NEW CONCEPTS IN THE IMMUNOPATHOGENESIS OF MULTIPLE SCLEROSIS

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1 NEW CONCEPTS IN THE IMMUNOPATHOGENESIS OF MULTIPLE SCLEROSIS Bernhard Hemmer*, Juan J. Archelos and Hans-Peter Hartung Multiple sclerosis (MS) is a commonly occurring inflammatory and demyelinating neurological disease. It has been considered to be an autoimmune disorder mediated by CD4 + type 1 T helper cells, but recent studies have challenged this idea by indicating a role for other immune cells. So, T- and B-cell responses in the brain of patients with MS involve the clonal expansion of lymphocytes and the antigen-driven maturation of the B-cell receptors, indicating that the immune response in MS engages a broad range of immune cells that target a limited number of brain antigens. At variance with the classical view, axons are not spared during the inflammatory process. Indeed, axonal damage determines clinical outcome to a large extent. Studies of the mechanisms of axonal damage and neurodegeneration in MS are in their infancy. Here, we summarize recent advances in our understanding of the pathogenesis of MS, and conclude with an outlook on how to capitalize on this knowledge to develop new therapeutic approaches. *Department of Neurology, Philipps-Universität, Marburg 35033, Germany. Department of Neurology, Karl-Franzens-Universität, A-8036 Graz, Austria. Department of Neurology, Heinrich-Heine-Universität, D Düsseldorf, Germany. Correspondence to H.-P.H. Hans-Peter.Hartung@ uni-duesseldorf.de DOI: /nrn784 Multiple sclerosis (MS) is one of the most common chronic and disabling disorders of the central nervous system (CNS), affecting % of Caucasians 1.The disease usually begins in young adulthood and affects women more frequently than men. In 80 90% of cases, MS starts with a relapsing remitting course (RR-MS). Over time, the number of relapses decreases, but most patients develop progressive neurological deficits that occur independently of relapses (the so-called secondary progressive phase). In 10 20% of patients, MS begins with a primary progressive course (PP-MS) without acute relapses. In general, the progression rate in RR-MS is comparable to that of PP-MS as soon as the patients enter the secondary progressive phase 2. Imaging studies have revealed differences between RR-MS and PP-MS. In patients that suffer from RR-MS, acute CNS lesions with spontaneous resolution are frequently detected, even in the absence of clinical attacks 3. These lesions are usually located in areas of white matter, and are often characterized by a disturbance of the blood brain barrier, local oedema and demyelination features that are compatible with an inflammatory process. By contrast, when progressing to the secondary phase and in patients with PP-MS, such inflammatory activity is much less conspicuous 3. Global brain atrophy, however, is more dominant in the progressive stage and seems to correlate with disability 4,5. These findings indicate that early in the disease, ongoing inflammatory activity is present in most patients and is responsible for the relapsing remitting course, whereas a distinct process might be operative in the progressive phase of the disease, when inflammatory activity diminishes despite faster evolution of disability. The prevalence of MS varies significantly depending on the genetic background of the patient 6. MS is highly prevalent in Caucasians, but only rarely observed in Asians or Africans. Even in areas with high MS prevalence, these ethnic groups are at much lower risk than Caucasians. Moreover, the risk of developing the disease is significantly higher in family members of patients with MS 7. By contrast, the prevalence in spouses and adopted family members does not differ from that of the general population. These findings argue for a strong genetic predisposition to MS, and have prompted a large number of linkage and association studies to identify disease loci and NATURE REVIEWS NEUROSCIENCE VOLUME 3 APRIL

2 Box 1 The acquired immune system T cells The T-cell population is the central arm of the acquired cellular immune response. Most T cells carry T-cell receptors (TCRs) to recognize antigens that are bound to major histocompatibility complex (MHC) molecules (human leukocyte antigens (HLA) in humans) 122. The antigens are usually derived from complex proteins that are processed by antigen-presenting cells (APCs). Through random recombination of the TCR genes, a very broad T-cell repertoire is generated, which is largely depleted from potentially autoreactive T cells by a maturation process in the thymus. In lymph nodes, naive T cells are approached by APCs, such as dendritic cells, which present antigens that are bound to MHC molecules on their surface. After recognition of specific peptide MHC complexes on the APC, the cell becomes activated, clonally expands and acquires effector functions (such as cytotoxicity or the ability to secrete cytokines). The cells leave the lymph nodes, migrate through the body and accumulate at sites where they encounter their specific peptide MHC ligands. On reactivation, the T cells mediate effector functions that lead to clearance of the antigenic source, and subsequently undergo apoptotic cell death. Only a few survive and remain in the immune system as a memory T-cell population. The population of T cells can be broken down into several subpopulations. T helper (T H ) cells, which are characterized by the expression of the CD4 receptor, usually recognize peptides from endocytotic sources in the context of MHC class II molecules. Cytotoxic T (T C ) cells, which express the CD8 receptor, mainly respond to cytosolic proteins in the context of HLA class I molecules. Additional T-cell subsets are characterized by the secretion of specific immune mediators (cytokines and chemokines). So, T H 1/ T C 1 cells secrete interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α). By contrast, T H 2/ T C 2 cells release IL-4, IL-5, IL-10 and IL-13. Last, T H 0/T C 0 cells secrete both T H 1 and T H 2 cytokines 124.T H 1 cells promote macrophage activation, facilitate the differentiation of cytotoxic cells and cause delayed hypersensitivity, whereas T H 2 cells help B cells and stimulate allergic