The Immunology of Multiple Sclerosis
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The Immunology of Multiple Sclerosis

Amit Bar-Or, M.D., F.R.C.P.C.Semin Neurol.  2008;28(1):29-45.  ©2008 Thieme Medical Publishers

Posted

Abstract and Introduction

Abstract

Recent years have witnessed a remarkable growth in literature related to the biology and treatment of multiple sclerosis (MS). The focus of this article is on aspects of the human immune response that have been implicated in the MS disease process and, as a corollary, represent rational targets for the development of safe and effective therapies. Much of the thinking about immune pathophysiology in patients with MS has been shaped by studies in animal models of central nervous system (CNS) inflammation. Translation to the human disease has continued to pose challenges. A simplified model of MS immune pathophysiology is presented to illustrate the basic principles by which peripheral immune activation, as well as compartmentalized immune responses within the CNS, is likely to impact the disease process and to identify the putative sites of action of current and future MS treatments.

Introduction

Clinicians are faced with an ever-growing body of animal and human studies that may, at times, appear to be in conflict. It can be challenging to distinguish between insights into disease mechanisms and/or therapeutic strategies that are plausible based on animal or in vitro studies, and those that emerge from direct observations from patients. Animal studies continue to provide important insights into fundamental mechanisms of immune-neural interaction, and the use of such models, including experimental autoimmune encephalomyelitis (EAE), has become an expected part of the drug development process. Nonetheless, agents applied to multiple sclerosis (MS) clinical trials based on promising animal model data have at times proved ineffective or even harmful. Some potentially useful MS therapies may never be pursued because of a lack of measured benefit in preclinical animal model studies. The translation of findings from animal modeling to understanding mechanisms of human disease and putative therapeutic targets has remained problematic. More effort is being made to study disease biology and treatment responses directly in patients. Even the latter are not foolproof, however, because not all biological responses induced by a therapeutic agent, even if reliably measurable, are necessarily relevant to the disease itself. A further challenge is inherent in our growing appreciation of the biological heterogeneity that exists as part of the MS spectrum, and the notion that the predominant pathophysiological process may differ across patients, or even within a given patient at different times, during their illness.

A more complete understanding of MS pathophysiology requires consideration of the neurobiological aspects of disease and how complex immune-central nervous system (CNS) interactions contribute to both tissue injury and repair—themes that are beyond the scope of this article. Here, we consider some of the more prevalent assumptions regarding immune contributions to the distinct phases of MS. The focus is studies of human immunology and studies in patients with MS, occasionally drawing from animal model findings to underscore principles of immune-CNS interactions that are relevant to the human condition. In this context, we consider how well MS meets the defining requirements of an autoimmune disease.

Initiation of Multiple Sclerosis: How Firm is the Autoimmune Hypothesis?

The normal adaptive immune system (comprising T and B lymphocytes) has the capacity to recognize and develop efficient memory responses to specific antigenic targets. This system must efficiently and reliably discriminate between foreign antigens and components of self, or self-antigens. Within the normal T-cell and B-cell repertoires are cells that can recognize and react to self-antigens. These autoreactive cells are normally kept in check as part of the state of tolerance to self-antigens, and do not mount vigorous immune responses that could be harmful to the host. Why cells that can potentially respond to “self” exist in all healthy individuals remains unknown; it is likely that this normal state of autoimmunity serves important homeostatic functions. Dysregulation of the mechanisms that maintain tolerance to a person's own tissue antigens is considered integral to the emergence and propagation of autoimmune diseases.^[1] Why some individuals develop autoimmune disease—and why there is selectivity toward particular target organs—is not known. The prevailing thought (Fig. 1) is that human autoimmune diseases (AIDs), including MS, manifest when certain environmental exposures (such as one or more common viruses) trigger immune dysregulation in genetically susceptible hosts (likely reflecting involvement of multiple risk-conferring and protective genes, each contributing relatively little to the overall susceptibility). The innate immune system, represented by a series of immune response elements capable of sensing and rapidly responding to perturbations in their environment, may be differentially expressed and triggered in different tissues. Innate responses are likely to have important influences on adaptive responses, at both the earliest time points in the disease process, as well as later in disease propagation and within the target organ.

