Therapeutic role of beta-interferons in multiple sclerosis

Pharmacology & Therapeutics 110 (2006) 35 – 56 www.elsevier.com/locate/pharmthera

Associate editor: M.M. Mouradian

Therapeutic role of beta-interferons in multiple sclerosis Adil Javed, Anthony T. Reder * Department of Neurology, MC-2030, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA

Abstract Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). In the last 12 years, there has been a proliferation of studies elucidating the immune mechanisms that mediate tissue damage in MS. Interferons (IFNs) have an important role in regulating innate and adaptive immune responses. They decrease pro-inflammatory responses such as the autoimmunity in MS, but other autoimmune responses such as systemic lupus erythematosus (SLE) may be exacerbated. This review offers a general overview of the biological properties of IFNs, effects on immune cells, and clinical effectiveness in MS treatment. IFN signaling is complex, from receptor binding events to the generation of effector mechanisms that dampen inflammation. Immune cell function is altered in MS. IFN treatment of MS patients ameliorates immune dysfunction, but not completely. The incomplete resolution of immune dysfunction by IFNs partly explains their significant, but modest therapeutic effects. This observation also suggests that there are immune mechanisms in MS that are resistant to IFN therapy. In MS, abnormalities may exist at several points along the IFN signaling pathway, including molecular defects in the IFN second messenger system. Currently, several studies are ongoing evaluating ways of potentiating IFN effects. IFNs were the first agents to show clinical efficacy in treatment of MS. More than a decade of experience with IFNs has showed continued clinical efficacy over time. In the near future, IFNs will continue to play a major role in MS. D 2005 Elsevier Inc. All rights reserved. Keywords: Multiple sclerosis; Interferon-h; IFNAR; STAT; Kinase; Immunology; Lymphocytes; Pivotal trials; MRI; Natalizumab; SLE; NAb Abbreviations: APC, antigen presenting cell; BBB, blood – brain barrier; CNS, central nervous system; DMA, disease modifying agent; EC, endothelial cells; EDSS, Expanded Disability Status Scale; GA, glatiramer acetate; IFN, interferon; IFNAR, type I interferon receptor; IRF, interferon regulatory factor; IVIG, intravenous immunoglobulin; MBP, myelin basic protein; MNC, monocytes; MOG, myelin oligodendrocyte glycoprotein; MRI, magnetic resonance imaging; MS, multiple sclerosis; PBMC, peripheral blood monocytes; PML, progressive multifocal leukoencephalopathy; PP, primary progressive; RR, relapsing remitting; SLE, systemic lupus erythematosus; SP, secondary progressive; STAT, signal transducers and activator of transcription; Th0, naı¨ve T cell; Th, T helper cell; Tr, T regulatory cell; Ts, T suppressor cell; VLA-4, very late antigen-4.

Contents 1.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Natural history of multiple sclerosis . . . . . . . . . . . 1.2. Pathology of multiple sclerosis lesions . . . . . . . . . . 1.3. Treatment options for multiple sclerosis . . . . . . . . . Interferons: pleotropic effects . . . . . . . . . . . . . . . . . . 2.1. Antiviral . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Antitumor. . . . . . . . . . . . . . . . . . . . . . . . . Molecular mechanism of action of interferons . . . . . . . . . . 3.1. Interferon receptors . . . . . . . . . . . . . . . . . . . . 3.2. Intracellular signaling. . . . . . . . . . . . . . . . . . . 3.3. Receptor cross talk . . . . . . . . . . . . . . . . . . . . Multiple sclerosis and molecular defects in interferon signaling .

* Corresponding author. Tel.: 773 702 6204; fax: 773 702 9076. E-mail address: [email protected] (A.T. Reder). 0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.08.011

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A. Javed, A.T. Reder / Pharmacology & Therapeutics 110 (2006) 35 – 56

5.

Immune system and multiple sclerosis . . . . . . . . . . . . . . . . . . . . . . . 5.1. Normal immune responses . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Immune dysregulation in multiple sclerosis . . . . . . . . . . . . . . . . . 5.3. Immunomodulatory effects of interferon-h in multiple sclerosis . . . . . . 6. Multiple sclerosis and systemic lupus erythematosus: polar opposites in responses 7. Therapeutic trials of interferons in multiple sclerosis . . . . . . . . . . . . . . . . 7.1. Short-term efficacy of interferons in multiple sclerosis . . . . . . . . . . . 7.2. Long-term efficacy of interferons in multiple sclerosis . . . . . . . . . . . 7.3. Interferons and progressive multiple sclerosis . . . . . . . . . . . . . . . . 7.4. Interferons and neutralizing antibodies . . . . . . . . . . . . . . . . . . . 7.5. Adverse effects of interferons . . . . . . . . . . . . . . . . . . . . . . . . 8. Interferons and combination therapy . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Role of natalizumab in multiple sclerosis . . . . . . . . . . . . . . . . . . 8.2. Combination of interferons with glatiramer acetate . . . . . . . . . . . . . 9. Future of interferon therapy in multiple sclerosis. . . . . . . . . . . . . . . . . . 10. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Multiple sclerosis (MS) is a chronic, demyelinating disease of the central nervous system (CNS). It affects more than 2 million people in the world and is a major cause of disability in the young adult population. The etiology of MS is unclear. However, both genetic susceptibility and environmental factors, including pathogens and diet, are thought to play a role in the pathogenesis. MS is an immune-mediated process. Its pathological hallmark is perivascular infiltration of immune cells across the blood –brain barrier (BBB). T cells, macrophages, monocytes, and B cells all play a major role. Immune cells invade into the brain parenchyma and cause demyelination by destroying the myelin, eliciting local CNS immune responses from resident microglia, or causing oligodendrocyte death by toxicity. The pathology of MS lesions has been carefully described in biopsied and postmortem tissue (Gay et al., 1997; Lucchinetti et al., 2000; Lassmann, 2004). These studies highlight the importance of inflammatory cellular and humoral immune mechanisms and oligodendrocyte degeneration. Magnetic resonance imaging (MRI) is an important diagnostic tool for assessing inflammatory activity in the CNS. MS lesions appear as ovoid T2 hyperintensities around the ventricles, usually in an orientation perpendicular to the ventricles (Fig. 1). T2 images on the MRI show inflammation and represent a combination of tissue edema and gliosis. T1 images are more useful for detecting acute MS lesions and chronic inactive lesions, and for evaluating brain atrophy (Fig. 1). An acute MS lesion enhances with contrast on a T1 scan. The chronic, inactive lesions on a T1 scan appear as hypointensities (‘‘black holes’’). Inflammatory MRI lesions usually precede the onset of clinical symptoms. It is estimated that MRI lesions occur 5 – 10 times more frequently than clinical relapses.

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42 42 42 43 45 45 46 47 48 48 48 49 49 50 50 50 51 51

There is no cure for MS, but immunomodulatory therapies are available that ameliorate the disease. These include the 3 different formulations of interferon-h (Table 1), glatiramer acetate (GA), and mitoxantrone. Natalizumab [anti-very late antigen-4 (VLA-4) antibody] was approved for MS in November 2004. It was suspended from the market pending further safety analysis. Of all the drugs available for MS, the interferons (IFNs) have been the best characterized.

Fig. 1. MRI scans of patients with RRMS. (A) FLAIR scan showing ovoid hyperintensities, as indicated by an arrow, representing inflammation. (B) Sagittal FLAIR image showing hyperintensities around the ventricle, often radiating outward at 45- angles. (C) T1 scan showing chronic, inactive lesions, ‘‘black holes’’ indicated by arrows. (D) Contrast T1 scan showing acute enhancing lesions, arrows. Note ‘‘open ring’’ sign of pericortical lesion, where gray and white matter are different immunologically.

A. Javed, A.T. Reder / Pharmacology & Therapeutics 110 (2006) 35 – 56 Table 1 Interferons approved for multiple sclerosis Drug

Trade name

Dose

Route

IFN-h-1b IFN-h-1a IFN-h-1a

Betaseron Avonex Rebif

250 Ag every other day 30 Ag once weekly 44 Ag 3 times a week

s.c. i.m. s.c.

Abbreviations: s.c., subcutaneously; i.m., intramuscularly.

1.1. Natural history of multiple sclerosis The clinical presentation of MS is heterogeneous. Patients commonly present with sensory disturbances, optic neuritis, ataxia, vertigo, or motor dysfunction. Less commonly, initial symptoms include bowel, bladder, and sexual problems. Younger patients commonly develop optic neuritis and sensory disturbances and older patients usually present with progressive motor symptoms. Fatigue is an early symptom and may even precede the onset of clinical signs of the disease. Cognitive dysfunction manifests later as the disease progresses. Depression is common in MS and its severity can vary with disease activity. The clinical course of MS has been classified into subtypes based on large cohort studies (Lublin & Reingold, 1996). There are 4 clinical courses of MS: relapsing remitting (RRMS), secondary progressive (SPMS), relapsing progressive (RPMS), and primary progressive (PPMS). RRMS is the most common form of MS, affecting up to 85% of the MS population. RRMS is more common in females than males, with a ratio of about 2:1. It usually occurs in earlier age groups. It is characterized by acute clinical exacerbations and inflammatory CNS activity followed by periods of clinical stability. The average number of clinical attacks is 1 every 2 years. Most patients with RRMS eventually develop a progressive course, known as SPMS. The hallmark of SPMS is slow clinical deterioration independent of clinical relapses. Patients with SPMS may or may not have relapses. From the natural history data, about 50% of the patients that start out with a RRMS course develop a SPMS course after 10 years of disease activity (Weinshenker et al., 1989). The relapsing progressive course is characterized by clinical deterioration with superimposed clinical relapses. In the primary progressive course, there is clinical deterioration without any identifiable relapses and remissions. The progressive course from onset of MS is seen in up to 15% of the MS population. The progressive MS course is usually seen in an older population and is slightly more common in males. 1.2. Pathology of multiple sclerosis lesions Histological analysis of MS lesions has shown heterogeneity of mechanisms involved in tissue damage. This heterogeneity in pathology likely explains differences in MS clinical subtypes as well as response to treatment. Pathology of MS lesions has been extensively studied in biopsied and postmortem tissue taken from patients with active MS (Lucchinetti et al., 2000). The pathological studies not only underscore the role of inflammation (both cellular and humoral) but also point