reactions. Importantly, T cells cannot recognize peptides in the absence of the appropriate MHC molecules, and the TCR does not change its molecular structure during repeated antigen exposure. B cells The B-cell population is the central arm of the humoral immune response. The cells recognize antigens by means of the B-cell receptor (BCR) 125. The BCR of each B cell is generated through a random rearrangement process of the BCR genes, in a similar way to TCR. B cells usually recognize soluble antigens and do not require antigen presentation by other cells. Immature B cells, as for T cells, undergo a selection process in the bone marrow to deplete autoreactive cells from the repertoire. After maturing, B cells express membranebound immunoglobulin-µ (IgM) and migrate to the periphery. On encountering highaffinity antigens, they migrate to the peripheral lymphoid tissue, proliferate, release soluble IgM and differentiate into memory B cells or plasma cells 126. During the differentiation process, an isotype Ig switch from IgM to IgG (immunoglobulin-γ) occurs. Repeated encounters with the same antigen by memory B cells induce a process of receptor maturation that is characterized by the introduction of random mutations in the BCR gene. This results in structural changes to the BCR, which usually lead to affinity maturation in some of the mutants. This process yields B cells that recognize the antigen more efficiently than the original B cell. The B cells with the receptors of highest affinity succeed in the expansion process and differentiate into plasma cells that secrete large quantities of antibodies. BCRs can recognize intact soluble molecules and undergo genetic maturation. These features complement the more diverse, but less flexible, T-cell repertoire. FREUND S ADJUVANT An oil emulsion that contains an immunogen, an emulsifying agent and mycobacteria, which enhance the immune response to the immunogen. ADOPTIVE TRANSFER An immune response involving the transfer of immunocompetent cells from a primed donor to a non-immune recipient. alleles. The results of genomic screens in MS indicate that a considerable number of different genes, each having a relatively small contribution, are involved in the susceptibility to MS 8,9. The large variations in susceptibility loci between the studies also indicate heterogeneity in disease alleles, even in Caucasian populations. The role of genetic factors is even more complex, as they seem to be involved not only in disease manifestation, but also in the clinical course of MS 10,11. Owing to the polygenic nature of genetic predisposition to MS, the identification of candidate genes has been extremely difficult 9. So far, only the human leukocyte antigen (HLA) class II alleles DR15/DQw6 (HLA-DRB1*1501; HLA-DQB1*0601), which code for molecules that participate in antigen recognition by T lymphocytes, have been consistently associated with MS in Caucasians 12. Epidemiological studies have provided evidence for an additional, environmental component in the disease process. For example, MS relapses are often associated with common viral infections 13, and migration from high- to low-risk areas before adolescence reduces the risk of developing MS 14. In areas with homogeneous ethnic populations, MS prevalence varies, being higher in areas with moderate climate 15. The impact of environmental factors is also supported by few reported MS epidemics. In particular, the case of the Farøe Islands where MS was unknown until 1940 and broke out shortly after British soldiers landed on its shores argues for the role of an environmental, possibly transmissible, agent in disease pathogenesis 16. Overall, clinical studies indicate that MS is characterized by at least two distinct phases, one that is dominated by acute relapses and one by steady progression. Both genetic and environmental factors seem to contribute synergistically to the manifestation and progression of the disease. From MS to EAE and back The classic EAE concept. The disease process in MS is focused on myelinated areas of the brain and spinal cord 17. Histopathological studies have shown that demyelination and inflammation involving B cells, T cells, macrophages and activated microglia are hallmarks of acute MS lesions. Tissue damage, including loss of neurons and oligodendrocytes, astrogliosis and remyelination, accompanies the inflammatory changes. The role of the immune system (BOX 1) in the pathogenesis of MS was emphasized by early reports of acute demyelinating episodes in humans after accidental immunization with myelin components (for example, after vaccination against rabies) 18. Following this lead, an animal model known as experimental autoimmune encephalomyelitis (EAE), was established 70 years ago 19 (FIG. 1). EAE is an inflammatory disease of the CNS, with variable degrees of demyelination, that is induced by immunization of susceptible animals with myelin antigens and FREUND S ADJUVANT 20. Although EAE can be elicited in most rodents and in some monkeys, susceptibility, disease course and severity are strain dependent. The role of an autoimmune response in this model was formally proven by ADOPTIVE TRANSFER experiments, which showed that immune cells from a diseased animal can transmit disease to naive animals 21. Myelin proteins were identified as the diseasecausing antigens in EAE, and cell separation before adoptive transfer indicated that mainly CD4 + T CELLS transmit the disease. With the introduction of T-cell cloning technology, it became possible to show that T cells that secreted T helper (T H ) 1 CYTOKINES such as interferon-γ (IFN-γ), tumour necrosis factor-β (TNF-β) and interleukin 2 (IL-2), and the pro-inflammatory cytokine TNF-α were more likely to transfer the disease 292 APRIL 2002 VOLUME 3