Figure 1.

Immune pathogenesis of multiple sclerosis. An interplay exists between different environmental exposures and multiple genes that influence susceptibility at the levels of both the immune system and the target organ. These complex gene-environment interactions, occurring in early life, ultimately converge and lead to insufficient function of immune regulatory mechanisms, with subsequent loss of tolerance to self-antigens and the development and/or propagation of target-directed autoimmune disease (AID), such as multiple sclerosis (MS).

The classical description of MS pathology involving perivascular immune cell infiltration at sites of CNS injury has long since supported an autoimmune disease etiology for MS. However, because one of the main roles of the normal immune system is to respond to all forms of tissue injury, the mere presence of immune mediators within MS lesions does not necessarily mean that the immune system was responsible for initiating the disease process. For a disease to have a frank autoimmune etiology, several criteria must be met. These include: (1) the presence of immune mediators within sites of pathology in patients who have the illness, (2) the absence of such mediators in persons or tissues without the illness, (3) the ability of the putative immune mediators to adoptively transfer the disease, and (4) the demonstration that removal of these mediators has therapeutic effects. Although MS fulfills the first requirement quite well, relatively few features of MS lesions have been established as specific to the MS pathologic state. Moreover, unlike passive-transfer experiments in myasthenia gravis, in which patient serum can induce disease in recipient animals, similar findings have not been replicated when transferring MS serum or cells into animals. Normal animals do not appear to acquire EAE spontaneously (i.e., without immunization with CNS antigen). Transgenic mice engineered to have all their T cells respond to a specific myelin antigen can develop CNS inflammatory demyelination spontaneously,^[2] and this occurs more efficiently when the animals' B cells are also modified to recognize the same antigen.^[3] The rather unique circumstance required to observe spontaneous EAE favors a complex, multifactorial etiology for the development of MS in the comparatively nonsterile and out bred human population.

The current ability of immune-directed therapies to favorably impact MS patients lends support for a pathogenic role of immune responses. However, the observation that even aggressive immune suppression or immune ablation does not appear to completely abrogate MS progression invokes pathogenic mechanism(s) that may be relatively independent of peripherally mediated autoimmune responses. These may reflect an important CNS-compartmentalized component of MS pathophysiology (local inflammation and/or degeneration) that can be relatively resilient to peripheral immune intervention and may be particularly dominant later in the disease process (Fig. 2). It has not been firmly established whether the very initial abnormalities in MS are inherent to the immune system or to the target organ.

Figure 2.

Peripherally mediated and central nervous system (CNS)-compartmentalized immune responses in multiple sclerosis (MS)—relationship to MS disease course. The common clinical course in MS (top panel) involves relapses and remissions in the earlier stages of illness, and subsequently progressive worsening occurs without clinically evident relapses and remissions. Imaging parameters applied during the clinical course (bottom panel) can track the accumulation of T2 hyperintense lesion volume (green) as a marker of disease burden, as well as identify time points (arrows) when gadolinium-enhancing lesions are present, reflecting local breach of the blood-brain barrier, a measure of active inflammation. However, an imaging:clinical dissociation exists because later in the disease course progression of disability occurs despite decreased accumulation of further T2 lesion burden and the appearance of fewer new gadolinium-enhancing lesions. This points to the presence of at least two distinct biological mechanisms underlying CNS tissue injury in MS (middle panel). Earlier in disease course, peripherally mediated waves of inflammation prevail, corresponding to some clinically manifest relapses and remissions. Later in disease, ongoing injury predominantly relates to CNS-compartmentalized processes that likely include local (CNS-compartmentalized) inflammatory responses and CNS degeneration.