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out the importance of degenerative processes in causing oligodendroglia death. Active MS lesions have been classified into 4 main patterns by Lucchinetti et al. (2000). The various patterns of lesions show inter-individual heterogeneity but are homogenous within an individual. A feature common to all 4 patterns is the predominance of T cells, macrophages, and activated microglia in the lesions. Patterns I and II are similar. Both types of lesions show perivenous distribution, have sharp borders, contain many surviving oligodendrocytes in the lesion center, and have extensive remyelination. A feature distinct to Pattern II is the presence of immunoglobulins (mainly IgG) and complement C9neo antigen at sites of myelin destruction. It is thought that tissue damage in Pattern I is caused mainly by toxic products released by activated macrophages, such as TNF-a. In Pattern II, tissue damage likely results from additional antibodymediated processes, including complement activation. Patterns I and II are reminiscent of lesions seen in autoimmune encephalomyelitis diseases, including acute disseminated encephalomyelitis (ADEM). Patterns III and IV are both characterized by oligodendrocyte dystrophy, with significant loss of oligodendrocytes and little remyelination effort. There is no immune complex deposition. Unique to Pattern III is that demyelinating lesions are not centered around blood vessels. They tend to be diffuse, with ill-defined borders. There is a rim of normal appearing myelin around inflamed blood vessels. Pattern III is also characterized by a preferential loss of myelin-associated glycoprotein (MAG) relative to other myelin proteins [myelin basic protein (MBP), PLP, CNP]. At sites of MAG loss, about 14– 37% of myelin oligodendrocyte glycoprotein (MOG+) or CNPase+ oligodendrocytes show histological evidence of death via apoptosis. In contrast to Pattern III, Pattern IV lesions have sharp borders. Also, oligodendrocyte death is due to necrosis and not apoptosis. Patterns III and IV are reminiscent of lesions produced by viral infections or toxic, metabolic, or ischemic insults. Attempts have been made to correlate the 4 histological patterns mentioned above with different clinical MS subtypes or variants of MS (Lucchinetti et al., 2000). However, it should be cautioned that the histological patterns are described in a small number of patients suffering from active disease, often with large lesions that prompted a biopsy. Hence, such a population may not be representative of MS population at large. Patterns I and II lesions are seen in all subtypes of MS (RR, SP, PP). Pattern III lesions are seen in both RR and SPMS. However, Pattern IV lesions were seen only in a few patients with PPMS. Devic’s neuromyelitis optica is a variant of MS characterized by antibody complement-mediated tissue destruction. This pattern of damage resembles Pattern II. Balo’s concentric sclerosis is characterized by histological pattern resembling Pattern III lesions. The different histological patterns seen in MS lesions have important implication for therapeutic interventions. Patterns I and II are characterized by inflammatory responses mediated by toxic cytokines or antibodies complement. Hence, drugs that inhibit these mediators of inflammation would be

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efficacious. For example, in patients with Devic’s disease, intravenous immunoglobulin (IVIG) or plasmapheresis is more beneficial than other therapies. Patients that have Pattern III or IV lesions may benefit more from drugs that enhance the growth potential of oligodendrocytes. It is interesting to note that loss of MAG is a prominent feature of lesions seen in progressive multifocal leukoencephalopathy (PML), which is caused by infection of oligodendrocytes with the JC virus. In Pattern III MS lesions, loss of MAG is also prominent. Hence, it may be speculated that latent infection of oligodendrocytes by JC virus or another oligotrophic virus plays an etiological role in MS. In a study of 121 patients with MS, 9% were found to have JC virus in the CSF, as measured by PCR (Ferrante et al., 1998). However, this high frequency of JC virus in MS has not been reported in any other study. Natalizumab was recently approved for treating MS (see Section 8.1). However, its use is linked to the development of PML. Natalizumab has been suspended temporarily pending further safety analysis. One mechanism whereby natalizumab use resulted in the emergence of PML complication could have been through reduction of immune surveillance, thus allowing activation of latent PML virus in oligodendrocytes or astrocytes.

both RRMS and SPMS (van de Wyngaert et al., 2001). However, cardiotoxicity limits the cumulative dosage of this drug. There are individual differences among clinicians as to the choice of ancillary treatments used with the DMAs. However, most clinicians use intravenous steroids as the first line therapy for an acute relapse. For frequent or severe relapses, no specific guidelines exist for the choice of secondary agents employed. Both pulse-dose steroids and intravenous immunoglobulins (IVIGs) administered monthly to every 3 months have been used for breakthrough relapses on DMAs with variable, often unsatisfactory, results. Azathioprine and methotrexate have long been used to treat MS, particularly prior to the advent of the first approved DMA (IFN-h-1b in 1993). They are rather mild chemotherapeutic agents and continue to be used by many as supplementary agents. Mycophenolate mofetil (CellCept) is becoming more popular as a supplementary agent, but without any substantial data to support its use. More cytotoxic agents, such as cyclophosphamide and mitoxantrone, are useful in treating rapidly progressive disease. However, the efficacy is often short lived. Long-term use of these agents is associated with risk of developing secondary malignancies. Statins have received much attention recently as a combination agent. MS trials are ongoing that will further elucidate its role in MS.

1.3. Treatment options for multiple sclerosis 2. Interferons: pleotropic effects Current disease modifying agents (DMAs) include the IFNs and glatiramer acetate. The DMAs are all approved by the FDA for treating RRMS and are considered first line therapy for RRMS. Of all the therapies used for treating MS, the DMAs are the main ones that have proven clinical safety and efficacy in treating RRMS. Other agents are also available for treating MS, usually when there are frequent relapses or rapidly progressive disease despite treatment with DMAs. Table 2 lists several of these therapies, categorized as immunomodulatory, immunosuppressive, or ‘‘other.’’ These ancillary treatments have been used alone but usually are given in combination with the DMAs. However, the data on the efficacy of these ancillary treatments are not impressive. Also, in the case of chemotherapies, systemic toxicity limits their use. Mitoxantrone is the only FDA-approved immunosuppressive agent that is efficacious in Table 2 Treatment options for MS Immunomodulatory

Immunosuppressive

Other

Interferon-ha Glatiramer acetatea

Steroids Azathioprine Methotrexate Cyclophosphamide Mitoxantronec Cladribine Mycophenolate mofetil

IVIG Plasmapheresis Statins Natalizumabb

Abbreviations: IVIG, intravenous immunoglobulin. a FDA approved for treating RRMS. b Anti-VLA-4 antibody; temporarily suspended from the market pending safety analysis. c FDA approved for treating RRMS and SPMS.

IFNs were discovered in 1957 (Isaacs & Lindenmann, 1957). Most of what we know about IFNs signaling and mechanism of action comes from tissue culture experiments. The term, interferon, originally described the biological activity of a soluble substance that ‘‘interfered’’ with viral replication in cultured cells. Other biological roles of interferons emerged later, including antiproliferative and immunomodulatory effects. IFNs are naturally occurring proteins produced by cells in response to antigenic stimulation with viral RNA, bacterial products, or tumor proteins. Viral and bacterial products induce IFNs by binding to receptors known as toll-like receptors (TLRs). IFNs are considered part of the innate immune system. However, they can influence adaptive responses of the immune system, including B and T cell differentiation. Early work identified 3 different classes of IFNs based on the cells of origin. Leukocytes exposed to viruses secreted IFN-a, stimulated fibroblasts produced IFN-h, and activated lymphocytes mainly secreted IFN-g (Friedman, 1981). Based on similar acid stability, homology, and function, the leukocyte and fibroblast IFNs were collectively called type I IFNs. Distinct from type I IFNs in terms of function and acid lability was IFN-g, also known as type II IFN. Later studies identified other type I IFNs, such as IFN-N, IFN-n, and IFN-H (Charlier et al., 1993; Rueda et al., 1993; LaFleur et al., 2001). Table 3 summaries some of the properties of IFNs. Some classes of IFNs contain several related genes. The IFN-a class makes up at least 18 nonallelic genes located on the short arm of chromosome 9 (Arnason & Reder, 1994). The N IFN class contains 6 nonallelic genes also located on chromosome 9 (Adolf,

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Table 3 Properties of interferons

Type I IFNs IFN-a IFN-h IFN-H IFN-N Type II IFN IFN-g

Cell origin

Cell target

Function

Lymphocytes, monocytes, macrophages Fibroblasts, epithelial cells Trophoblasts

Almost all cells

Viral resistance, class I MHC expression, inhibition of cell proliferation; autoimmunity (SLE) Similar to IFN-a Ovum implantation, antiviral and antiproliferative activities Antiviral, antiproliferative, antitumor

Virally infected leukocytes T cells, NK cells, macrophages

Almost all cells Endometrial cells; most cells with systemic administration Almost all cells Hematopoietic cells, epithelial cells, endothelial cells, tumor cells

1987). IFN-h and IFN-g exist in only 1 allelic form and are located on chromosomes 9 and 12. 2.1. Antiviral After viral infection, virtually all nucleated cells respond by increasing the synthesis of IFNs, which in turn stimulate the production of multiple gene products that impair viral replication. Type I IFNs and to a lesser extent IFN-g inhibit viral replication in infected cells, at the level of transcription and translation. Inhibition of viral replication after IFN treatment requires transcription and protein synthesis (Pestka et al., 1987; Samuel, 1991). The enzyme 2V,5V-oligoadenylate synthetase (2V,5V-OAS) is induced in response to all IFNs and catalyzes the polymerization of ATP (Lengyel, 1982; Pestka et al., 1987). The 2V,5V-oligomers of ATP activate a cellular RNase that cleaves viral RNAs and thereby prevents viral translation of proteins (Kerr & Brown, 1978; Kerr & Stark, 1992). IFNs also activate protein kinase R (PKR), a double-stranded RNA-dependent protein kinase (Clemens et al., 1993). This enzyme phosphorylates the a-subunit of the translation initiating factor, eIF-2 (Pestka et al., 1987; Katze, 1992). Inhibition of eIF2 decreases viral and to a lesser extent host protein synthesis. Another protein induced by IFNs with antiviral actions is the Mx protein. Mx proteins bind GTP and have intrinsic GTPase activity. They have potent antiviral activity against negativestranded RNA viruses, such as influenza (myxovirus) (Staeheli et al., 1993). Viruses also induce interferon regulatory factor-1 (IRF-1), a DNA-binding factor that recognizes positive regulatory elements in the promoters of IFN-h and some of the IFNinducible genes mentioned above. Constitutive expression of IRF-1 confers viral resistance (Pine, 1992). 2.2. Antitumor IFNs are effective against a variety of tumors, including multiple myeloma, chronic myelogenous leukemia, hairy cell leukemia, Kaposi’s sarcoma, lung cancers, ovarian cancers, and malignant melanoma (Dorr, 1993; Byhardt et al., 1996; Ulmer et al., 2002; Wall et al., 2003). The antitumor effects of IFNs are mediated directly by effects on proliferation, cell cycle, or induction of apoptosis, and indirectly by immune activation, including stimulation of MHC expression, cytotoxic T cells,