3 MAG MOG Surface MOG P P Myelin sheath OA PLP PLP MBP MBP PLP PLP NA P NA Neuron An autoimmune response to myelin antigens would explain many of the features of MS, but the available association studies have not shown convincingly that the frequency or function of myelin-specific CD4 + T cells are different in patients with MS and healthy people 28,34. Similarly, a pathogenetic role for myelin antibodies has not been established unequivocally 35.However,the EAE model has provided the basis for most MS therapies during the past decade. Approaches that focus on silencing the immune response to myelin antigens, shifting immune responses from T H 1 to T H 2 or antagonizing TNF-α, have all been shown to be highly efficient in most EAE models 36. CD4 + T CELLS A subset of T lymphocytes that carry the CD4 receptor and are essential for turning on antibody production, activating cytotoxic T cells and initiating other immune responses. CYTOKINES In general terms, cytokines are proteins made by cells that affect the behaviour of other cells. They are produced mainly by the immune system. T H 1 CELLS A subset of T cells that secrete inflammatory cytokines. CD8 + T CELLS A subset of T lymphocytes that carry the CD8 receptor, such as cytotoxic T cells. The CD8 protein is the co-receptor for class I molecules of the major histocompatibility complex. INNATE IMMUNITY The early response of a host to infection. One of its main features is the pro-inflammatory response induced by antigenpresenting cells macrophages, dendritic cells and, in the brain, microglial cells. This response is followed by an adaptive response that is mediated by clonal selection of lymphocytes, which leads to long-term immune protection. Oligodendrocyte NA OA P Antigens resident in neurons Antigens resident in oligodendrocytes Proteins encoded by foreign DNA Figure 1 Possible target antigens in the white matter. Proteins of the myelin sheath, oligodendrocytes and neurons are possible targets of the immune response in multiple sclerosis. Among the candidates are myelin and neuronal antigens, and also proteins that are introduced into those cells by infectious agents. MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein. than other myelin-specific T cells 22. By contrast, T cells that secreted IL-4, IL-5, IL-10 and IL-13 seem to protect animals from the disease 23. These findings established the widely accepted idea that EAE is a prototypic autoimmune disorder that is caused by a T H 1 CELL response to myelin antigens. Because EAE shares some clinical and pathological features with MS, the model was applied to the human disease. Further support for the idea that the disease results from a T H 1-driven immune process was provided by a clinical trial showing that administration of the T H 1 cytokine IFN-γ exacerbates MS 24. The impact of EAE on MS. To strengthen the idea that MS is caused by a T H 1 response to myelin, several laboratories have characterized immune responses to myelin antigens in patients with MS. Although higher antibody titres to some myelin antigens are observed in the CNS and serum of patients with MS, such autoantibodies are also found in healthy people CD4 + T cells that are specific for a variety of myelin antigens were isolated from patients with MS and from healthy donors, showing that autoreactive T cells are part of the normal T-cell repertoire and are not necessarily harmful 28,29. Investigations of antigen recognition of myelin-specific T cells showed that these cells can potentially cross-react with foreign antigens such as viral peptides 30,31. This cross-recognition termed molecular mimicry has provided an attractive model to explain how self-reactive T cells could become activated by infectious agents to mediate an autoimmune process 32. After the initial CNS tissue damage by autoreactive T cells, the release of myelin antigens could promote immune responses to additional myelin epitopes, starting a cascade of events that culminates in chronic disease 33. Broadening the EAE model. When similar approaches entered clinical trials in humans, first observations in genetically modified animals began to question the validity of the T H 1 model 37. Mice lacking IFN-γ develop an even more severe disease 38,39, and overexpression of T H 1 cytokines in the CNS ameliorates EAE 40. By contrast, disruption of the IL-4 gene does not alter the disease course 41. Although overexpression of TNF-α in the CNS results in demyelination in most animal models 37, TNF-α knockout mice still develop EAE in some cases with a disease course that is even more severe than that of their wild-type littermates Because permanent overexpression or disruption of immune genes can significantly influence the development of the lymphoid and nervous systems, the results from genetic animal models of neuroinflammation have been interpreted with caution 46. During the mid-1990s, other studies of EAE reported on the encephalitogenic potency of T H 0 cells, which secrete both T H 1 and T H 2 cytokines 47.Moreover, it was even possible to induce EAE with myelin-specific T H 2 cells in a transgenic mouse model 48.Histopathological studies supported this observation by showing the presence of T H 1 and T H 2 cytokines in EAE and MS lesions 49,50. Recently, myelin-specific CD8 + T CELLS were found to induce progressive EAE 51,52. Lesions in these models were located primarily in the brain not in the spinal cord as seen in most EAE models that are induced by CD4 + T cells and featured only little inflammation, but massive perivascular cell death and demyelination. Similarly, the role of antibodies in EAE is well established. B-cell-deficient mice develop EAE but remission of disease is incomplete 53. Antibodies cannot passively transfer EAE to naive animals by themselves. However, EAE severity in some models is significantly enhanced by co-administration of myelin-specific antibodies after induction of disease 54. Finally, the role of the innate immune system has been clarified in EAE. Although T cells are a cell population that transfers the disease, they are strictly dependent on INNATE IMMUNITY in the CNS. In particular, microglial cells provide the proinflammatory milieu that is required for efficient T-cell activation 55. The CD4 + T H 1 model was also questioned by EAE treatment studies. The potency of antigen-specific intervention therapies was not strictly correlated with the extent of skewing immune responses from T H 1 to NATURE REVIEWS NEUROSCIENCE VOLUME 3 APRIL

