Introduction

Multiple sclerosis is an autoimmune disease affecting central nervous system myelin. Myelin antigen-specific autoreactive cells are activated in the periphery then migrate to the central nervous system where they initiate an inflammatory response.
Several relatively abundant CNS proteins including myelin basic protein (MBP) (Ben-Nun et asl. 1981, Vandenbark et al. 1985), proteolipid protein (PLP), myelin oligodendrocyte-associated protein (MOG) and S-100 can be recognized in the central nervous system (CNS) by activated T cells. MBP and PLP can easily induce experimental inflammatory CNS disease in animals when injected with adjuvant, while CNS inflammation with MOG and S-100 appear to require adoptive transfer of antigen reactive T cells activated in vitro to naive animals. Further, while activation of T cells with reactivity to one CNS antigen may initiate autoimmune disease, with time, T cells with reactivity to other antigens will become activated and these cells are capable of autoimmune tissue destruction. T cells recognizing MBP and PLP exist in normal humans.
From investigation of the animal model for MS, it has been learned that for these cells to be pathogenic they must be activated in vivo. While there are no differences in the frequencies of MBP and PLP-reactive T cells after primary antigen stimulation, the frequency of MBP or PLP but not tetanus toxoid (TT) reactive T cells generated after primary rIL-2 stimulation are significantly higher in MS patients as compared to control individuals (Zhang et al. 1984). Thus, there is an absolute difference in the activation state of myelin reactive T cells in the central nervous system of patients with MS.
If circulating, autoreactive T cells are present in the circulation of normal individuals, how do they become activated? Possible mechanisms in the absence of autoantigen involve immune activation associated with infections which include: 1) molecular mimicry, 2) activation by superantigens.
1. Molecular mimicry implies that epitopes of an infectious agent such as bacteria or viruses which induce an immune response in the host are present on self-proteins of the host. The immune response directed against such cross-reactive epitopes of the foreign invader may possibly result in the activation of autoreactive T cells and autoimmunity. An example of this is the Hepatitis B virus polymerase, which cross-reacts with an epitope of myelin basic protein. Injection of this hepatitis virus protein with adjuvant into rabbits induces an inflammatory CNS disease as evidenced by histological analysis.
2. Superantigens, which bind to class II antigens and specific T cell receptor (TCR) V? segments may activate T cells. Such superantigen activation may occur during the course of bacterial or viral infections, and injection of superantigens can trigger relapsing EAE in susceptible mice. As superantigens drive T cell expansion which use specific TCRs, it is possible that the oligoclonal T cells in the blood and CSF of MS patients are driven by exposure to a superantigen. This also raises the possibility that the oligoclonal immunoglobulin bands found in MS CSF are also related to T cell activation by superantigen.
The past decade has provided a rich interaction between the fields of neurology and immunology. This has given rise to improved understanding of the pathogenesis of multiple sclerosis and the development of new therapies that target specific immune pathways. The demyelinating process in multiple sclerosis (MS) involves T-cells, immunoglobulins and complement, but recent evidence shows that cytokines, chemokines, adhesion molecules, metalloproteinases, nitric oxide and oxygen metabolites, all participate in the effector stages of the disease, and can therefore be potential therapeutic targets.