Activation, growth, and differentiation of T and B cells; synergistic effects with IFN-a/h against viruses

natural killer cells, and monocyte function. IFN-a/h affects all phases of the cell cycle, but particularly lengthen or block G1 phase (Stark et al., 1998). Thus, IFN-induced growth arrest can eventually lead to cell death. Apoptosis plays an important role in elimination of damaged cells. One mechanism of apoptosis is through activation of caspases, which induce the morphological characteristic of apoptotic cells (Ashkenazi & Dixit, 1998; Earnshaw et al., 1999). Both types I and II IFNs can activate caspases and cause cell death via apoptosis, resulting in DNA fragmentation, mitochondrial changes, and plasma membrane changes (Chawla-Sarkar et al., 2001; Chen et al., 2001; Morrison et al., 2001; Thyrell et al., 2002). IFN-g can induce apoptosis as well, in cells expressing high levels of IFN-g receptors (Ahn et al., 2004). In addition to having beneficial effects on controlling tumor growth, this action of IFN is important in suppressing inflammatory responses (see Section 5.3). IFNs activate immune effector cells. IFNs increase MHC expression on antigen presenting cells (APCs) and target cells, hence aiding in the process of recognition of transformed cells. IFNs promote body defenses by enhancing the activities of NK cells, cytotoxic T lymphocytes, macrophages, and B cells. These cells in turn help eliminate damaged cells. 3. Molecular mechanism of action of interferons IFNs produce their biological effects by binding to multisubunit receptors on the cell surface (Fig. 2). IFN receptors have inherent enzymatic activity. Activation of this enzymatic activity leads to auto- or cross-phosphorylation of the receptor subunits. The phosphorylated subunits then serve as docking sites for cytoplasmic transcription factors. These transcription factors are in turn phosphorylated, dimerize, and then become active transcription factors. They translocate to the nucleus and bind to enhancer elements, thereby stimulating gene expression. The IFN signaling cascade can be influenced by a variety of other receptors. The pleotropic effects of IFNs may be explained in part by the cross talk between IFN signaling cascade with a variety of different receptor systems. 3.1. Interferon receptors Type I IFNs (a, h, n, H , and N) compete for binding to the same receptor, designated type I interferon receptor (IFNAR)

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Fig. 2. IFN signaling pathway. Type I IFNs (IFN-a/h) bind to a common receptor, consisting of 2 subunits IFNAR-1 and IFNAR-2. Ligand binding causes activation of JAK1 and TYK2 and tyrosine residues on the subunits through autophosphorylation. Following this, cytoplasmic STAT proteins are activated via phosphorylation by the JAK1/TYK2 enzymes. The IFNAR-1 serves as a docking site for efficient dimerization of STAT2 and STAT1. Dimerization of STAT proteins occurs through the SH2 binding domains. STAT2:STAT1 heterodimers further bind to IRF-9, collectively called ISGF3. This then translocates to the nucleus and binds to the enhancer element ISRE, thereby stimulating gene expression. Serine/threonine kinases phosphorylate the Ser-727 residue and further potentiate the effects of the ISGF3 transcription factor. IFN-a/h also stimulate the production of STAT1:STAT1 homodimer (AAF), which binds to a different enhancer element, GAS. This is a minor pathway in type I IFN signaling. IFN-g can influence the IFN-a/h pathway by enhancing the production of STAT1 through stimulating AAF production. Through feedback inhibition, AAF induces IRF-2 which in turn inhibits the actions of ISGF3. SHP1 is a tyrosine phosphatase that terminates signaling through the IFNAR. Abbreviations: IFN, interferon; IFNAR, IFN-a/h receptor; tyrosine-P, tyrosine phosphorylation; Ser-P, serine phosphorylation; SH2, src homology domain; IRF-9, IFN regulatory factor-9; ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated response element; GAS, IFN-g activated site; AAF, IFN-a activated factor; IFNGR, IFN gamma receptor; MxA, myxovirus A protein; PKR, double-stranded RNA-activated protein kinase; S/T kinase, serine/threonine kinase; SHP1, protein – tyrosine phosphatase.

(Branca et al., 1982; LaFleur et al., 2001; Imakawa et al., 2002). However, type I IFNs bind to this receptor in different orientations, leading to differential second messenger and gene activation (Vassileva et al., 2003). All human tissues express IFNARs. The IFNAR is composed of 2 subunits, IFNAR-1 and IFNAR-2 (Uze et al., 1990; Domanski et al., 1995; Lutfalla et al., 1995). Based on the homology of their primary amino acid sequences with other receptor types, IFNAR-1 and -2 are classified as helical cytokine class II receptors (hCRII) (Bazan, 1990b; Kotenko et al., 2003; Sheppard et al., 2003). Other members of the hCRII family include IFN-g, IL-10, and tissue factor receptors (Bazan, 1990a; Ho et al., 1993). The IFNAR-1 protein is composed of 557 amino acids and has a small cytoplasmic end, consisting of only 100 amino acid residues (Uze et al., 1990). Intracellularly, the IFNAR-1 contains 4 tyrosine residues at amino acids 466, 481, 527, and 538, which can be potentially phosphorylated. The IFNAR-2 exists in long and short variants: BL and BS, arising from alternate splicing of the same gene (Colamonici & Domanski, 1993; Domanski et al., 1995; Lutfalla et al., 1995; Domanski & Colamonici, 1996). The exact role of the BS form has not been fully elucidated. The BL is composed of 515 amino acid residues and

has 7 intracellular tyrosine residues, which can be potentially phosphorylated at amino acid positions 269, 306, 316, 318, 337, 411, and 512. Based on transfection and binding studies, it appears that IFNAR-2 is necessary for the initial binding by IFNs, and IFNAR-1 is necessary for transformation into a high-affinity binding structure (Colamonici et al., 1994b; Cohen et al., 1995; Ghislain et al., 1995; Russell-Harde et al., 1995; Domanski & Colamonici, 1996). The high-affinity structure then initiates the full biological response (Fig. 2). 3.2. Intracellular signaling Binding of type I IFNs to the IFNAR results in rapid phosphorylation of the IFNAR-1 and -2 subunits (Platanias & Colamonici, 1992; Platanias et al., 1994). This phosphorylation is mediated by the Janus kinases, TYK2 and JAK1 (Domanski & Colamonici, 1996; Stark et al., 1998). IFNAR-1 is associated with TYK2 (Colamonici et al., 1994a, 1994c). IFNAR-2 is associated with JAK1 (Novick et al., 1994). Once the IFNAR subunits are phosphorylated, they serve as docking sites for other regulatory proteins, such as signal transducers and

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activators of transcription (STAT). The JAK1/TYK2 associated with IFNAR can phosphorylate STAT 1, 2, and 3 (Darnell et al., 1994; Ihle & Kerr, 1995; Schindler & Darnell, 1995; Stark et al., 1998; Tanabe et al., 2005). The phosphorylated tyrosine (Y) residue 466 on the IFNAR-1 is necessary for binding to STAT1 (Leung et al., 1995; Yan et al., 1996). However, this association occurs only in the presence of STAT2 (Leung et al., 1995). After STAT proteins are phosphorylated, 2 divergent pathways emerge, with STAT2 playing an important role. In 1 pathway, the phosphorylated STAT2 forms a complex with STAT1. The STAT2:STAT1 heterodimer in turn associates with the DNA binding protein called IFN regulatory factor 9 (IRF-9; p48) (Ghislain & Fish, 1996). The STAT2:1:IRF-9 complex is a transcription factor (IFN-stimulated gene factor, ISGF3), which translocates to the nucleus and binds to the IFN-stimulated response element (ISRE) of multiple genes (Fig. 2) (Ghislain & Fish, 1996; Li et al., 1996). In the other pathway, the activated STAT proteins do not form a complex with IRF-9. The activated STAT2:1 and STAT2:3 heterodimers and the STAT1:1 homodimer (IFN-aactivated factor, AAF) translocate to the nucleus and bind to the IFN gamma-activated sequence (GAS) response element (Fig. 2) (Li et al., 1996; Ghislain et al., 2001; Takaoka & Taniguchi, 2003; Brierley & Fish, 2005). IFN-induced activation of ISRE and GAS enhancer elements through their respective transcriptional factors turns on a wide variety of genes (Der et al., 1998). Even though IFN-a and IFN-h bind to a common receptor, they induce a unique set of genes (Der et al., 1998). This may be due to differences in the conformational states of the receptor induced by IFN-a versus IFN-h. IFN-g activates transcription factors (STAT1:1) that also bind to GAS enhancer elements. It also induces genes that are different from those induced by IFN-a/h (Der et al., 1998). One way of turning off the IFN-induced JAK-STAT signaling pathway is by dephosphorylating the tyrosine kinases. SHP1 is a tyrosine phosphatase that is mainly expressed in hematopoietic cells. It is associated with the IFNAR-1 subunit and dephosphorylates JAK1, thus inhibiting signal transduction (Arnason & Reder, 1994; Decker, 1997). 3.3. Receptor cross talk Type I IFNs activate STAT proteins by phosphorylating tyrosine residues on STAT 1, 2, and 3 (Fasler-Kan et al., 1998). STAT4 is phosphorylated in humans. Some mouse strains, however, lack the terminal portion of STAT2, which induces phosphorylation of STAT4 (Farrar et al., 2000). Some of the STAT proteins also require phosphorylation of the serine residues for maximal transcriptional activity. In vertebrates, the C-terminus of STATs 1, 3, and 4 is conserved between residues 720 and 730. STAT5A and STAT5B contain a conserved sequence in the PSP motif. These conserved sequences are collectively referred to as P(M)SP-STATs and represent potential serine phosphorylation sites (Wen et al., 1995; Yamashita et al., 1998; Decker &

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Kovarik, 2000). STAT1 is phosphorylated on serine 727 independently of tyrosine phosphorylation (Wen et al., 1995; Zhu et al., 1997; Kovarik et al., 1998; Goh et al., 1999; Kovarik et al., 1999). Serine 727 sits in a transcription activation domain. Phosphorylation at serine 727 leads to binding to several nuclear proteins such as MCM5, which is necessary for full transcriptional activity (Zhang et al., 1998). Serine phosphorylation of STAT1 plays a major role in IFNg-induced responses. In cell lines with a mutation in the serine phosphorylation site (S727A) of STAT1, the transcription activity of IFN-g is reduced by 80% (Wen et al., 1995). Serine phosphorylation of STAT1 also occurs in response to stimulation of the IFN-a/h receptor (Goh et al., 1999). It is not clear to what extent serine phosphorylation of STAT1 is necessary for mediating the responses of IFN-a/h receptor in immune cells. However, it may be very important in MS, as discussed below. Depending on the cell line used, serine 727 phosphorylation is potentially mediated by several distinct kinases, including mitogen-activated protein kinases (MAPK), phosphatidylinositol 3-kinase (P13K), double-stranded RNA-activated protein kinase (PKR), and proline-rich tyrosine kinase 2 (Pyk2) (Decker & Kovarik, 2000; Nguyen et al., 2001; Brierley & Fish, 2002). Particularly, the MAPK pathways are involved in serine phosphorylation of the PMSP motif in STAT 1, 3, 4, and 5 (Decker & Kovarik, 2000). The MAPK pathway is activated by a wide variety of receptors involved in growth and differentiation, including tyrosine kinase-linked receptors, integrins, and ion channels. Hence, there is cross talk between the IFN signaling cascade and various other receptors. The intricacy of these pathways in part explains the pleotropic effects of IFNs in cells. 4. Multiple sclerosis and molecular defects in interferon signaling There is increasing evidence that type I IFN signaling is suboptimal in MS. Decreased levels of IFN-a and -h are released by immune cells from MS patients in response to viral or mitogenic challenges in vitro (Reder & Arnason, 1985; Hertzog et al., 1991; Wandinger et al., 1997; Feng et al., 2001). Also, there are low levels of IFN-stimulated gene products in MS. IFNs stimulate their own secretions, and IFN-h-induced IFN-h levels are also low in MS. After type I IFNs bind to their receptor, there is a rapid formation of ISGF3 complex and transcription of several genes, including IRF-1 (a positive regulator of IFN genes), IRF-2 (a negative regulator of IFN genes), IFN-h, 2V,5V-OAS, and MxA (Decker, 1997). Levels of these gene products are low in MS (Feng et al., 2002a). Peripheral blood monocytes (PBMC) taken from MS patients have lower levels of 2V,5V-OAS mRNA and MxA protein than controls. Clinically active MS patients have the lowest resting levels (Feng et al., 2002a). PBMC from MS patients also have significantly reduced levels of IRF-1 and IRF-2 mRNA (Feng et al., 2002a). In addition, the IRF-1/IRF-2 mRNA ratio is lower in MS than healthy controls. Because IRF-2 is an inhibitor of several IFN-a/h gene products and its mRNA is