4 Primary Autoimmune (e.g. EAE model) Target antigens Secondary Infectious (e.g. MHV model) Myelin antigen Priming Release Virus SUBACUTE SCLEROSING PANENCEPHALITIS A form of encephalitis that develops after the reactivation of a latent measles virus. It is characterized by progressive motor and mental deterioration, accompanied by myoclonus. HTLV-I-ASSOCIATED MYELOPATHY Also known as tropical spastic paraparesis, this progressive neurological disorder is associated with the human T lymphotropic virus type I, and is characterized by spasticity and hyperreflexia. HUMORAL RESPONSE An immune reaction that can be transferred with immune serum. In general, this term refers to immune resistance that results from the presence of specific antibodies. Lymphoid tissue Central nervous system Migration Targeting Tissue damage Lymphocytes Secondary Degenerative (e.g. axotomy model) Figure 2 Possible mechanisms leading to neuroinflammation. Neural antigens are released into lymph nodes, where they are presented to B and T cells by antigen-presenting cells (such as dendritic cells). T and B cells with high-affinity receptors for these antigens are expanded and released from the lymph nodes. The cells migrate through the body and accumulate at sites where they re-encounter their priming antigen. On reactivation, these cells recruit effector functions. In autoimmune models, peripheral immunization with central nervous system (CNS) antigens can induce an autoimmune response that targets CNS antigens and leads to primary CNS inflammation. Introduction of non-self antigens (for example, viral infection) or acute brain damage (such as stroke) precipitates the release of antigens to the periphery, possibly priming a secondary immune response against those antigens. EAE, experimental autoimmune encephalomyelitis; MHV, mouse hepatitis virus. T H 2. Likewise, therapeutic approaches to EAE in monkeys induced a T H 2 shift but resulted in severe relapses 56. However, the most striking arguments for the limitation of the CD4 + T H 1 model came from clinical trials in humans. Surprisingly, two studies showed that inhibition of TNF-α by a blocking antibody or soluble TNF receptor exacerbates MS 57,58. Global depletion of CD4 + T cells failed 59, and most antigen therapies that specifically targeted the CD4 + myelin response were inefficient or even worsened the disease 60. This was particularly true for therapies that induced a T H 2 shift in myelin-specific T cells, such as altered peptide ligands or oral administration of myelin 61,62. Reading the new tracks in MS Rethinking CNS inflammation. The difficulties in nailing down the immune response in humans, together with the disappointing results from clinical trials, have called into question whether MS is primarily an autoimmune disease against myelin antigens (FIG. 2). As inflammation is seen in many infectious and degenerative disorders of the CNS, the immune response in MS could also be a secondary event (BOX 2). Indeed, the idea that MS is caused by an infectious agent has been controversial for almost a century. However, the hypothesis is still attractive in view of other infectious diseases of the CNS that cause inflammation and demyelination in humans, such as SUBACUTE SCLEROSING PANENCEPHALITIS or HTLV-I-ASSOCIATED MYELOPATHY 63. In both disorders, the cause is a primary infection of the CNS with intact or mutant virus, whereas an inadequate immune response probably contributes to disease progression 63. The Theiler s murine encephalomyelitis virus and the mouse hepatitis virus (MHV) induce demyelinating encephalitis in mice. In particular, the MHV model shares many features with MS 64. Mice naturally infected with MHV, which is a common enteric pathogen, rarely develop spontaneous acute encephalomyelitis. But direct inoculation of the CNS with a low dose of virus usually leads to a chronic demyelinating encephalomyelitis. During the first phase of disease, the virus replicates rapidly in the CNS and induces variable degrees of demyelination. After a few days, increasing mononuclear cell infiltrates that target the virus infection are observed. Predominantly CD8 + T cells, but also CD4 + T cells, are crucial for controlling the virus 65, whereas B cells and antibodies seem to be less important in the acute disease stage 66. Although efficient immune responses are mounted in MHV-infected animals, CNS sterility is not achieved. Persistent latent virus infection is associated with continuing demyelination foci, although the virus cannot be recovered from the infected brains. In this phase, the HUMORAL RESPONSE is essential for controlling virus reactivation 67.However, T- and B-cell responses might contribute to the disease mechanism that is involved in chronic tissue destruction 68,69. The extent of disease and the outcome depend largely on the MHV strain and on the genetic background of the mouse. In contrast to EAE, the immune response in MHV infection is secondary to an immune response that specifically targets viral antigens in the CNS (FIG. 2). The quest for a pathogen that causes MS has been continuing for almost a century. Neither the search for infectious particles in lesions nor the transmission of brain tissue to laboratory animals has so far identified a pathogen that is relevant to MS. Similar to autoantibody studies, many researchers have reported elevated antibody titres to a broad range of pathogens in patients with MS, although these studies are not always confirmed. This also applies to recently proposed pathogens, such as Chlamydia pneumoniae, human herpesvirus type 6 and EPSTEIN BARR VIRUS The inconsistencies between studies might be a result not only of different experimental setups, but also of possible heterogeneity in exogenous factors that are involved in the predisposition to MS. However, clear evidence that any pathogen has a role in the pathogenesis of MS is lacking so far. Neurodegenerative disorders, traumatic CNS tissue damage and stroke as well as infectious diseases cause inflammation of the CNS 75,76. Studies of these disorders have shown that loss of CNS tissue integrity is associated with microglial activation, cytokine production, gliosis and infiltration of leukocytes. These 294 APRIL 2002 VOLUME 3