Therapies targeting T cell costimulatory signal blockade

Background: CD4+ T cells play a critical role in initiating the immune response leading ultimately to the effector mechanisms mediating autoimmune disease. For T cells to become activated, the T cell receptor (TCR) must recognize the target antigen in the form of a processed peptide presented on MHC molecules expressed on the surface of antigen-presenting cells (APCs). In the case of autoimmunity the antigenic peptide is derived from specific autoantigens after processing and presentation by self-APCs. TCR recognition of a peptide+MHC complex provides a signal (signal 1), which results in initial T cell activation. Full activation however, does not occur unless the T cell receives a costimulatory signal (signal 2) provided by the interaction of specific receptors on T cells with their ligands on APCs. The best-characterized costimulatory signal is that provided by CD28 on T cells interacting with the B7 {CD80 (B7-1) and CD86 (B7-2)} family of molecules on APCs. Blockade of this pathway induces a state of antigen-specific T cell anergy in vitro, and induces tolerance in experimental autoimmune and transplantation models in vivo (Bluestone 1995; Sayegh and Turka 1998). Another recently characterized T cell costimulatory pathway is provided by interaction of CD40 on the surface of APCs/B cells with CD40 ligand (CD40L) on the surface of activated T cells. CD40 may provide a direct costimulatory signal for full T cell activation. There is also evidence that engagement of CD40 and CD40L leads to upregulation of B7 expression on APCs. In addition, CD40-CD40L interaction is important in B cell and monocyte/macrophage activation. CD40L-CD40 interaction is essential for B cell survival and Ig switching. Ligation of CD40 molecules triggers IL-12 production in monocytes/dendritic cells.
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP) or its major encephalotigenic peptide and adjuvant. It has been used as a model for the study of MS, and many treatments for MS were initially tested in the EAE model. The effects and mechanisms of inhibiting EAE by blockade of the CD28-B7 costimulatory pathway by the fusion protein CTLA4Ig or anti-B7 monoclonal antibodies (mAbs) have been investigated by several laboratories (Khoury, Akalin et al. 1995). Similarly, blockade of CD40-CD154 interactions was shown to protect mice from clinical EAE (Schaub, Issazadeh et al. 1999). Furthermore, CD40 was identified in MS brain lesions (Gerritse, Laman et al. 1996), and expression of CD40 in the central nervous system (CNS) of mice correlates with the bouts of clinical symptoms during the course of EAE. Blockade of CD28-B7 or CD40-CD154 pathways was successful in preventing disease or ameliorating ongoing disease in numerous other autoimmune disease models (Durie, Fava et al. 1993; Finck, Linsley et al. 1994; al. 1996). Furthermore, the approach of costimulatory signal blockade was also successful in preventing transplant rejection (Sayegh and Turka 1998)

Clinical trials: Based on the success of this approach in animal models, clinical trials have been initiated. A phase I clinical trial of CTLA4Ig (Bristol-Myers Squibb) treatment in psoriasis was completed and showed the treatment to be safe and demonstrated a hint of efficacy in controlling clinical and histologic disease (Abrams, Lebwohl et al. 1999). A phase I clinical trial of CTLA4Ig (RG2077 from Repligen Corp.) in MS is in the planning stages, and a phase II trial with the Bristol-Myers Squibb product is also being planned.
Anti-CD154 trials were initiated in several autoimmune diseases by Biogen, but the trials were all halted after the occurrence of thrombotic events in lupus patients. A phase I study of anti-CD154 from IDEC in MS was completed. It included 12 patients with relapsing-remitting disease; the primary outcome measure was safety. The study was designed as a dose escalation and showed this antibody to be safe as administered. A phase II study in MS is scheduled to start shortly with this material.

Therapies targeting cytokines

Background: IL-12 is a heterodimeric cytokine produced mostly by phagocytic cells and induces cytokine production, primarily of IFN-?, from T cells. Several studies in humans and in the mouse have assigned a role to IL-12 as the promoter of Th1 cell generation, acting in antagonism with IL-4, the major promoter of Th2 responses. Administration of IL-12 to mice after transfer of encephalitogenic cells resulted in increased severity and duration of EAE; treatment with anti-IL-12 antibodies substantially reduced the incidence and severity of adoptively transferred EAE (Leonard, Waldburger et al. 1995). IL-12 message has been detected in MS brains (Windhagen, Newcombe et al. 1995), and we have found increased anti-CD3 induced IL-12 secretion in patients with progressive disease (Balashov, Smith et al. 1997). Elevated serum levels of IL-12 have been reported in the chronic progressive form of MS (Nicoletti, Patti et al. 1996). We have recently reported that IL-12 production is elevated in monocytes from MS patients and that treatment with cyclophosphamide/methylprednisolone monthly boosters normalized IL-12 production (Comabella, Balashov et al. 1998). Furthermore, we found that IL-12 production was linked to disease activity, with higher production in patients with active disease (Comabella, Balashov et al. 1998) and in patients with gadolinium enhancing lesions on MRI (Makhlouf, Weiner et al. 2001). Because of its key role in MS and EAE, treatments targeting IL-12 are of potential interest in MS.