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increased in MS lesions (Whitney et al., 1999), it likely plays an important role in decreasing IFN-induced signaling in MS. Phosphorylation of STAT1 on serine residue enhances signaling through the IFN-a/h pathway (Section 3.3). In clinically active MS (especially in progressive disease), PSer-STAT1 levels are low in unstimulated PBMC relative to controls. However, in clinically stable MS, the P-Ser-STAT1 levels are identical to the controls (Feng et al., 2002a). This implies that disease activity is related to decreased phosphorylation of the serine residue, possibly from decreased activity of serine phosphokinases during active MS. There are also abnormalities in the tyrosine phosphorylation of STAT1. During normal IFN signaling, SHP1 phosphatases remove P-Tyr-STAT1, allowing recycling of substrates for further IFN signaling. In clinically stable MS patients, IFNstimulated levels of P-Tyr-STAT1 in PBMC are significantly increased compared to controls. Correlated with this are reduced levels of SHP1 phosphatase (Feng et al., 2002a). Interestingly, in clinically active MS, P-Tyr-STAT1 and SHP1 levels are unchanged relative to the controls. Thus, the defect in the IFN signaling in MS is unique to the P-Ser-STAT1 pathway and it may be partially compensated by the P-Tyr-STAT1 pathway, at certain times in the disease process. Treatment of patients with IFN-h partially reverses the IFN-signaling defects. In response to IFN-h therapy in vivo, levels of IRF-1, IRF-2, and 2V,5V-OAS in PBMC increase, but only up to levels seen in unstimulated controls (Feng et al., 2002a). In normal controls, however, IFN-h treatment stimulates these products by several-fold (Witt, 1997). PBMC from patients with active MS not receiving IFN-h therapy have low levels of P-Ser-STAT1 under basal conditions. Stimulation of these cells with IFN-h in vitro increases the levels of P-Ser-STAT1, but only up to onefourth of the normal controls (Feng et al., 2002a). These observations show that IFN-h partially ameliorates the defects in IFN signaling in MS. They also suggest that IFN signaling and gene activation pathways are partially resistant to IFN therapy. 5. Immune system and multiple sclerosis In MS, the immune response is directed against CNS antigens. The identity of CNS antigens remains elusive. Some evidence supports the role of myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) in eliciting autoimmune responses. There is speculation as to the initial event that triggers the autoimmune response. An antecedent viral infection is often thought of as a trigger. Suspects include human herpes virus 6, Epstein –Barr virus, varicella-zoster virus, and herpes simplex virus. However, no single virus has been clearly identified as a culprit. The viruses could trigger an immune response through antigenic mimickery, a mechanism whereby a viral protein that closely resembles the host’s endogenous protein is able to stimulate self-reactive T lymphocytes. Generally, MS patients are resistant to viral infections. However, a viral infection in patients often provokes a relapse.

5.1. Normal immune responses After thymic selection, T cells are released into the periphery in 2 main subtypes: CD4+ and CD8+ cells. Twothirds of the circulating T cells are CD4+ subtype and are MHC class II restricted. One-third of the circulating T cells are CD8+ and are MHC class I restricted. Human CD8+ cells are divided about evenly into cytotoxic CD28+ cells and suppressive CD28 cells. Prior to antigenic presentation, CD4+ T cells circulate in a naı¨ve state, designated naı¨ve T cells (Th0). T cells encounter antigens presented by various antigen presenting cells (APCs), usually macrophages, natural killer (NK) cells, or even B cells. Upon antigenic stimulation in the peripheral lymphoid tissues, Th0 cells differentiate into Th1 and Th2 cells (Mosmann & Sad, 1996). In the presence of IL-12 released by APCs, Th0 cells differentiate into Th1 cells. These cells release proinflammatory cytokines, such as IFN-g, IL-2, and TNF-a. In the presence of IL-4, Th0 cells differentiate into Th2 cells, which secrete the anti-inflammatory cytokines IL-4, IL-5, and IL-10. Both types of T helper (Th)-mediated immune responses exert a reciprocal inhibitory influence on the actions of each other. Inflammatory cell responses are inhibited by 2 types of T cells: CD4+25+ T regulatory cells (Tr) and CD8+CD28 T suppressor cells (Ts). The CD8+ Ts cells act on already activated CD4+ T cells that recognize self-antigens expressed on APCs in association with Qa-1 MHC class Ib molecules (HLA-E in humans) (Chess & Jiang, 2004). Therefore, the CD8+ Ts cells inhibit the inflammatory response mediated by potential autoreactive CD4+ T cells (Chess & Jiang, 2004). 5.2. Immune dysregulation in multiple sclerosis In MS, the aberrant inflammatory response is mediated by overactive Th1 cells and their cytokines, such as IFN-g, IL-2, and TNF-a (Misu et al., 2001). Th2 cytokines are generally thought to be low in MS. Of the cytokines producing CNS damage in MS, IFN-g has received the most attention. IFN-g is produced primarily by Th1 lymphocytes and NK cells. It causes activation and proliferation of self-reactive T cells in MS, hence leading to demyelination. Clinical observations support a pathogenic role of IFN-g in MS. Levels of IFN-g and mRNA transcripts increase in peripheral blood lymphocytes prior to and during clinical exacerbations (Beck et al., 1988; Link et al., 1994). At the onset of early MS (clinically isolated syndromes), there are significantly higher levels of IFN-g and TNF-a mRNA in whole blood samples taken from patients (Kahl et al., 2002). Treatment of MS patients with IFN-g increases clinical relapses (Panitch et al., 1987b). IFN-g treatment also causes an increase in circulating monocytes expressing class II (HLA-DR) molecules, enhances proliferative responses of peripheral blood leukocytes, and increases NK cell activity (Panitch et al., 1987a, 1987b, 1987c). Histological studies show IFN-g in active, chronic MS lesions (Traugott & Lebon, 1988a, 1988b).

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MS patients also have higher levels of other proinflammatory cytokines such as IL-2 and TNF-a (Gallo et al., 1991; Maimone et al., 1991; Sharief & Hentges, 1991). During active MS, circulating IL-2 or its soluble receptor levels are increased, indicating enhanced Th1 cell activity (Gallo et al., 1988; Adachi et al., 1990; Trotter et al., 1991). TNF-a levels are elevated in the circulation and CSF of MS patients during active disease (Maimone et al., 1991; Sharief & Hentges, 1991). MS patients also have higher levels of IL-12 and IL-18, both of which stimulate IFN-g production (Windhagen et al., 1995; Balashov et al., 1997; Karni et al., 2002). IL-12 is produced by antigen presenting cells, such as monocytes and macrophages. It is a key cytokine in the differentiation of Th1 cells. Th1 cytokines are thought to produce CNS damage through several mechanisms. For instance, IFN-g induces expression of MHC class II molecules on lymphoid cells. By inducing the expression of MHC class II molecules on antigen presenting cells, IFN-g enhances T cell and APC interaction, thus favoring T cell proliferative responses. IFN-g also increases the expression of adhesion molecules on endothelial cells (EC; Duijvestijn et al., 1986; Tsukada et al., 1993). Stimulation of adhesion molecules on the endothelial cells would enhance migration of self-reactive T lymphocytes into CNS. IFN-g activates monocytes, macrophages, and microglial cells. Macrophages are the most prominent cells found in active MS plaques. By activating macrophages, IFN-g enhances the cytotoxic potential of macrophages and destruction of CNS tissue (Arnason & Reder, 1994). IFN-g also stimulates the production of TNF-a from macrophages (Collart et al., 1986; Arnason & Reder, 1994). This cytokine is toxic to oligodendroglia in vitro (Selmaj & Raine, 1988). 5.3. Immunomodulatory effects of interferon-b in multiple sclerosis IFN-h inhibits the inflammatory response in MS at several steps. It decreases the expression of MHC molecules on APCs, inhibits T cell activation, and decreases the release of inflammatory cytokines. It enhances T cell suppressor function, which is important in attenuating inflammation. IFN-h also stimulates the production of growth factors from immune cells, which may help repair damaged tissue. During T cell activation, T cell receptors recognize antigens in association with MHC class II molecules. This serves as the first signal in T cell activation. IFN-h alters the expression of MHC class II molecules in MS. This may have a therapeutic effect. Individuals expressing HLA-DR15Dw2 and DQw6 alleles have increased susceptibility to MS (Dyment et al., 1997; Compston, 2000). In MS, the expression of MHC class II molecules increases in monocytes during a relapse (Armstrong et al., 1987). IFN-a/h inhibits the effects of IFN-g on MHC class II expression in macrophages and monocytes (Ling et al., 1985; Inaba et al., 1986; Fertsch et al., 1987; Panitch et al., 1989). It is likely that part of the anti-inflammatory effects of IFN-h is mediated by reducing IFN-g-induced expression of MHC class II molecules. These inhibitory effects of IFN-h