5 Box 2 Immune defence in the central nervous system The brain has long been considered to be an immunologically privileged site. This idea is based on the observation that tissue transplants in the central nervous system (CNS) are not commonly rejected by the immune system. An anti-inflammatory and pro-apoptotic environment in the brain, and the limited access of brain-derived antigens to the lymphoid organs, were used to explain the lack of an effective immune response to antigens in the brain. However, recent studies in infectious and autoimmune models have challenged this view. Systemic inflammation and tissue damage lead to activation of microglia, which are the main arm of the innate immune system in the CNS. Activation of microglia results in release of inflammatory mediators and upregulation of immune receptors (for example, major histocompatibility complex (MHC) molecules) on other CNS cells, preparing ground for the acquired immune response in the CNS 78,127.Proteins released by tissue damage are quickly drained into local lymph nodes 128. In the presence of antigen-presenting cells, such as dendritic cells, efficient B- and T-cell responses are initiated. After priming, these cells cross the blood brain barrier, migrate to the site of antigen exposure and develop effector functions 129. Homing to the CNS, adhesion to the endothelial lining and penetration of the blood brain barrier encompass a highly coordinated sequential interaction of adhesion molecules that are reciprocally expressed on invading leukocytes and endothelium, the discharge of matrix metalloproteinases by T cells and the T-cell perception of chemokines in the nearby CNS parenchyma Likewise, B cells differentiate on re-encountering their antigen in plasma cells and release large amounts of antibodies in the CNS. Similarly, CD4 + and CD8 + T cells become activated on encountering their antigen on appropriate MHC molecules, and release cytokines or directly target the presenting cell. Although CD8 + T cells can recognize antigen that is presented by neurons and glial cells, CD4 + T cells are dependent on activated microglial cells or infiltrating macrophages. These cells phagocytose cell debris, efficiently capture antibody antigen complexes by their Fc receptors, and present those antigens on MHC class II molecules to CD4 + T cells. On activation, they secrete mediators of inflammation such as tumour necrosis factor-α. Both the innate and acquired immune system synergistically target the antigenic source and remove it from the CNS tissue. This view is consistent with clinical observations that infections of the CNS induce vigorous immune responses, clearing pathogens from the site. Priming of T cells in the peripheral lymphoid tissue is also essential for disease induction in active immunization and adoptive-transfer experimental autoimmune encephalomyelitis models 133. Accordingly, CNS inflammation in experimental animal models is blunted after the removal of nuchal lymph nodes 134. EPSTEIN BARR VIRUS A herpesvirus that is the main cause of mononucleosis and is associated with several cancers, particularly lymphomas, in immunosuppressed people. OLIGOCLONAL Describes cells or members of a clone that share a specific feature. INTRATHECAL Within the meninges. HEAVY CHAIN All immunoglobulins have two types of chain heavy (50 70 kda) and light (25 kda). The basic immunoglobulin unit consists of two heavy and two light chains. CLONAL EXPANSION The proliferation of antigenspecific lymphocytes from a single cell in response to antigenic stimulation. This expansion precedes their differentiation into effector cells. findings indicate that brain damage and release of antigens to the periphery might prime an immune response that seems to target the antigen source in the brain (FIG. 2). The immune system in these disorders might contribute to the disease process 76.Peripheral leukocyte depletion or inhibition of leukocyte migration reduces tissue damage in stroke. As acute CNS tissue damage does not usually result in sustained CNS inflammation, the possible autoimmune response is apparently well controlled and terminated after the initial event 77. Some of the features of MS, such as the early loss of neurons and oligodendrocytes, are compatible with a primarily neurodegenerative cause of the disease. But an additional regulatory defect in the immune system must be present to explain the prominent and sustained immune response that occurs in the CNS of patients with MS. A possible explanation would be a change in the inflammatory milieu that is introduced by alteration of the local innate immune response 78. Alternatively, insufficient control of the cellular immune response in the CNS, accomplished by redistribution of immune cells to the lymphoid system or by local apoptotic cell death of immune cells, might be responsible Although studies of immune responses in infectious or degenerative diseases similar to the studies of autoimmune mechanisms have provided important insights into the mechanisms of neuroinflammation, they have not provided consistent clues to the aetiology of MS. Therefore, recent immunological research has focused on the inflammatory response that is detectable in the CNS of patients with MS to clarify the nature of the local immune response and the possible targets. Such approaches are reasonable because the immune cells that are enriched in the CNS of patients with MS probably have a causative or protective role in disease pathogenesis. Rethinking T- and B-cell responses in the CNS. It has been known for a long time that high concentrations of antibodies are found in the brain and cerebrospinal fluid (CSF) of patients with MS 82. These high concentrations are caused by a local OLIGOCLONAL immunoglobulin-γ (IgG) response that mainly involves the IgG1 and IgG3 isotypes 35,83,84. Indeed, the occurrence of an oligoclonal INTRATHECAL antibody response is still an important diagnostic marker for chronic CNS inflammation in MS. Interestingly, the antibody response seems to be stable over long periods and to involve a limited number of clonotypes 85. These findings were recently complemented by investigations of antibody-secreting B cells in the CNS and CSF of patients with MS These studies uniformly showed preferential use of specific HEAVY-CHAIN genes or CLONAL EXPANSION of B cells that were, largely, not observed in the peripheral blood of the patients 87,89. Expanded B cells in CNS lesions have extensive replacement