Thus, although not straightforward indicators that faulty immune regulation is the primary initiating event or a secondary response in MS, the presence of inflammatory mediators at sites of CNS pathology and the observations that immune manipulation in patients can both aggravate or diminish disease activity argue that immune responses do play an important role in MS pathophysiology and are not merely epiphenomena of CNS tissue injury. How such immune mediators may contribute to MS relapses, remissions, and disease progression is considered in the following sections.

Peripheral Immune Activation: The Presumed Trigger of Multiple Sclerosis Relapses

In MS, the immune system is separated from the CNS target by the blood-brain barrier (BBB) (Fig. 3), representing the typical three-compartment model of immune-mediated disease. A key event in the triggering of an MS relapse is thought to be the activation outside the target organ (Step 1) of T cells that can recognize antigens in the CNS. Through a sequence of subsequent events, these activated immune cells then undergo more efficient adhesion to the endothelial barrier (Step 2), attraction (Step 3), and active invasion (Step 4) into the target organ where they may become reactivated (Step 5) and subsequently participate in the disease process (Step 6). The differentiation state and functional response profile of the invading cells (defined in part based on the profile of cytokines they produce, their cytotoxic machinery, etc.) determines whether they contribute to injury or, possibly, counter the injury process. Immune responses may also be propagated within the CNS itself by previously infiltrating peripheral cells that then become chronically activated and/or by chronically activated CNS resident cells such as microglia. Such CNS-compartmentalized inflammation may become relatively independent of ongoing peripheral events. This simplified model is used as a framework to discuss how abnormalities at each of these steps may contribute to the MS process and how targeting one or more of these steps may be of therapeutic benefit.

Figure 3.

Immune responses contributing to multiple sclerosis (MS) propagation. Immune cells activated in the periphery, including T cells, myeloid cells, and B cells, (Step 1) upregulate surface molecules that enable them to more efficiently adhere (Step 2) to the endothelial cells of the blood-brain barrier (BBB) and respond to local chemokine gradients (Step 3). Active secretion of matrix proteases (Step 4) facilitates immune cell invasion into the CNS where they may become reactivated (Step 5) and impact on the biology of central nervous system (CNS) elements (Step 6). These steps also represent rational therapeutic targets for MS therapies. Immune responses may also be propagated within the CNS by previously infiltrating peripheral cells that then become chronically activated and/or by chronically activated CNS-resident cells such as microglia. This CNS-compartmentalized inflammation may take place independent of ongoing peripheral events. Therapeutic targeting requires treatments that result in modulating inflammation within the CNS.

The concept that triggering an autoreactive immune response in the periphery could translate into a CNS inflammatory attack in humans was first considered following Pasteur's introduction of a neural tissue- containing vaccine as a therapy for rabies that resulted in episodes of acute disseminated encephalomyelitis (ADEM) after vaccination.^[4] This led to the development of the commonly used EAE model of MS in which peripheral immunization with myelin components (including myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], and proteolipid protein [PLP]) can induce inflammatory demyelination of the CNS.^[5-7] EAE can also be induced in naïve animals by means of “adoptive transfer” of activated (but not resting) myelin reactive CD4* T cells from animals with established disease.^[8] The particular phenotype of EAE, including anatomic predilection (spinal cord or optic nerve) and clinical patterns (relapsing and chronic), depends on the genetic background of the animal strain, the specific antigen selected, and the immunization regimen.^[7] The experience with EAE and other animal models established that peripheral activation of CNS autoreactive T cells can result in a CNS inflammatory disease. Based on these animal studies, initiation of the MS process (or at least an MS relapse) is thought to involve activation of auto-aggressive, CNS-directed T cells in the periphery.