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probably occur during the early phase of immune cell responses, at the level of MHC-mediated APC and T cell interaction. It is important to note that IFN-g and its receptor (IFNGR) also have an important role in inhibiting T cell responses. IFNg is pro-inflammatory when the surface expression of IFNGR is low. IFN-g induces apoptosis in immune cells that express high levels of IFNGR (Ahn et al., 2004). In untreated MS patients, the levels of IFNGR-alpha chain (binding subunit) are increased and IFNGR-beta chain (signaling subunit) are decreased in peripheral blood monocytes. IFN-h regulates IFNGR expression. With IFN-h treatment, there is an increase in IFNGR-beta/alpha ratio at 3 months but a decrease at 12 months. The initial rise in the IFNGR-beta levels after IFN-h may be responsible for allowing IFN-g to remove activated T cells through apoptosis. Supporting this is the observation that after IFN-h therapy, there is an initial lymphopenia observed in the peripheral blood of treated patients. In addition to signal 1, several co-stimulatory molecules on APCs serve as second signals, including CD80 (B7-1) and CD86 (B7-2), which bind to CD28 on T cells. In EAE, CD80 on APCs induces Th1 cell responses, whereas CD86 generates Th2 cells (Freeman et al., 1995; Kuchroo et al., 1995). In MS patients, the number of CD80+ and CD86+ lymphocytes is significantly increased during active disease but not in stable disease (Genc et al., 1997). Treatment of patients with IFN-h significantly decreases the number of CD80+ lymphocytes, possibly reducing Th1 cell function (Genc et al., 1997). IFN-h also reduces HLA-DR+ and CD71+ lymphocytes, thereby further blocking the activation of T cells via TCR and transferrin receptor stimulation (Genc et al., 1997). Important cytokines involved in Th1 cell development include IL-2 and IL-12. IL-2 is also known as T cell growth factor and T cells are the main source of its production. It has autocrine and paracrine actions on T cells and neighboring cells. It is necessary for the proliferation and differentiation of T cells and B cells, and for monocyte and macrophage activation. IL-2 stimulates T cells to produce IFN-g (Kasahara et al., 1983; Vilcek et al., 1985). The number of IL-2 producing cells is increased in MS (Lu et al., 1993). During active MS, circulating IL-2 or its soluble IL-2 receptor levels in the serum and CSF are increased, indicating enhanced Th1 cell activity (Gallo et al., 1988; Adachi et al., 1990; Trotter et al., 1991). Type I interferons inhibit the production of IL-2. Incubation of ConA-activated cells in culture with IFN-h decreases IL-2 receptor expression and accumulation of IL-2 (Noronha et al., 1993). IL-12 is necessary for differentiation of Th1 cells from Th0 cells. The EAE model supports the role of IL-12 in demyelination. During the clinical phase of EAE, there is an increase in IL-12 expression in the CNS and a decrease during the recovery phase (Bright et al., 1998). During the height of clinical disease in EAE, the IL-12 level is high in serum and its expression is increased in lymphoid organs (Bright et al., 1998). Also, administration of rIL-12 increases proliferation of MBP-specific T cells and production of IFN-g (Bright et al., 1998). A clinical trial of anti-IL-12 antibody is underway to fully assess its benefits in MS.

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IFN-h has inhibitory effects on IL-12 production. IFN-a/h inhibits the production of IL-12 in mouse splenic leukocytes induced by bacterial antigen (Cousens et al., 1997). IFN-h inhibits CD40-mediated IL-12 p40 secretion in human DCs and Th1 cell development (McRae et al., 1998). In human monocytes, IFN-a/h also inhibits the production of IL-12 subunits, p40 and p70 (Karp et al., 2000). Lymphotoxin (LT, also known as TNF-h) and TNF-a play an important role in tissue damage in MS. LT is produced by both T and B lymphocytes. TNF-a is produced by activated monocytes, macrophages, DCs, and T cells. Both LT and TNFa are toxic to oligodendroglia in culture (Selmaj et al., 1991; Soliven et al., 1991). During MS relapses, spontaneous LT production by T cells is increased (Glabinski et al., 1991). Type I interferons reduce production of LT by mitogen-stimulated PBMC from MS patients (Abu-Khabar et al., 1992). Elevated levels of TNF-a are also detected in the serum and CSF during periods of disease activity in MS (Hauser et al., 1990; Maimone et al., 1991; Sharief & Hentges, 1991). IFN-h inhibits TNF release from PBMC and DC/T cells in culture (Arnason & Reder, 1994; McRae et al., 1998). Suppression of immune response is important in maintaining homeostasis between activation and inhibition of inflammation. During the course of an inflammatory response, both Th1 and Th2 cells play an important role. In the initial phases of inflammation, Th1 responses are dominant. In the recovery phase, Th2-mediated responses become important. IFN-h treatment inhibits Th1-mediated responses by decreasing IFN-g secretion by T cells and stimulating the secretion of anti-inflammatory cytokines, such as IL-4 and IL-10 (Panitch et al., 1987a; Noronha et al., 1993; Shakir et al., 1994; Kozovska et al., 1999; Ersoy et al., 2005). This would favor development of a Th2-mediated response. However, there is some evidence that type I IFNs induce Th1 responses, just like IFN-g (O’Shea & Visconti, 2000). The Th2 cytokine, IL-10, has several immunoregulatory properties, including suppression of antigen-specific proliferation of Th1 cells (De Waal Malefyt et al., 1991). Again, the picture is not simple. IFN-h induces IL-10 in activated T cells but inhibits production in activated monocytes (Feng et al., 2000). Another cell type that limits inflammation is the CD8+CD28 T suppressor cell (Ts). ‘‘Knockout’’ mice lacking CD8 cells develop the first attack of EAE after immunization and recover normally, indicating that CD8 T suppressor cells are not involved in recovery from the initial bout of inflammation (Jiang et al., 1992; Koh et al., 1992). Wild-type mice are refractory to recurrent attacks of EAE; however, CD8 / mice develop recurrent EAE (Koh et al., 1992). This observation points to an important role of Ts cells in controlling relapses in late adaptive immune responses. The inhibitory effects of Ts cells are mediated by several cytokines, including IL-4, TGF-h1, IL-10, and IFN-a/h (Arnason & Reder, 1994). In MS, CD8+CD28 T suppressor cell function is decreased (Arnason & Reder, 1994). Ts cell activity is reduced in comparison to normal individuals (Fig. 3) (Noronha et al., 1990, 1992). Ts activity also varies with the disease state. During MS attacks, Con-A-activated Ts cell activity is reduced,

45

% Suppression

44

40 35

Na ve IFN in vitro

30 25 20 15 10 5 0

MS

NL

Fig. 3. T suppressor cell activity is reduced in MS compared to normals in the naı¨ve state. In vitro stimulation of cells with IFN-h enhances CD8+28 suppressor activity in MS patients, but less so than controls.

but it recovers during remission (Antel et al., 1979; Arnason & Reder, 1994). In chronic progressive MS, Ts cell activity is persistently low (Antel et al., 1986). In vitro stimulation with IFN-h enhances Ts activity in cells taken from MS patients, however, not to the same level as normal controls (Fig. 3). CD4+CD25+ T regulatory cells (Tr) also have an important role in suppressing inflammation. In contrast to the CD8 Ts cells, CD4 Tr cells inhibit T cell function early in the course of the immune response (Gavin & Rudensky, 2003; Chess & Jiang, 2004). In MS, CD4+CD25+ T regulatory (Tr) cell function is reduced (Viglietta et al., 2004). Ongoing studies are further elucidating the role of Tr cells in autoimmunity, including MS and systemic lupus erythematosus (SLE). During the course of inflammation, protective mechanisms emerge to repair myelin damage. Intense inflammation is often associated with a vigorous remyelination effort. Growth factors are released during the course of inflammation either by the infiltrating immune cells or the surrounding brain tissue. Brainderived neurotrophic factor (BDNF), a member of the neurotrophin family, prolongs neuronal survival in various models of nerve injury (Ebadi et al., 1997; Kishino et al., 1997; Terenghi, 1999). BDNF production is increased during nerve injury and inflammation (Cho et al., 1997; Tonra et al., 1998; Batchelor et al., 1999). BDNF is also a protective agent for oligodendrocytes. It stimulates oligodendrocyte proliferation and myelination of regenerating axons in a spinal cord injury model (McTigue et al., 1998). Sources of BDNF include neurons, astrocytes, T cells, and to a lesser extent B cells and monocytes (Kerschensteiner et al., 1999; Ziemssen et al., 2002; Kerschensteiner et al., 2003; Meeuwsen et al., 2003; Hohlfeld, 2004). Lymphocytes taken from MS patients produce more BDNF than controls (Petereit et al., 2003). BDNF and its receptor are present in MS lesions, suggesting their involvement in neuroprotection (Ziemssen et al., 2002; Hohlfeld, 2004). However, there is no clear evidence that chronic therapy with IFN-h enhances BDNF production in MS (A. Reder and K. Hamamcloðlu, unpublished work) (Petereit et al., 2003). Other growth factors such as leukemia inhibitory factor (LIF) and nerve growth factor (NGF) are elevated by IFN-h (Byskosh & Reder, 1996; Boutros et al., 1997). There is evidence that glatiramer acetate treatment increases BDNF production in T lymphocytes (Aharoni et al., 2003; Hohlfeld, 2004).

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6. Multiple sclerosis and systemic lupus erythematosus: polar opposites in responses to interferons Autoimmune diseases arise from an inflammatory response to self-antigens. The autoimmune responses may be classified as primary versus secondary, or systemic (e.g., SLE) versus organ specific (MS). There is often an overlapping spectrum of disorders affecting multiple organs (MCTD, autoimmune endocrinopathies). Autoimmunity is caused by B cells that produce autoantibodies (SLE, MG) and immune complexes (SLE, RA), and by T cells (RRMS), and by innate immune cells such as macrophages that attack normal tissues (progressive MS). MS and SLE are 2 autoimmune diseases that seldom if ever occur simultaneously. Despite this, both diseases affect women more than men, patients are relatively young, there is familial aggregation, there is linkage to HLA-DR2 and EBV, and function of CD8 suppressor T cells is defective. Herein, it is proposed that MS and SLE are disparate because of opposite responses to IFN signaling. Clinically, important differences are seen between MS and SLE. In MS, sun exposure seems to be protective, minocycline inhibits EAE, and pregnancy decreases MS exacerbations. In SLE, however, sun exposure triggers SLE symptoms, minocycline can induce an SLE-like syndrome, and pregnancy may increase symptoms and disease activity. In MS, IFN-a decreases disease activity. In SLE, however, IFN-a increases disease activity. IFN-a treatment induces antinuclear antibodies in 22%, autoimmune disorders in 19%, and overt SLE in 0.7% of patients with carcinoid tumors (Ro¨nnblom et al., 1991). IFN-a therapy also can induce IDDM and rheumatoid arthritis. IFN-h therapy rarely induces autoimmune diseases, with autoimmune thyroiditis possibly being more frequent. There are occasional reported cases in the literature for IFN-h-induced lupus-like illnesses (Nousari et al., 1998). There are key differences in the immunopathology of MS and SLE. In MS, CNS destruction is largely mediated by CD8+ T cells and macrophages, with regulatory control by CD4+ T cells, and possibly with an additive effect of B cells, antibodies, and complement. True autoimmunity to CNS antigens could derive from an initial response to 1 antigen followed by epitope spreading, from a bystander effect following an unregulated reaction against a virus or antigen, or from familial proclivity for hyper-reactivity to CNS proteins. Antibodies in MS are directed against myelin components, autoantigens, and viruses but are usually ‘‘nonsense.’’ About 56% of MS plaques (type II pattern) contain deposits of antibodies against CNS antigens (Lucchinetti et al., 2000). The role of these antibodies is unknown and may be an epiphenomenon, or destructive, or even regenerative. B cells in MS are activated by unknown factors (possibly IL-6 or Epstein– Barr virus). There is a selective B cell proliferation, enough to induce germinal-like centers in the meninges, which may be partly responsible for intrathecal production of oligoclonal Ig bands (Prineas, 1979; Serafini et al., 2004). In SLE, autoantibodies are generated after abnormal processing of apoptotic bodies by macrophages and also