mutations that are clustered in the hypervariable region of the B-cell receptor genes (BOX 1). Accumulation of B-cell clonotypes and antigen-receptor maturation is seen only after repeated exposure of memory B cells to the same antigen, which strongly argues for an antigendriven response. Such an immune response, involving oligoclonal IgG synthesis and clonal B-cell responses, is not unique to MS, but can be detected in a variety of acute and chronic infectious CNS disorders. In acute disorders, the intrathecal immune response vanishes after resolution of the infection, whereas in chronic infections, continuous intrathecal antibody production is observed. In chronic CNS infections, the intrathecal IgG synthesis is specific for the infectious agent, such as defective measles virus in subacute sclerosing panencephalitis 90. Similar studies were carried out to dissect the local T-cell responses in the CNS of patients with MS. Early studies indicated overexpression of particular T-cell receptor (TCR) genes in MS brain lesions 91. By analysing single cells from lesions or CSF of patients with MS, two groups 92,93 recently reported extensive clonal expansion of T cells. In contrast to the commonly held view, clonal expansion was seen predominately in CD8 + and, to a much lesser extent, in CD4 + T-cell populations 92,93. In the lesions of one patient, up to 30% of all T cells were derived from a single CD8 + T cell, as shown by the analysis of the molecular structure of their rearranged TCR 92. T cells expanded from lesions or CSF were not found at high frequency in peripheral blood, indicating that a NATURE REVIEWS NEUROSCIENCE VOLUME 3 APRIL

6 REVIEWS B cell Lymphoid tissue B cells CNS Plasma cell Neuron Antibodies T helper cell Cytokines Antigens Oligodendrocyte Dendritic cell Microglia CD8+ T cells CD4+ T cells T cells Figure 3 Immune responses in multiple sclerosis. Hypothetical view of immune responses in acute multiple sclerosis lesions. Independent of the causative event, two steps are required to induce an immune response in the central nervous system (CNS): a pro-inflammatory milieu in the CNS, leading to upregulation of major histocompatibility complex (MHC) molecules, co-stimulatory receptors and inflammatory cytokines and an antigen-driven acquired immune response. T- and B-cell responses are primed in the peripheral lymphoid tissue by antigens that are released from the CNS or by cross-reactive foreign antigens. Dendritic cells that present neural antigens are strong stimulators of T-cell responses. After clonal expansion, T and B cells infiltrate the CNS. Clonally expanded B cells re-encounter their specific antigen, mature to plasma cells and release large amounts of immunoglobulin-γ (IgG) antibodies. These antibodies bind soluble or membrane-bound antigen on expressing cells. Clonally expanded CD8+ T cells also invade the brain and could encounter their specific peptide ligand, presented by glial or neuronal cells on MHC class I molecules. The recognition of specific MHC peptide complexes on these cells prompts direct damage to expressing cells. CD4+ T cells migrate into the CNS and encounter antigens that are presented by microglial cells on MHC class II molecules. Reactivation of these cells leads to heightened production of inflammatory cytokines. These cytokines attract other immune cells, such as macrophages, which contribute to inflammation through the release of injurious immune mediators and direct phagocytic attack on the myelin sheath. specific migration of these cells might have occurred into the CNS compartment. Serial studies showed the persistence of CD8+ T cells in the CSF over a period of months. The clonal accumulation of CD8+ T cells in the CNS, together with the findings in B cells, strongly argue for an antigen-specific and highly focused immune response in the CNS of patients with MS (FIG. 3). COMPLEMENT CASCADE The complement system is a set of plasma proteins that attack extracellular pathogens. The pathogen becomes coated with complement proteins that facilitate pathogen removal by phagocytes. Complement components are also involved in inflammation and tissue destruction. 296 APRIL 2002 VOLUME 3 Learning from lesion pathology. Important new implications for understanding MS have come from histopathological studies. These studies have shown a high degree of heterogeneity in lesion pathology94. Pioneering work by Lassmann and co-workers pointed out that the composition of acute demyelinating lesions varies greatly among patients with MS95. The authors defined four types of lesion that differed in the extent of cellular infiltrates, antibody deposition, de- and remyelination, the magnitude of COMPLEMENT activation and the degree of oligodendrocyte loss. Different acute demyelinating lesions of any given patient showed a similar composition, indicating that lesion heterogeneity does not reflect different stages, but rather distinct pathogenetic mechanisms. This heterogeneity is at least partly reflected by the cellular composition of the CSF of patients with MS96. Histopathological studies have also addressed the process of de- and remyelination in MS lesions. In acute lesions, inflammatory changes are associated with demyelination and oligodendrocyte loss. Although most of the damage is a direct result of the coordinated immune attack that is mediated through specific cellular and humoral mechanisms, nonspecific immune mediators, neurotransmitters and toxic substances (such as nitric oxide) might significantly facilitate this process97 (FIG. 4). During the resolution of lesions, oligodendrocyte progenitor cells enter the area of demyelination, expand and develop into myelinating oligodendrocytes98,99. These cells myelinate axons and

