS (Hoehn et al, 1995; Söderstrom et al, 1995).

6.3 Strategies for glucocorticoid treatment
in MS

The definite aim of MS therapy is to inhibit the development of irreversible disability and associated handicap. Methylprednisolone treatment can increase the speed of recovery after an attack of MS, and two recent meta-analyses conclude that there is good evidence supporting the use of glucocorticoid treatment in attacks of MS but no evidence that the final outcome is better after methylprednisolone treatment. (Brusaferri and Candelise, 2000; Miller et al, 2000b). This does not rule out that such an effect could exist because studies of sufficient size and duration have not been conducted. The lack of a persistent effect of methylprednisolone in optic neuritis does not exclude a persistent effect in MS because spontaneous improvement is much more pronounced in ON than in attacks of RRMS. In a recent MRI study it was reported that methylprednisolone treatment results in the development of less severe changes in magnetization transfer ratios (Richert et al, 2001). This finding and a more pronounced clinical response to treatment observed in patients with active Gd-enhanced MRI suggests that treatment with high-dose methylprednisolone could prevent the development of more destructive lesions in patients with active Gd-enhanced MRI, and it was recently reported that treatment with intravenous high-dose methylprednisolone at 4 months intervals slowed the development of hypointense lesions on T1-weighted MRI and persistent disability in RRMS (Zivadinov et al, 2001). The role of glucocorticoids in chronic progressive MS is not clear. The role of inflammation in the pathogenesis of PPMS and SPMS is controversial, and myelin-reactive T cells from patients with SPMS were reported to have an impaired sensitivity to glucocorticoid-induced apoptosis (Correale et al, 2000). Nevertheless some clinical benefit has been observed after treatment of these patient categories with high-dose methylprednisolone, suggesting some contribution of glucocorticoid-responsive inflammatory responses to the pathogenesis in PPMS and SPMS (Milligan et al, 1987; Cazzato et al, 1995; Goodkin et al, 1998).

There is a need for studies establishing optimal treatment regimens for glucocorticoid treatment in MS. Unresolved questions relate to the optimal dosage, whether tapering is beneficial, and possible differences in the efficacy of different glucocorticoids and inter-patient differences in the biological response to glucocorticoids. It has been suggested that higher doses of methylprednisolone are associated with more pronounced improvement in paraclinical measures of disease activity than are the commonly used doses of 500-1000 mg daily, but it is not clear if these differences translate into meaningful differences in clinical efficacy (Oliveri et al, 1998; Fierro et al, 2002). It is commonly assumed that different glucocorticoids have similar biological effects, but there is evidence that this is not the case. The induction of responses such as the induction of gene expression and apoptosis, repression of the transcription factor NF- κ B, and inhibition of lymphocyte proliferation in vitro varies widely between different glucocorticoids, but it is not known if these differences are important in mediating in vivo effects of glucocorticoids in inflammatory disease states (Fauci, 1976; Langhoff and Ladefoged, 1983; Hofmann et al, 1998). In addition, the response of inflammatory cells to glucocorticoid treatment is not constant but is modulated by the activation status of the cell (Heijnen and Kavelaars, 1999). There are no major changes in glucocorticoid binding to glucocorticoid receptors in mononuclear cells from patients with MS, but it has been reported that the response to glucocorticoid treatment is impaired in patients who have MBP-reactive T cells in peripheral blood that are non-responsive to glucocorticoid suppression of antigen-induced proliferation (Webb et al, 1974; Bergh et al, 1999).

Currently available disease modifying drugs have only limited efficacy in RRMS and there is clearly a need for better treatments. The role of glucocorticoids in combination therapy with disease modifying drugs is an active research area. A temporary suppression of Gd-enhanced MRI disease activity is observed after treatment with methylprednisolone whereas treatment with IFN- β and glatiramer acetate results in long term suppression of Gd-enhancement. However, all three drugs promote recovery of MRI lesions as assessed by T1 hypointensity or magnetization transfer (Comi et al, 2001; Filippi et al, 2001; Richert et al, 2001). The suppression of Gd-enhanced MRI disease activity by methylprednisolone is more pronounced and of a longer duration in IFN- β -treated patients, suggesting that the combination of IFN- β therapy and regular treatment with high-dose methylprednisolone could be of benefit in patients with RRMS (Gasperini et al, 1998). Glucocorticoids and IFN- β could have synergistic effects due to different effects on immunopathogenetic mechanisms, and it has been suggested that glucocorticoids could lower the prevalence of neutralizing antibodies to IFN- β (Pozzilli et al, 2002). Definite clinical trials are, however, needed to establish the role of glucocorticoids in combination therapy.

A major challenge in MS research is understanding the impact of histopathology and immunogenetic heterogeneity on the pathogenesis of different subtypes of MS. Parallel studies of immune activation, immune regulation, immunogenetics and histopathology together with studies addressing phenotypic differences in subgroups of patients with MS are expected to lead to an improved insight into the pathogenesis of MS. Such studies will help in defining the role of glucocorticoid treatment given alone or in combination with other drugs in the future management of patients with MS.

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