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possibly by an abnormal response to viral infections. Processed nucleosomes are presented to B cells that generate autoantibodies. The antigens and antibodies are many but are largely directed against ubiquitous components of nucleus (nuclear DNA, RNA, and nucleoproteins; e.g., ANA, dsDNA, Sm, snRNP) that affect multiple organs. IFN responses are different between MS and SLE. In MS, IFN-stimulated gene expression is low. In SLE, type I IFN and IFN-inducing factors are elevated, especially during exacerbations (Bengtsson et al., 2000). In RNA microarrays of mononuclear cells (MNC) from children with very active SLE, 15 genes were highly up-regulated compared to agematched controls. Fourteen of these were type I IFN-stimulated genes (Bennett et al., 2003). In adults with lupus, 26/48 patients had increased levels of IFN-inducible genes, and the up-regulation predicted more severe SLE disease (Baechler et al., 2004). As a readout for activation of the IFN system, these microarray profiles were more sensitive than serum cytokine levels. The innate immune system is abnormal in SLE. Plasmacytoid dendritic cells (pDC) produce excessive, sometimes massive, amounts of IFN-a (some of it a mysterious acidlabile form) and possibly smaller amounts of IFN-g (Baechler et al., 2004). Immune complexes with ssCpG sequences bind toll-like receptor-9 (TLR-9) on B cells and dendritic cells and induce IFN-a production. In active SLE sera, there is also an ‘‘IFN-inducing factor,’’ likely comprised of antibody-DNA immune complexes (Vallin et al., 1999). This factor, in combination with IFN-a-2b or GM-CSF, induces huge amounts of IFN-a by pDC. IFN-a in turn induces monocytes to differentiate into DC and this leads to increased APC function (Blanco et al., 2001). Serum IFN-a and IFN-inducing factors increase during SLE flares (Bengtsson et al., 2000). Moreover, in cutaneous SLE, infiltrating cells express high levels of MxA, a protein specifically induced by type I IFNs (Wenzel et al., 2005). Clinically, high levels of IFN-a may account for flu-like symptoms common in SLE. Often patients are seen that have do not clearly fit the diagnosis of MS. This is particularly true of patients who have elevated ESR, mild hematological and liver abnormalities, MRI T2 lesions that are not clearly periventricular, absence of oligoclonal bands, recurrent attacks of myelitis, and borderline elevation of anti-nuclear antibodies. These patients should be treated cautiously as they may have SLE. Based on the effects of IFN in SLE, treatment of such patients with IFNs for a questionable diagnosis of MS may provoke a severe inflammatory response. 7. Therapeutic trials of interferons in multiple sclerosis In MS, disability accrues slowly. According to the natural history data, the median time to reach disability to a point requiring assistance from disease onset is about 14 years (Weinshenker et al., 1989). In evaluating any therapy for MS, the ultimate goal is to prevent long-term disability. Randomized, placebo-controlled, clinical trials, due to both ethical reasons and costs, are conducted over a short time period, at

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most 2– 3 years. Open-labeled trials analyzing therapeutic interventions are fraught with biases, hence providing at best speculations on long-term efficacy of agents. 7.1. Short-term efficacy of interferons in multiple sclerosis Three pivotal trials in MS have established the efficacy of IFNs in MS: IFNB multiple sclerosis study group, Multiple Sclerosis Collaborative Research Group (MSCRG), and Prevention of Relapses and disability by Interferon-h 1a Subcutaneously in Multiple Sclerosis (PRISMS) study (Ebers et al., 1998; Jacobs et al., 1996; Sibley et al., 1993). All were phase III, randomized, placebo-controlled, double-blinded trials involving patients with RRMS. IFN formulations used in these trials were IFN-h 1b given subcutaneously every other day (qod), IFN-h 1a given intramuscularly once a week, and IFN-h 1a administered 3 times a week (tiw), respectively. The results of these trials are summarized in Table 4. Betaseron (IFN-h 1b) was the first disease-modifying agent to show therapeutic efficacy in MS. In comparison to the natural IFNs, it lacks glycosylation sites and has 2 amino acid substitutions to optimize manufacturing from its Escherichia coli source. The trial consisted of 3 treatment arms, placebo, IFN-h given at 1.6 million international units (MIU, 50 Ag), and 8 MIU (250 Ag) s.c. qod. At the conclusion of 2 years, the annualized relapse rates for the placebo, 1.6 MIU, and 8 MIU were 1.27, 1.17, and 0.84, respectively. The 2 doses of IFN-h 1b had a statistically significant dose-related effect on the reduction of relapse rate. Disability was analyzed at the end of the 3rd year, the extension year. A trend towards decreased worsening in the confirmed Expanded Disability Status Scale (EDSS) score was seen in the 8 MIU treatment arm relative to the placebo ( P = 0.161). IFN-h 1b had the greatest efficacy on MRI parameters (Paty & Li, 1993). The MRI lesion area was significantly reduced by the 8 MIU dose of IFN-h 1b and there was a median reduction of 75% in the rate of new lesion formation per year as compared to placebo. The MRI outcome measures favored the higher dose of IFN-h 1b over the lower dose. In a later MRI study, 8 MIU of IFN-h 1b given every other day decreased the number of contrast-enhancing lesions by 91% compared to baseline at 6 months (Stone et al., 1997).

Avonex (IFN-h 1a) was the second agent to show benefit in the treatment of RRMS. This form of IFN is produced in mammalian COS cells and is glycosylated. Patients were randomized to receive either placebo or IFN-h 1a at 6 MIU (30 Ag) i.m. once a week. The trial was terminated early because patient withdrawal from the study was fewer than expected. Of the 301 patients randomized, only 172 completed 2 years of protocol. The unique, primary end point of this study was the effect on sustained worsening of disability. When analyzing the results based on intent-to-treat analysis, IFN-h 1b therapy reduced disability progression by 37%, an effect that was significant. However, when the patient population who completed the 2 years of therapy was analyzed (only 172 of the 301), the proportion of patients with sustained disability progression was not statistically different between the IFN-h 1a and placebo (21.1% vs. 33.3%). In the Avonex trial, annual relapse rate was reduced by 18% compared to controls in the intent-to-treat analysis. For patients who completed the 2 years, the annual relapse rate was significantly reduced by 32%. On MRI, the number and volume of contrast enhancing lesions were significantly lower in the IFN-h-1a-treated group. The change in T2 lesion volume from baseline was statistically lower in the IFN-h-1a-treated group after 1 year but not in the second year. The number and volume of contrast enhancing lesions were significantly reduced by IFN-h 1a at years 1 and 2. Rebif (IFN-h 1a) was the third agent to receive FDA approval for the treatment of RRMS. Patients were randomized to receive placebo or IFN-h 1a at 6 MIU (22 Ag) or 12 MIU (44 Ag), given s.c. tiw. The relapse rates were reduced by 27% and 33%, respectively, by the 2 doses as compared to placebo. There was no statistical difference between the 6 MIU and 12 MIU doses. The number of T2 active lesions on the MRI was reduced by 67% and 78% with the lower and the higher dosages. Disability measures (progression) were also significantly reduced by both doses, 22% and 30%. The PRISMS-4 trial was an extension to the fourth year (Hughes et al., 2001). Patients who received placebo treatments were randomized after 2 years of the study to receive either 6 MIU or 12 MIU of IFN-h 1a (cross-over design). Patients in the treatment arm continued further therapy. The beneficial effects of IFN-h on clinical and MRI measures were

Table 4 Results of pivotal trials in MSa Drug

RR reduction

MRI measureb

Sustained disability

Betaseron (250 Ag, qod) Avonex (30 Ag, weekly) Rebif (44 Ag, tiw) Avonex (30 Ag, weekly) (CHAMPS trial)

, 34% , 32%d , 33% [, 44%f]

New T2 lesions, , 75% Number Gd+, , 52% Active T2 lesion, , 78% Number Gd+, , 67%g

, 29% ( P = 0.161)c , 37% ( P = 0.024)e , 30% ( P = 0.01) NA

Abbreviations: NA, not applicable; CHAMPS, Controlled High Risk Avonex Multiple Sclerosis Prevention Study; RR, relapse rate; Gd, gadolinium. a Data are not meant to be compared across trials. b Data compared to controls. c 3-year data. d Patients who completed 2 years of study (see text). e Data based on intention to treat analysis (see text). f Decreased rate of developing clinically definite MS over 3 years. g Data at 18 months.

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maintained during the extension period. More importantly, patients who continued therapy from the start had better clinical and MRI outcome measures than the cross-over group. Several important points are apparent from the pivotal trials. First, in both the IFN-h 1b multiple sclerosis study and PRISMS, there was a dose-dependent effect of IFN treatment on relapse rate and MRI outcome measures. This points out the better efficacy of higher doses of IFNs, when given frequently. Second, the PRISMS trial points out the importance of starting therapy early in MS. In the extension part of the trial, patients who remained on IFN treatment from the start had better outcome measures than those who were in the cross-over group. Third, all of the pivotal trials showed substantial effects of IFNs on MRI outcome measures. However, the effects on relapse rate and disability progression were modest and the latter outcome measure was the least affected. This implies that even though the IFNs are good at controlling inflammation in the brain, they are relatively weak in influencing the neural degeneration responsible for disability. The dose – response effects of IFNs as suggested by the pivotal trials were confirmed later by other studies: Once Weekly Interferon for MS Study (OWIMS) Group, Evidence for Interferon Dose-Effect: European-North American Comparative Efficacy (EVIDENCE), and Independent Comparison of Interferon (INCOMIN) (Durelli et al., 2002; Freedman et al., 1999; Panitch et al., 2002). The OWIMS study was a 48-week study comparing the efficacy of IFN-h 1a, 22 or 44 Ag weekly, relative to placebo in RRMS. It showed benefit on MRI measures, but not on disability. The EVIDENCE trial compared the efficacy of weekly IFNh 1a given at 30 Ag i.m. to 44 Ag of IFN-h 1a injected s.c. tiw. After 48 weeks of treatment, the relapse rate and MRI measures were significantly better with the subcutaneous high dose. Disability measures were not different. The INCOMIN trial showed greater efficacy of IFN-h 1b given qod versus IFN-h 1a given once weekly on relapse rate, MRI measures, and confirmed disability progression over 2 years. These comparative trials indicate that at least for the short term, higher and more frequent doses of IFNs appear to have better efficacy on MRI measures, relapse rate, and perhaps on disability. The pivotal trials highlight the importance of starting treatment early. However, there is a controversy whether therapy needs to be initiated even prior to the diagnosis of clinically definite MS (CDMS). In the Controlled High Risk Avonex Multiple Sclerosis (CHAMPS) trial, treatment was given at the time of the first clinical attack (e.g., optic neuritis, myelitis, or posterior fossa lesion) (Jacobs et al., 2000). Patients were followed for 3 years. The rate of developing CDMS was significantly lower in the IFN-treated group than placebo (44% reduction). There was also a significant reduction in MRI measures. In the Early Treatment of MS (ETOMS) trial, patients were given 22 Ag of IFN-h 1a once weekly or placebo for 2 years (Comi et al., 2001). The rate of developing a second attack was reduced by 34% and MRI measures were better in the treated group. In both trials, more