7 Damage Protection/repair Immune cells/microglia, astroglia a CD8 + T cell b Glutamate, neurotoxins c Antibody complement d Cytokines, chemokines e Neurotrophins, growth factors f Oligodendrocyte progenitors Neuronal stem cells FAS Apoptosis Cell lysis NMDA receptor Structural alterations tcc Loss of function Repair Remodelling Replacement Neurons/oligodendrocytes Figure 4 Molecular interactions in central nervous system inflammation and repair. The mechanisms of direct neuron and oligodendrocyte damage and repair are shown. They include: a direct antigen-specific attack of CD8 + T cells, with the discharge of cytotoxic granules and the ligation of the FAS molecule; b release by glial cells of excitatory amino acids and neurotoxins, which bind to glutamate receptors or directly target the cells; c binding of a specific antibody, leading to complement activation and formation of the membrane-attacking terminal complement complex (tcc) and also, possibly, promoting remyelination; d release of cytokines, matrix metalloproteinases and metabolites from macrophages, microglia, T cells and astroglia that are involved in inflammation, neurodegeneration and neuroprotection; e release by glial cells and CD4 + T cells of neurotrophins, which are involved in neuroprotection and regeneration; and f migration of oligodendrocyte progenitor cells and neuronal stem cells to the lesion, which replace damaged oligodendrocytes and neurons. NMDA, N-methyl-D-aspartate. ANTIGEN-PRESENTING CELLS Specialized cells that present specific antigens to T cells. Macrophages and dendritic cells are the main antigen-presenting cells, but in the CNS the microglia have this role. DENDRITIC CELLS Also known as interdigitating reticular cells because of their branched morphology, dendritic cells are the most potent stimulators of T-cell responses. MAJOR HISTOCOMPATIBILITY COMPLEX (MHC). There are two classes of MHC molecules. MHC class I molecules are present at the surface of most cells and present proteins generated in the cytosol to T lymphocytes. MHC class II molecules are expressed only at the surface of activated antigenpresenting cells, and they present peptides degraded in cellular vesicles to T cells. FAS A transmembrane protein that mediates apoptosis and might be involved in the negative selection of autoreactive T cells in the thymus. repair the local damage. The ability of the cells to remyelinate seems to be most efficient in acute lesions but disappears over time. In chronic lesions, oligodendrocytes are still encountered, but little remyelination is observed. These findings indicate that active oligodendrocytes might still be present even in chronic lesions, but do not succeed in remyelinating axons 100,101. The pathological studies have also refocused our attention on the neurodegenerative aspects of MS pathogenesis. In particular, axonal degeneration in the chronic progressive phase of disease is a crucial factor responsible for the irreversible long-term disability of patients with MS 4. Elegant histopathological studies of MS lesions showing axonal transection, formation of axon spheroids and axonal sprouting highlighted the extent of neuronal damage that occurs even in the early stages of MS 102.The axonal damage, however, can also be observed in the absence of inflammation and demyelination, questioning whether immune-independent mechanisms are also involved in the neurodegenerative damage in MS 103,104. In summary, these studies have led us to revise our view of the immunological mechanisms in MS, and have pointed out that non-immune-mediated processes might also be substantially involved in disease pathogenesis. The emerging synthesis Complex but focused. Recent findings in MS have shown that considerable heterogeneity exists in terms of clinical and pathological changes. This heterogeneity is also reflected in the polygenic nature of the disease. The level of complexity might be increased further by various exogenous factors that contribute to disease onset and course. In contrast to the high degree of phenotypic complexity, the nature of the acquired immune response in the CNS of patients with MS seems to be less complex (FIG. 3). A large proportion of locally accumulated T cells originates from single cells. Similarly, B cells are clonotypically expanded in the brain of patients with MS. Their B-cell receptors (BCRs) are antigen maturated as a probable result of repeated exposure to the same antigen. Although the primary event that paves the way to the manifestation of MS is still unknown, it is almost certain that the initial and ongoing activation of immune cells takes place in the lymphoid tissue (FIG. 3). Proteins from cross-reactive antigens (autoimmune hypothesis), a brain-resident pathogen (infection hypothesis) or CNS proteins after primary degeneration (degeneration hypothesis) are released into the periphery (TABLE 1). They reach lymph nodes and spleen centres of the immune system to set off an acquired immune response. T-cell antigens are probably processed and presented by ANTIGEN-PRESENTING CELLS, such as DENDRITIC CELLS (DCs). DCs can load endocytotic and cytosolic proteins onto MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) class I and II molecules, allowing the priming of CD4 + and CD8 + T cells. Soluble proteins that are captured by B cells will promote efficient priming of T cells and of B cells. Specific recognition of the antigens by single T and B cells will result in clonal expansion and acquisition of effector functions. Primed cells will migrate through the body and accumulate in the CNS at sites where they encounter their target antigens (FIG. 3). These will be displayed on neurons, glial cells and, in particular, activated microglial cells, which orchestrate the development of a NATURE REVIEWS NEUROSCIENCE VOLUME 3 APRIL