Clinical trials: Salbutamol (albuterol in the USA) a ?2-agonist selectively inhibits the production of IL-12 by human monocytes in vitro and in vivo in healthy subjects, through increased intracellular cAMP. In animal models of autoimmune disease, ?2-agonists were shown to suppress chronic-relapsing EAE in Lewis rats, and to suppress collagen-induced arthritis, a murine model for rheumatoid arthritis (Malfait, Malik et al. 1999). Furthermore, ?2-adrenergic receptor expression is increased on peripheral blood mononuclear cells (PBMC) of patients with MS (Arnason, Noronha et al. 1988), and is correlated with clinical and MRI disease activity. We have shown that oral administration of Salbutamol decreases the percentage of IL-12-producing monocytes in patients with progressive MS (Makhlouf, Weiner et al. 2001). We are currently testing the efficacy of Albuterol as an add-on therapy to glatiramer acetate in a phase II study.
Another class of drugs, the phosphodiesterase inhibitors (PDEI), also target cAMP and increase its intracytoplasmic level by inhibiting its degradation by PDE. Rolipram, a type IV PDEI, is the most extensively studied: it is shown to suppress IL-12 in mice, to prevent EAE in rats (Sommer, Loschmann et al. 1995) and in non-human primates (Genain, Roberts et al. 1995). An additional protective mechanism for Rolipram in EAE in mice is its ability to reduce the BBB permeability. Although mostly known as an antidepressant in humans, Rolipram has, like SB, a therapeutic potential in Th1–mediated autoimmune (Bielekova, Goodwin et al. 2000), and is now being tested in a clinical trial in MS patients

Therapies targeting adhesion molecules:

Background: Adhesion molecules promote cell-cell and cell -extracellular matrix interactions, and as such, are involved in many steps of the immune response, in particular in the migration of inflammatory cells through the blood-brain barrier (BBB) into the CNS. They are classified into three families according to their structure: immunoglobulin (Ig) superfamily members, integrins, and selectins. The expression of intercellular cell adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 is upregulated on brain microvessel endothelial cells in active lesions of MS (Cannella and Raine 1995), concomitantly with upregulation of the expression of their respective receptors on leucocytes (Leukocyte function antigen (LFA)-1 for ICAM-1, and very large antigen (VLA)-4, also named alpha 4-integrin, for VCAM-1).
Anti-alpha 4 integrin monoclonal antibody treatment reduced cellular infiltration in CNS and inhibited development of EAE in rats and guinea pigs, and could also reverse the ongoing disease process in the guinea pig. Anti ICAM –1 monoclonal antibody inhibits EAE in rats (Archelos, Roggenbuck et al. 1993), although in another study, neither anti- ICAM-1 nor anti LFA-1 could alter the course of EAE (Cannella, Cross et al. 1993). However, ICAM-1 deficient mice develop more severe EAE than in controls, suggesting that ICAM-1 plays an important role in down-regulating autoimmune inflammation in the CNS (Samoilova, Horton et al. 1998).
Circulating ICAM-1 is the most studied adhesion molecule in MS: its serum levels are elevated in active RR-MS, and sICAM-1 levels correlate with magnetic resonance imaging disease activity (Giovannoni, Lai et al. 1997; Khoury, Orav et al. 1999).

Clinical trials: In 1999, a randomized, double-blind placebo-controlled trial of a humanized anti-alpha 4 integrin antibody was performed on 72 patients with RR and SP MS (Tubridy, Behan et al. 1999): this study showed a significant reduction in the number of new active lesions on magnetic resonance imaging (MRI). The drug was given intravenously and was well tolerated, but the study was not designed to look at the effect on the relapse rate. More recently, the results of a phase II clinical trial with a humanized anti-alpha-4 integrin antibody (Natalizumab) were reported at the annual meeting for the European Congress on Treatment and Research in Multiple Sclerosis. A placebo-controlled trial of 213 patients (relapsing remitting or secondary progressive) was conducted at 26 sites in the US, Canada and the UK by Elan pharmaceuticals. The study included 2 dose groups (3mg/Kg, and 6mg/Kg) and a placebo group. Treatments were administered intravenously at 4–week intervals for six months. The primary analysis showed that patients treated with Natalizumab for six months had fewer gadolinium-enhancing lesions than patients receiving placebo. Phase III clinical trials with Natalizumab as a single agent or as an add-on to Avonex have been initiated.