To become activated, T cells recognize antigens that bind with sufficient affinity and avidity to their T-cell receptor (TCR). The appropriate antigen fragment must be “presented” to the TCR within the pocket of the major histocompatibility complex (MHC) molecule, expressed on the surface of antigen-presenting cells (APCs) (Fig. 3, Step 1). Effective APCs include dendritic cells, monocyte/macrophage B cells, and CNS microglial cells. Appropriate binding of the antigen-MHC to the TCR delivers an activating signal to the T cell (termed “signal 1”). However, this signal 1 alone is usually not sufficient for full T-cell activation. Costimulatory signals (collectively referred to as “signal 2”) are also required and may be delivered by APCs that express costimulatory molecules on their surface. Typically, a T cell stimulated only via signal 1 (in absence of costimulation) may become anergic, resulting in an inability to subsequently respond to the antigen. In contrast, a T cell receiving both signal 1 and signal 2 may become fully activated, resulting in proliferation, secretion of cytokines and other effector molecules, acquiring cytotoxic capacity, and so forth. These functions may, in turn, impact the target organ directly or through modulation of other immune cell functions. When APCs are exposed to factors such as infectious particles, for example through toll-like receptors (TLRs), various other inflammatory mediators, or injured tissue, they become activated and upregulate their surface expression of costimulatory molecules.^[9]

Several families of costimulatory molecules and their receptors are thought to play key roles in regulating immune responses in both health and disease. One important costimulatory pathway is represented by the CD28 or CTLA-4 molecules on T cells, being engaged by the CD80 (B7.1) or CD86 (B7.2) costimulatory molecules expressed on APCs. Engagement of CD28 by B7 molecules stimulates the T cell, while the same B7 molecules engaging CTLA-4 mediates an inhibitory signal. Other costimulatory molecules, with either proinflammatory or antiinflammatory effects, have been described and implicated in MS.^[10-19] Whether a T cell becomes productively activated or suppressed depends on the integration of signals mediated by the TCR and the profile of costimulatory molecules available in the particular microenvironment.^[20-22] Targeting costimulatory pathways provides an attractive therapeutic approach.^[23-25]

The normal immune system has the capacity to generate very different response profiles, depending on the local needs. For example, the same naïve T cells can differentiate into functionally distinct subsets defined based on their distinct effector cytokine responses. In addition to integrating signals mediated by the TCR and the costimulatory profile, the presence of distinct cytokines during T-cell activation provides important contextual cues that can define the subsequent T-cell response. Cytokines released by APCs, including interleukin (IL)-12 and IL-23, can preferentially generate T-helper type 1 cells (Th₁) that subsequently secrete IFNγ.^[26] These T cells are effective as part of the normal antiviral response.^[27-29] T cells that initially become activated in the presence of IL-4 and in the absence of IL-12 or IL-23 subsequently produce cytokines, including IL-4, IL-5, and IL-13. These are defined as “Th₂ cells.” As part of the normal immune response, Th₂ cells and their effector cytokines are important in containing parasitic infections. Additional types of effector T cells, such as ThIL-17 cells, are being identified based on distinct effector profiles and molecular mechanisms that generate them.^[30-32] Furthermore, several CD4 and CD8 regulatory T-cell (Treg) subsets, including thymic-generated natural Tregs (nTregs), inflammation-induced Tregs (iTregs), and human leukocyte antigen-E (HLA-E) expressing CD8 T-cell subsets, are viewed as critical regulators of normal immune responses.^[33-41]