8 Table 1 Current hypotheses for the pathogenesis of multiple sclerosis Hypothesis Pros Cons Autoimmune Focused on myelinated areas Immune responses to myelin of the brain antigens have not been Findings in EAE associated with disease HLA association onset or disease progression Response to immunosuppression and immune modulation Infectious HLA association No pathogen yet identified Response to interferons Similar oligoclonal IgG banding pattern in infectious diseases Findings in infectious CNS disease models Degenerative Early neuronal loss HLA association Little inflammation seen in Extent and chronicity of progressive phase inflammation CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; HLA, human leukocyte antigen; IgG, immunoglobulin-γ. EXPRESSION LIBRARY A gene library that allows the cloning of genes on the basis of the transfection of a large number of cells with cdna in an expression vector and subsequent screening for a functional property. PHAGE DISPLAY LIBRARY A gene library that encodes fusion proteins of a foreign sequence and a coat protein of a phage. When cloned, the phage is said to display the foreign protein. COMBINATORIAL PEPTIDE LIBRARY A collection of large numbers of peptides that is a very useful in the fields of drug discovery and structural biology. MICROARRAY A device that is used to interrogate complex nucleic acid samples by hybridization. It makes i t possible to count the number of different RNA or cdna molecules that are present in a sample as a preparative stage for their subsequent characterization. pro-inflammatory milieu. On reactivation by the antigen, they initiate their effector functions, release antibodies and cytokines, and recruit to the lesion other inflammatory cells, such as macrophages. Once the combined attack of the acquired and innate immune responses has cleared the target antigen from the site of the lesion, the cells will either undergo activation-induced cell death or redistribute to other tissues. In parallel, repair mechanisms are initiated that result in remyelination. This cascade of events is initiated during the course of MS at different locations in the CNS. As the oligoclonal IgG banding patterns persist in the CSF of patients with MS and show little clonotypic variation over time, it is tempting to speculate that at least the focus of the humoral response does not change during the course of disease. The players and their targets. What are the targets of the focused immune response in patients with MS? As inflammation is not observed in other tissues, CNS antigens or antigens from CNS-resident infectious agents are good candidates. The dominance and persistence of an intrathecal IgG1 and IgG3 antibody response, together with the locally expanded T cells, are consistent with an ongoing immune response against protein antigens. However, the expression pattern of MHC molecules in the CNS tissue has hampered our development of models of lesion pathogenesis. MHC class II molecules are expressed only on activated microglia and infiltrating immune cells (for example, macrophages and B cells). By contrast, all CNS cells can express MHC class I molecules, which are recognized by CD8 + T cells 105. In vitro studies have shown that both oligodendrocytes and neurons can efficiently present antigens to cytotoxic CD8 + T cells, leading to activation of T cells and damage to the presenting cell 106,107. Furthermore, CD8 + T cells can attack neurites, leading to spheroid formation that is similar to that seen in MS lesions 108. Given the MHC expression profile and the clonal expansion of CD8 + T cells in the CNS, these cells are are probably crucial in the early development of lesions. This might provide a simple explanation not only for the loss of oligodendrocytes and the extent of demyelination, but also for the neuronal damage 104.The recognition characteristics of CD8 + T cells indicate that either endogenously expressed proteins or, indeed, exogenous antigens (for example, after infection of the target cells) are targeted. Although a role of the CD8 + response is strongly supported by these findings, no study has been carried out so far to investigate the peptide specificity of locally expanded T cells. The B-cell response in the CNS of patients with MS similar to the CD8 + T-cell response fulfils all the criteria for a central involvement in disease pathogenesis. IgG1 antibody binding to cell surfaces activates the complement cascade, thereby directly damaging the expressing cell. Binding of antibodies is solely dependent on the antigenic protein itself, so all cells that express the specific proteins are targets. Findings in EAE and infectious disease models indicate that antibodies are most important in the chronic phase of disease. This could also apply to MS, although direct evidence is lacking. Several studies have investigated the focus of the local humoral immune response in MS. Using EXPRESSION or PHAGE DISPLAY LIBRARIES, antigen mimics were identified, although their pathogenic role in MS has not yet been established 35,109,110. The role of CD4 + T cells although suggested as key players by studies of EAE is much less clear. The lack of substantial clonal expansion does not necessarily mean that these cells are not involved in the pathogenesis of MS. It is well established that the capacity of CD8 + T cells to expand clonally is much higher than for their CD4 + counterpart 111. CD4 + T-cell responses in the lesions could still target specific antigens, but in a much broader manner, therefore escaping clonotype analysis. Alternatively, the cells could be nonspecifically recruited to sites of inflammation, or even be involved in regulation or priming of CD8 + T cells and B cells. Finally, recent work indicates that immune cells might contribute to myelin regeneration and, possibly, neuroprotection in CNS lesions 112. There is good evidence for an involvement of growth factors, such as platelet-derived growth factor, insulin-like growth factor, brain-derived neurotrophic factor and neurotrophin 3, in myelin regeneration. Immune cells release certain neurotrophins, leading to a local accumulation in the CNS tissue. Studies in infectious and autoimmune diseases also indicate that antibodies can contribute to regeneration by promoting remyelination 113. However, additional studies are necessary to establish the contribution of immune cells to CNS tissue repair in human diseases. Beyond immune pathogenesis. Although the coordinated immune attack in the CNS is clearly the most important factor in the early relapsing remitting phase of MS, other non-immune-mediated mechanisms could dominate the chronic progressive phase. This assumption is supported by the histopathological findings and by the failure of most immunosuppressive and immunomodulatory treatments in patients in which the disease has entered the progressive phase. The prominent loss of axons and impaired myelin repair that are seen in this phase resembles what is observed in some neurodegenerative disorders. Although little is known about this progressive phase, it is likely that general disease mechanisms observed in neurodegeneration are applicable. 298 APRIL 2002 VOLUME 3