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than 50% of the patients did not have a second clinical attack by the trial end. Most patients who converted to CDMS did so within the first year. Hence, early treatment may be beneficial only in a minority of patients. Most patients can be carefully monitored over time clinically and by MRI. Rather than starting DMAs early, frequent follow-up of patients with clinically isolated attacks and lack of other prognostic factors, such as absence of > 1 T2 lesions and CSF oligoclonal bands, may avoid unnecessary side effects of drugs and costs. IFNs have robust responses on reducing contrast-enhancing lesions. Contrast-enhancing lesions represent a disruption of the blood –brain barrier (BBB) that may be an early event in the pathogenesis of permanent lesions. High-dose intravenous steroids rapidly decrease enhancing lesions in MS and have a positive effect on the duration and severity of clinical relapse (Barkhof et al., 1991; Miller et al., 1992). IFNs also have a fairly rapid effect, with a reduction in enhancing lesions seen within weeks of starting therapy (Stone et al., 1995; Pozzilli et al., 1996). Measuring contrasting– enhancing and T2 lesions as surrogate markers of drug efficacy should be done with caution. These measurements are nonspecific because they represent a variety of pathological processes. Also, they correlate poorly with clinical relapses and disability progression. Recent studies have shown beneficial effects of IFNs on brain atrophy and T1 holes, which represent neurodegenerative processes. Hence, they correlate better with disability (Losseff et al., 1996; Truyen et al., 1996). However, brain atrophy measures should also be done with caution. When T2 burden of disease decreases, there can be resultant atrophy, at least in the short term as retracted gliotic scars replace areas of inflammation. IFN-h 1b decreases the rate of brain atrophy during years 2 and 3 of therapy in RRMS and reduces the development of T1 lesions in SPMS (Barkhof et al., 2001; Frank et al., 2004). In a 2-year trial, IFN-h 1a given at 30 Ag per week trended towards reduction of T1 lesion volume as compared to placebo (Simon et al., 2000). In a different trial, the same treatment significantly reduced the rate of brain atrophy in RRMS patients, as measured by brain parenchymal fraction (BPF) (Rudick et al., 1999). IFN-h 1a given 3 times a week has similar effects on T1 lesion volume and brain atrophy in RRMS (Gasperini et al., 2002). 7.2. Long-term efficacy of interferons in multiple sclerosis Long-term efficacy of IFNs in MS is difficult to assess. There are no long-term, placebo-controlled, double-blinded trials of IFNs in MS, mostly due to ethical considerations. In the pivotal trials, patients assigned to the placebo arm were crossed-over to treatment arms after 2 years. Long-term followup of these patients has been done in an open-label manner, introducing examination biases. Another major problem in long-term follow-up studies was the significant number of patients who dropped out or were lost to follow-up, potentially selectively biasing the study population. Therefore, in extension portion of the trials and long-term follow-up studies, best efforts must be made to identify clinical and MRI measures

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both in the drop out population and the remaining study population. The London, Ontario cohort of patients in IFNB multiple sclerosis study has the longest follow-up, 12-year data (Rice, 2001). Of the 31 patients enrolled in the pivotal trial, 61% were still on IFN-h 1b and 39% had withdrawn. Most of the patients who withdrew had disease progression (7/12). Patients continuing IFN treatment had decrease in the T2 burden of disease compared to untreated, age-matched controls. Similar results were seen in the long-term follow-up study of Rebif in MS, 7- to 8-year data (Li et al., 2004). In 68% of the original cohort, patients treated with higher dose of IFN (44 Ag tiw) had the lowest MRI disease burden, compared to the lower dose of IFN (22 Ag tiw) or cross-over controls. Even though these data show benefit of IFN on MRI parameters and 32% of the patients remain to be examined, it needs to be determined whether disability measures also remain stable over a longer time period. 7.3. Interferons and progressive multiple sclerosis Two large studies have provided conflicting but explainable results on the efficacy of IFNs in SPMS. In European Study Group on Interferon-h 1b in Secondary Progressive MS trial, IFN therapy delayed the time to confirmed progression of disability significantly, about 9 –12 months (Kappos et al., 1998). It also reduced the relapse rate, T2 lesion volume, and contrast enhancing lesions. In the North American Study Group on Interferon-h 1b in Secondary Progressive MS, similar effects were seen on relapse rate and MRI measures (Panitch et al., 2004). However, IFN did not reduce time to confirmed disease progression. The difference in the efficacy of IFN in disability progression can be explained by several factors. At baseline, the European trial (EU) had fewer number of relapse-free patients. Also patients in the EU trial had more frequent relapses in the prior 2 years, mean of 1.7 versus 0.82. Hence, more patients in the EU trial had more active disease. The NA trial required a 1-point increase in EDSS score in the prior 2 years; in the EU trial, either 1-point increase in EDSS score or 2 relapses in the previous 2 years were sufficient. End-point measurements were also more stringent in the NA trial, requiring 6 months for assessing sustained progression versus 3 months in the EU trial. Based on the EU trial, IFN therapy in patients with more active disease (frequent relapses) may be beneficial in delaying disability progression. The NA trial suggests little utility in treating SPMS without relapses. In PPMS, there is no clear evidence for the efficacy of IFNs on disability progression. A 2-year trial of IFN-h 1a given either at 30 or 60 Ag once weekly failed to show any effect on clinical outcomes (Leary & Thompson, 2003). Recently, a European pilot study using IFN-h 1b over 2 years showed some efficacy in PPMS on the MSFC scores and MRI measures (Montalban, 2004). IFN-h 1b treatment did not have any effects on disability progression as assessed by EDSS scores. It remains to be determined whether these differences will hold in a larger trial or over a longer time period.

7.4. Interferons and neutralizing antibodies IFNs are immunogenic proteins. Antibodies that develop in response to IFN treatment have been characterized as binding or neutralizing. Persistent neutralizing antibodies (NAbs) are associated with a reduction in the efficacy of IFNs, on both clinical and MRI measures (Rudick et al., 1998; Li et al., 2001). However, several characteristics of NAbs make it difficult to assess their role in clinical efficacy of IFNs. First, the development of NAbs during IFN therapy is often transient, developing within 3– 18 months after therapy and disappearing over months or years in most cases (Rice, 1997; Reske et al., 2004; Sibley et al., 1996; Vartanian et al., 2004). This makes it difficult to assess the apparent effects of NAbs seen in short-term studies. Second, there is a volatility in the development of NAbs, some patients revert back-and-forth from positive to negative states (Petkau et al., 2004). Hence, it is difficult to correlate NAb status and clinical efficacy. Third, there is about a 6-month lag in the onset of clinical efficacy after starting IFN therapy and a 6-month delay after stopping treatment. Any effect of NAb should also be delayed. Fourth, high titers of NAbs are seldom observed in patients who worsen on IFN therapy (Hurwitz & Polman, 2003). Hence, clinical disease worsening is not clear evidence for the presence of high titers of NAbs. These surprising data raise the issue of cause versus correlation. Fifth, in the long-term follow-up studies, NAbs disappeared over time (Rice et al., 1999). Finally, there is a dose-dependent effect of IFNs on the prelavance of NAbs. In the PRISMS studies, NAbs developed in 24% of patients receiving 22 Ag of IFN-h, but only in 12– 14% receiving 44 Ag of IFN-h (PRISMS, 1998; PRISMS-4, 2001). In the pivotal IFN-h 1b study, relapse rates during NAbpositive periods were also higher for the low-dose IFN-h-1btreated patients than those receiving a high-dose therapy (Petkau et al., 2004). This implies that 1 way of circumventing the development of NAbs is by using high-dose IFNs. There are no clear guidelines as to whether NAbs need to be tested in patients on IFNs. Up until recently, some clinicians were testing for NAbs after 12 months of therapy. Many experts do not recommend routine testing of NAbs, regardless of disease status because of cost and uncertain utility. Currently, the issue of NAbs is being critically reviewed in order to establish more clear guidelines. 7.5. Adverse effects of interferons IFNs are generally well tolerated, as discussed in several reviews (Cirelli et al., 1997; Walther & Hohlfeld, 1999). The most common side effects include local site injection reactions, fever, chills, headache, and myalgias (‘‘flu-like’’ symptoms). Some patients, usually with pre-existing spasticity, have increased spasticity on the day after the injection. This may be from loss of spinal inhibition by inflammatory cytokines or a direct effect of IFNs on neurons. Most side effects usually develop after 6 hr of injection and resolve by 12 hr. With repeated use, the ‘‘flu-like’’ side effects dissipate. The use of acetaminophen or ibuprofen prior to IFN injection

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is usually helpful in decreasing ‘‘flu-like’’ symptoms. Also, when starting therapy, a slow dose escalation, especially for the high-dose IFN preparations, is helpful in alleviating the above symptoms. Laboratory abnormalities are can be associated with IFN therapy. Common ones include leukopenia and elevated liver transaminases (AST and even better, ALT). These lab abnormalities are often transient and stabilize over time. However, some patients may have a persistent leukopenia, usually >3000 cells/AL. Liver transaminases usually increase within 1 month of starting therapy and decline thereafter. This is due to either direct effects of IFN on hepatocytes or secondary to IL-6 induced by IFNs. Some patients may continue to have slightly elevated liver transaminases. These patients are typically asymptomatic and continue IFN therapy. Repeated elevation of liver transaminases more than 3 times the normal value warrants dose reduction or possible discontinuation of the drug. Although hepatic dysfunction with IFN therapy is typically asymptomatic, fulminant cases of hepatic failure have been described (Durelli et al., 1998; Yoshida et al., 2001; Tremlett & Oger, 2004). Therefore, routine liver function monitoring is recommended, especially if concomitant drugs with potential liver toxicity are used, and because random liver toxicity cannot be predicted. Development of autoimmunity is rare with IFN-h (Walther & Hohlfeld, 1999). Autoimmune thyroiditis has been noted with IFN-h therapy. At baseline, MS patients have a higher incidence of pre-existing thyroid disease, particularly Hashimoto’s thyroiditis (Karni & Abramsky, 1999). IFN therapy has been associated with early thyroid dysfunction, especially in patient with pre-existing thyroiditis (Monzani et al., 1999; Monzani et al., 2000). However, thyroid dysfunction is often subclinical and spontaneous resolution occurs in most patients, without the need for withdrawing IFN therapy (Monzani et al., 2004). Thyroid function should be checked prior to initiating IFN therapy and over time, especially if malaise and fatigue symptoms become prominent. 8. Interferons and combination therapy The therapeutic trials of IFNs demonstrate a modest efficacy of these agents in treating MS. Hence, there have been continued attempts to improve the efficacy of drugs for treating MS. Several studies have demonstrated partial efficacy of multiple agents in MS, including statins, immunosuppressive agents, immunoglobulins (IVIG), and more recently PPAR agonists (e.g., rosiglitazone, pioglitazone). Clinical practices vary, but some clinicians have combined IFN therapy with immunosuppressive agents and IVIG, especially in patients with aggressive disease. The efficacy of combining IFNs with statins or PPAR agonists remains speculative at this point. Moreover, it is not certain whether such a combination is safe. The 2 agents that have received the most attention in terms of their concomitant use with IFNs are natalizumab and glatiramer acetate.