Therapies targeting matrix metalloproteinases:

Background: Matrix metalloproteinases (MMPs) are a family of zinc-containing endo-proteinases that share structural domains but differ in substrate specificity, cellular sources, and inducibility. MMPs can degrade any protein component in the extracellular matrix, including but not limited to, membrane bound adhesion molecules, cytokine precursors and receptors, and pro-forms of MMP. Most of MMP are secreted by a wide range of cell types as proenzymes that need to be cleaved in order to get activated. Except for the membrane-type MMP, all other MMP are secreted into the extracellular space including in the CNS, where their lytic activity has to be finely regulated to avoid potential tissue destruction.
In animal models, the injection of MMP-7, -8 and –9 in the brain parenchyma of rats is followed by breakdown of the blood-brain barrier and leukocyte recruitment into the CNS (Anthony, Miller et al. 1998). MMP-7 and –9 mRNA expression is dramatically upregulated at the peak of clinical disease in EAE, and some MMP inhibitors (MMPI) can suppress the development of EAE in rats (Hewson, Smith et al. 1995), or reverse ongoing clinical EAE in mice (Gijbels, Galardy et al. 1994).
There is evidence that MMPs are also involved in the BBB breakdown in MS patients: thus, MMP-9 is increased in the CSF of MS patients during clinical relapses (Leppert, Ford et al. 1998). High serum MMP-9 levels are significantly associated with more T1-weighted gadolinium-enhancing MRI lesions. Treatment with high-dose methylprednisolone (which is known to downregulate MMP) is shown to reduce both MRI gadolinium enhancing lesions and CSF level of MMP-9 in MS patients (Rosenberg, Dencoff et al. 1996).

Clinical trials: There are no ongoing trials with MMPI in MS, but such drugs are currently being tested in other autoimmune diseases and cancers (Brown 2000). Naturally occurring MMPI, called tissue inhibitors of metalloproteinases (TIMP), are involved in the regulation of MMP expression, and it has been suggested that an abnormality in the inhibitory response to MMP might play an etiological role in the chronicity of multiple sclerosis (Lee, Palace et al. 1999).

Therapies targeted to neuroprotection

Background: Axonal pathology appears early in the disease course of multiple sclerosis (MS), and may play a critical role in disease progression (Trapp, Peterson et al. 1998). However, it is still unclear whether axonal pathology is the primary event or whether it is a consequence of neuronal toxicity. Neuronal toxicity may be mediated by components of the immune system or through excitotoxicity. L-Glutamate (Glu) is the most widespread excitatory transmitter system in the vertebrate CNS. Glu mediates its effects through two general classes of receptors, those that form ion channels or "ionotropic" such as the kainate, AMPA, and NMDA receptors, and those that are linked to G-proteins or "metabotropic". Glu is not only produced by neurons and glial cells, but also by cells of the immune system, like macrophages and T-cells, and Glu receptors are expressed both in neuronal and glial membranes. It was reported that activated immune cells release large amounts of Glu in the murine CNS (Piani, Frei et al. 1991), and like neurons, oligodendrocytes are highly sensitive to AMPA/kainate receptor-mediated death. Furthermore, Glu degradation is downregulated in astrocytes during EAE, due to glutamine synthetase and glutamate dehydrogenase reduced expression, thus leading to an increase of Glu in the CNS (Hardin-Pouzet, Krakowski et al. 1997). Recently, NBQX an AMPA/kainate receptor antagonist, was shown to improve clinical EAE and increase oligodendrocyte survival, without reducing the lesion size nor the degree of CNS inflammation, both in SJL mice and in Lewis rats (Smith, Groom et al. 2000), suggesting that Glu excitotoxicity is an important mechanism in autoimmune demyelination.

Clinical trials: In humans the level of Glu was increased in the CSF of patients during acute attacks of MS (Stover, Pleines et al. 1997). Serum Glu is also elevated during relapses (Westall, Hawkins et al. 1980). AMPA antagonists are now being tested in stroke patients. There are no clinical trials of Glu receptors antagonists in MS patients

Conclusions:

Modern biotechnology and improved understanding of the immunopathology of MS have led to the development of new therapeutic targets for the disease. Most of the strategies outlined in this chapter are in the early phases of clinical investigation. Although it is not always straightforward to extrapolate from animal studies to humans, EAE and other animal models of MS have made it possible to bring MS patients new effective treatments and new hopes for their disease.


Samia J. Khoury, M. D.
Associate Professor of Neurology Harvard Medical School
Co-Director, Partners MS Center
Director, Clinical Immunology Laboratory Center for Neurologic Diseases, Brigham and Women's Hospital

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