As a corollary to the normal functions of the cell subsets noted in the previous paragraph, a dysregulation in the body's normal balance between effector (Th₁,Th₂, ThIL-17) responses, or deficiencies in regulatory functions, can result in disease states. The roles of natural killer (NK) cells, NK T cells, and γδ T cells in MS are also under investigation.^[42,43] Abnormal activation or regulation of Th₁, and more recently ThIL-17 responses have been implicated as pathogenic in several target directed autoimmune diseases, including MS and its EAE animal model.^[31,44,45] In EAE, proinflammatory CNS-autoreactive T cells can adoptively induce disease, whereas transfer of Th₂ cells reactive to the same CNS antigens, or TGFβ-producing Th₃ cells, does not usually induce disease. Moreover, the Th₂ or Th₃ cells may protect the animal from getting the disease. By extension, it has been suggested that in MS, CNS-directed Th₁ or ThIL-17 responses may be proinflammatory and cause damage, whereas Th₂ or Th₃ responses may be antiinflammatory and possibly protective.^[24,46-49] Abnormalities in regulatory cell subsets have been implicated in several autoimmune diseases including MS, such as deficiencies in CD25^hiFoxP3* CD4 T cells (nTregs),^[36,50-52] IL-10-producing T regulatory type 1 (Tr1) cells,^[37,53] and HLA-E-restricted CD8 suppressors.^[38,39,54]

Several established MS therapies, including β-interferons and glatiramer acetate, are thought to mediate part of their benefit by shifting immune responses in patients from Th₁ to Th₂ responses (Th₂-immune deviation), and/or by promoting function of regulatory immune cells. Similar strategies continue to be pursued.^[24,25,55] It should be kept in mind that unchecked Th₂ responses may contribute to asthma or atopic conditions, and under certain situations even to exacerbation of CNS inflammation.^[56,57] With regards to enhancing regulatory T-cell functions, it is of interest whether such a strategy will impact already differentiated autoaggressive cells.^[58,59] Whether CNS antigen-specific regulation can be selectively enhanced in MS is relevant because it also remains to be seen whether over-aggressive induction of broad regulatory immune functions may result in insufficient antiviral or antitumor host responses.

Target-specific regulation of the immune response is predicated on defining the specific CNS antigens involved in the MS disease process. To date, no single CNS antigen has been established as the major MS target. It is likely that the predominant targets differ across patients and, indeed, change within the same individual over time, for example from one relapse to another (epitope spread). Multiple CD4 T-cell targets have been implicated in MS including myelin antigens such as MBP, MOG, and PLP.^[60-65] As noted previously, the mere presence of CNS-reactive T cells is not sufficient to cause disease.^[66-68] Studies comparing properties of CNS-reactive T-cell responses between MS patients and healthy controls have generally revealed considerable overlap in frequencies and functional profiles. On the average, it appears that myelin-reactive T cells from patients can be found circulating in a higher state of activation,^[69,70] require less costimulation,^[71,72] tend to be of higher avidity,^[70] and preferentially belong to the memory T-cell pool.^[73-75] There is also some evidence that MBP-reactive T cells isolated from MS patients are more likely to produce proinflammatory (Th₁), rather than antiinflammatory mediators.^[76,77] A limitation of such studies in adults with MS relates to the long duration between sampling for immune studies and biological disease onset. Identification of abnormal CNS-directed immune responses in adult patients may, at least in part, be influenced by epiphenomenon of chronic immune dysregulation. Studies in pediatric-onset MS capture a unique and early window of the autoimmune disease pathophysiology. A recent comparison between childhood-onset CNS inflammatory disease and type-1 diabetes (T1D) reveals abnormally enhanced T-cell responses to selected target and environmental antigens. Results suggest that abnormal T-cell responses are not always restricted to disease target antigens, pointing to a more general state of dysregulation. However, a marked degree of specificity is also seen in the study, with abnormal responses to particular mil-antigen peptides in children with one autoimmune disease but not the other.^[78]

Much of the focus over the years has tended to center on the role of CNS autoreactive CD4 Th cells, in part because of technical difficulties in maintaining CD8 cytotoxic T cells in culture. Pathologically, however, a prominence of CD8 over CD4 T cells is reported within MS lesions.^[79,80] Newer cellular and molecular approaches are being developed.^[81,82] It can now be demonstrated that specific CD8 (rather than CD4) T cells are expanded and persist long term within the CNS compartment of patients.^[79,83] Higher frequencies of CNS-specific autoreactive CD8 T cells have also been reported in the circulation of MS patients compared with controls.^[84-86] It is noteworthy that CNS-reactive T-cell responses may not always be detrimental.^[87-91] Indeed, immune responses in MS are not all bad or all good—adaptive response to injury and promotion of repair represent major roles of the normal immune response. The challenge is to appropriately develop and apply immune-modulating therapies that minimize CNS-directed injury and maximize beneficial responses.