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8.1. Role of natalizumab in multiple sclerosis Natalizumab (Tysabri, formerly known as Antegren) is a human IgG4k monoclonal antibody (MAb) with short segments of murine protein at the antigen recognition site (CDR). It was approved by the FDA in November 2004 for treatment of RRMS after an impressively short trip from lab bench to bedside. It is also potentially effective in Crohn’s disease and rheumatoid arthritis. Natalizumab binds to a4h1 integrins (also known as VLA-4) on the surface of immune cells. VLA-4 is expressed on T cells, B cells, and monocytes. It interacts with VCAM-1 on endothelial cells of the BBB. Interaction of VLA-4 with VCAM-1 is involved in migration of immune cells across the BBB. By binding to VLA-4, natalizumab blocks the entry of immune cells into the CNS. Natalizumab inhibits migration through endothelial cells by the vast majority of T cells and about 75% of monocytes (Deloire et al., 2004). In a 25-month, phase III MS trial of 942 RRMS patients, monthly infusions of 300 mg natalizumab reduced new contrast enhancing MRI lesions by 92%, MRI T1 black hole development (32% with natalizumab compared to 74% in placebos) (Dalton et al., 2004), relapse rate by 66%, and progression by 42% (progression confirmed by consistent neurological exam scores, 3 months apart) (Phillips et al., 2005). The impact on progression was critical, as this is the most important criterion for an MS therapy. Another trial evaluated 1196 patients who had 1 exacerbation in the prior year during therapy with Avonex alone (weekly intramuscular IFN-h 1a). In combination with IFN, natalizumab reduced relapse rate by 53%, compared to Avonex alone (Rudick et al., 2005). The superiority of natalizumab versus placebo in this group of patients was not studied and is difficult to forecast. Patients in this study were weighted toward poorer IFN-h 1a responses ( 1 attack in the prior year on Avonex); those with good clinical responses were excluded. (Potentially, data from earlier trails for comparison with a similar group of Avonex-treated patients with  1 attack versus matched placebos could suggest the relative advantage of Avonex plus natalizumab over Avonex alone and over placebo). Natalizumab was also tested in Crohn’s disease and rheumatoid arthritis with promising preliminary results. Most adverse effects of natalizumab were minimal (Rudick & Sandrock, 2004). There were rare allergic reactions to the infusion, no tuberculosis, and statistically nonsignificant suggestions of a slight increase of infections and cancer. Metastasis and invasion are enhanced by integrins on cancer cells, so VLA-4 blockade is potentially therapeutic in some cancers. NAb to natalizumab developed in 6% over 2 years. When persistent, NAb abolished efficacy. Unexpectedly, a form of encephalitis caused by the JC virus, progressive multifocal leukoencephalopathy (PML), developed in 2 patients in the Avonex plus natalizumab group (after 28 and 37 natalizumab infusions), and in 1 Crohn’s patient who had received multiple treatments of immunosuppressive drugs and 8 natalizumab infusions. Investigators and patients were aware of a potential risk of CNS lymphoma and systemic

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infections with this experimental drug, but concern had been allayed by the minimal side effects over 5 years of human trials. After the PML cases arose, administration of the drug was put on hold, as were important trials of natalizumab in primary progressive MS, Crohn’s disease, and rheumatoid arthritis, and in other MS trials with small protein blockers of VLA-4/VCAM interactions. This complication has far-reaching implications. Future therapy with this effective agent, if available, will require weighing of risk/benefit ratios by well-informed patients and close monitoring. This may include frequent neurological exams and MRIs, and possibly testing for JCV titers in blood or CSF. Understanding the mechanism of how PML developed will lead to better understanding of immune surveillance of CNS by T cells, synergy between IFNs and anti-VLA-4 agents, and the importance of VLA-4 in the brain, other immunologically privileged sites, and peripheral organs. In addition, it remains to be determined whether the combination of natalizumab with immunosuppressive agents or IFNs is safe. Of note, PML was only seen in patients who were also taking immunosuppressive drugs (Crohn’s patients) or Avonex. IFNs also affect T cell migration into the CNS. IFN-h causes a pronounced reduction in matrix metalloproteinase (MMP) secretion by T cells, down-regulates IFN-g-induced adhesion molecules on endothelial cells, and counteracts the increased microvascular permeability induced by LPS (Yong et al., 1998; Kuruganti et al., 2002). These actions prevent immune cell penetration through endothelial cells barriers. It is possible that effects of IFN on T cell migration synergize with those of natalizumab, resulting in poor immune surveillance of the CNS. This would allow emergence of opportunistic infections, such as PML. 8.2. Combination of interferons with glatiramer acetate Glatiramer acetate (GA) is mixture of randomly arranged polypeptides composed of 4 amino acids. It is thought to suppress inflammation by inducing a Th2 cell response. Its immunomodulatory effects are believed to occur both in the periphery and CNS. The therapeutic efficacy of GA has been established by a large, multi-center, phase III trial (Johnson et al., 1995). The combination of IFNs with GA for MS therapy remains speculative at this time. One mechanism of action of GA is to deviate the immune response to a Th2 type. An important site of this action may be the CNS. IFNs decrease the frequency and size of contrast-enhancing lesions in MS, hence decreasing the permeability of the blood – brain barrier (BBB). By affecting the BBB, IFNs restrict entry of lymphocytes into the CNS. It is possible that combination of IFNs with GA would restrict the entry of GA-specific T cell in to the CNS, thereby limiting the effectiveness of GA in MS. Results from in vitro studies do not necessarily suggest antagonistic effects of IFNs and GA on T cells (Prat et al., 2005). IFNs and GA selectively affect different subpopulations of T cells. In cultured endothelial cells (ECs), both Th1 and Th2 cells migrate across the cell layer, with Th2 cells having a

higher migration rate. When lymphocytes and ECs are treated with IFN alone, migration of Th1 cells is significantly reduced and Th2 migration is unchanged. Treatment with GA alone increases Th2 migration without affecting Th1 migration. Similar effects IFN and GA may be seen in vivo with selective penetration of Th2 lymphocytes that may be helpful in curtailing inflammation. Combination of IFNs with GA has been reported to be safe (Lublin et al., 2001). The efficacy of this combination is currently being studied in the Combi-Rx trial sponsored by the NIH. 9. Future of interferon therapy in multiple sclerosis Several trials have shown better efficacy of high-dose IFNs in MS compared to low-dose IFNs, at least in the short-term analysis. The BEYOND trial is currently being conducted, examining the efficacy of higher doses of IFN in MS (comparison of standard 250 with 500 Ag given qod). Preliminary results indicate that higher doses of IFN are well tolerated and show an additional benefit on MRI parameters. In order to improve the pharmacokinetic properties of IFNs, pegylated IFNs have been developed. Pegylation involves attaching PEG (polyethylene glycol) moieties to IFNs. Pegylated products have slower systemic clearance, prolonged halflives, and reduced antigenicity compared to the recombinant IFNs (Moreno-Otero, 2005). They can therefore be dosed once weekly. Pegylated IFNs have enhanced efficacy when compared to the conventional IFNs in the treatment of Hepatitis C infection (Lindsay et al., 2001; Fried et al., 2002). Pegylated IFN-h is currently being studied in MS. Recently, intranasal administration of IFN-h was examined as a method for targeting drugs to the CNS in animals (Ross et al., 2004). Nasal administration resulted in 5 – 10 times higher levels of IFN-h in the brain and 10 – 40 times higher levels in the cervical lymph nodes than the intravenous (IV) route. IFNh delivered intranasally stimulated tyrosine phosphorylation of IFN receptors, demonstrating biological activity of this route of administration. It remains to be determined whether intranasal drug delivery to the CNS will prove useful in MS. It is unclear whether the therapeutic effects of IFN-h are on peripheral or central immune mechanisms. It is presumed that systemic IFN-h (subcutaneous or intramuscular) predominantly affects peripheral immune cells. Very little IFN-h penetrates the normal BBB, but more may cross the BBB in MS. The CNS is an obvious site of intervention to decrease inflammation. Intranasal IFN-h could potentially decrease APC function of CNS microglia and inhibit IFN-g secretion. However, an IFN-h-induced decrease in IL-10 secretion by monocytes or microglia in the CNS could be deleterious (Feng et al., 2002b). 10. Conclusions The efficacy of IFN-h in MS has been well established by large, phase III trials. IFNs are clinically effective in reducing

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relapse rate, CNS inflammation as measured by MRI, and disability progression. The clinical effects of IFN-h in MS are significant but nonetheless modest. This implies that there must be molecular mechanisms that are either not affected by IFNs or are resistant to the effects of IFNs. There could be several sites within IFN response system responsible for the ineffectiveness of IFNs in MS. IFNs produce their effects by binding to surface receptors and initiating a complex cascade of intracellular secondary messengers. In clinically active MS, there are molecular defects in this pathway causing low levels of PSer-STAT1. IFN-h treatment partially ameliorates this defect. Investigation of compounds that synergize with IFN and enhance STAT1 phosphorylation may prove to be useful in MS. The immunopathology of MS is complex. Myelin destruction is mediated by Th1 cells and macrophages/microglia, along with their pro-inflammatory cytokines such as IFN-g, IL2, and IL-12. T cell suppressor function, which would normally limit the inflammatory response, is subnormal in MS. IFN-h inhibits Th1-mediated responses and leads to the secretion of anti-inflammatory cytokines such as IL-10 and TGF-h. It also partially enhances T cell suppressor function. Future clinical trials aim at enhancing the efficacy of currently approved IFNs. A phase III study, BEYOND, is underway evaluating the safety and efficacy of high-dose IFNs. Interestingly, treatment of certain cell lines with mitogens such as vitamin A or poly (I:C) enhances phosphorylation of STAT1. Pegylated and intranasal formulations of IFNs may turn out to be promising in the future. A decade of clinical experience with IFNs has shown continued clinical efficacy of these agents in MS. In view of the several ongoing trials with IFNs, it is likely that they will continue to be used to treat MS either as single agents or in combination with other drugs. Acknowledgments A. Javed is a Sylvia Lawry Physician Fellow of the National Multiple Sclerosis Society. References Abu-Khabar, K. S., Armstrong, J. A., & Ho, M. (1992). Type I interferons (IFN-a and -hb) suppress cytotoxin (tumor necrosis factor-a and lymphotoxin) production by mitogen-stimulated human peripheral blood mononuclear cells. J Leukoc Biol 52, 165 – 172. Adachi, K., Kumamoto, T., & Araki, S. (1990). Elevated soluble interleukin-2 receptor levels in patients with active multiple sclerosis. Ann Neurol 28, 687 – 691. Adolf, G. R. (1987). Antigenic structure of human interferon omega 1 (interferon alpha II1): comparison with other human interferons. J Gen Virol 68(Pt 6), 1669 – 1676. Aharoni, R., Kayhan, B., Eilam, R., Sela, M., & Arnon, R. (2003). Glatiramer acetate-specific T cells in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factor in situ. Proc Natl Acad Sci U S A 100, 14157 – 14162. Ahn, J., Feng, X., Patel, N., Dhawan, N., & Reder, A. T. (2004). Abnormal levels of interferon-gamma receptors in active multiple sclerosis are normalized by IFN-h therapy: implications for control of apoptosis. Front Biosci 9, 1547 – 1555.

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