The most direct evidence in patients with MS that peripherally activated CNS-autoreactive T cells participate in the disease process is available through careful immune studies of patients treated with an altered peptide ligand (APL) of MBP. The use of APLs represents a form of antigen-specific therapy in which a peptide antigen is altered by replacing one or more of its amino acids. T cells recognizing a particular antigen may also recognize an APL of that antigen, although the T-cell response to the APL may be considerably different than to the native antigen. In EAE, an APL of MBP led to a Th₂ shift in MBP reactivity associated with improved clinical outcomes.^[92,93] Subcutaneous injection of the same APL in a Phase II placebo-controlled trial of MS was found to induce the anticipated Th₂ responses to MBP, and suggested possible benefit on magnetic resonance imaging (MRI) outcomes.^[94] However, in another trial, a higher dose of the same APL was associated with significant increases in MRI activity in several patients, with or without clinical correlates.^[95] In these patients, elegant immune monitoring identified a strong induction of both APL-reactive T-cell responses and enhanced MBP-directed responses, both in the periphery and in the cerebrospinal fluid (CSF) of patients. These MBP-reactive T cells were notable for their production of high levels of the Th₁ cytokine, IFNγ. Although further insights are required to strike the optimal balance of efficacy and safety with APL therapy, these studies provide the most direct evidence that myelin antigens such as MBP are likely targets in MS, and that peripheral modulation of CNS-directed immune responses can translate into modulation of immune responses within the CNS. Indeed, newer antigen-specific approaches are being pursued, including DNA-vaccine technology, that enable presentation of myelin antigens to the immune system of patients in an immune downregulatory context.^[96,97] The biological proof-of-principle for an MBP-based DNA vaccine in MS has recently been generated in a Phase I/II study.^[98]

Where and how do CNS-reactive T cells become activated in the periphery of patients with MS? Such activation may occur when circulating CNS autoreactive cells encounter fragments of CNS self-antigens that are released, or carried, by APC, for example into the cervical lymph nodes that drain the brain.^[99-101] “Molecular mimicry” represents another mechanism by which CNS autoreactive T cells can become activated when they encounter fragments of foreign (nonself) antigens that are sufficiently similar to their cognate CNS self-antigen. Some human MBP-reactive T cells have been shown to respond to antigenic fragments derived from common viruses (including herpes simplex, Epstein-Barr, and influenza viruses) presented by the same MHC molecules.^[102] Extending the concept of molecular mimicry is the phenomenon of “structural equivalence,” wherein two different MHC molecules (encoded by the individual's two MHC alleles) bind two distinct antigens (self and foreign), creating two antigen-MHCs that may be sufficiently similar in structure that they are both recognized by the same CNS-reactive T cell.^[103] The following sections focus on the molecular mechanisms that regulate the infiltration of peripherally activated immune cells across the BBB and into the CNS.

Immune Cell Interaction with the Blood-Brain Barrier: Adhesion, Attraction, and Invasion

On activation, immune cells upregulate the expression of a series of molecules that are tightly regulated to facilitate cellular migration across endothelial barriers and into the target organ (Fig. 3).^[104,105] Upregulated selectins and integrins on the activated immune cells interact with ligands expressed on the endothelial cells of the BBB, resulting in immune cell “rolling” and “tethering/arresting,” respectively (Fig. 3, Step 2). Chemokines are secreted by endothelial cells, by injured tissue, or by the activated immune cells themselves, and can promote integrin activation and further contribute to cell arrest.^[106] Chemokines also attract cells expressing the corresponding chemokine receptors toward the source of the chemical gradient (Step 3). Production of tissue lytic enzymes (Step 4), such as the matrix metalloproteinases (MMPs), results in breakdown of the basement membrane and facilitates infiltration of immune cells into the CNS parenchyma.^[107] Selected studies implicate these molecular steps in disease pathogenesis and potential therapeutic targets.

Implicating Adhesion Molecules in Multiple Sclerosis. In MS lesions, endothelial cells have been shown to express abnormally elevated levels of adhesion molecules, including intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. These elevated levels correlate with the extent of immune cell infiltration.^{[105,108-110]} Their ligands for these adhesion molecules (lymphocyte function-associated antigen [LFA]-1 and very late antigen [VLA]-4, respectively) have been identified on the perivascular inflammatory cells within MS lesions.^[111] These observations and proof-of-principle studies in animal models provided the basis for development of antiadhesion molecule therapies in MS, including the anti-VLA-4 antibody, natalizumab. The clinical trial program of natalizumab confirmed impressive efficacy results with this strategy; however, enthusiasm has been tempered by the emergence of cases of progressive multifocal leukoencephalopathy (PML).^[112] Observations in a small number of immune monitoring studies in patients have demonstrated that: (1) the effects of natalizumab infusion do not appear to fully saturate the VLA-4 expressed on circulating immune cells, and that the degree of saturation varies across patients;^[113] (2) trafficking of CD4 T cells into the CNS is more significantly impacted than CD8 T cells, resulting in a reversal of the CD4/CD8 ratio in the CSF;^[114,115] and (3) natalizumab infusion alters not only the trafficking capacity of immune cells but also their activation threshold.^[113] In this regard, it is interesting to note that the adhesion molecules VCAM-1 and LFA-1 are also expressed on glial cells within MS lesions,^[116] possibly pointing to additional roles for these molecules in antigen presentation and T-cell costimulation.^{[111,117,118]}

Selective Expression of Chemokines and Chemokine Receptors in Multiple Sclerosis. A complex network of chemokines and chemokine receptors regulates movement of immune cells within and between tissues. Such chemokine-chemokine receptor interactions are also involved in the trafficking of immune cells into the CNS as part of normal immune surveillance, and also in context of the CNS inflammation of patients with MS (Fig. 3, Step 3).^[119,120] Studies in both EAE and MS have implicated certain chemokines in the selective CNS recruitment of T cells and monocytes/dendritic cells.^[121] In active MS lesions, RANTES (regulated on activation, normal T-cell expressed and secreted) (CCL5), which is chemotactic for lymphocytes and monocytes expressing chemokine receptor (CCR)5, is upregulated at the lesion edge;^[122] whereas MCP-1 (CCL2), a chemokine that attracts monocytes expressing CCR2, is expressed by local astrocytes.^[123] Compared with neurological controls, MS patients' CSF contains elevated levels of interferoninducible protein (IP)-10 (CXCL10) and CCL5, both of which are chemotactic for activated T cells expressing the chemokine receptor CXCR3.^[124] An increased frequency of CD4* and CD8* T cells from the CSF of MS patients were seen to express CXCR3. Tissue-based studies confirmed increased expression of CXCR3 as well as CCR5 on immune cells infiltrating around vessels within MS lesions. Together these observations suggest that CCL2/CCR2, CXCL10/CXCR3, and CCL5/CCR5 interactions may be selectively involved in MS pathogenesis. Some reports suggest preferential contribution of selected chemokines to the trafficking of proinflammatory Th₁ T-cell populations,^{[122,124-127]} although this may not always be the case for individual T cells.^[128] Careful studies of cell subsets demonstrate that the majority of CSF T cells in MS patients are memory T cells that express CCR7, consistent with a central memory phenotype of cells that participate in immune surveillance. Within the MS lesions themselves, T cells tend not to express CCR7, consistent with an effectormemory phenotype. Many cells with a maturing dendritic cell phenotype (expressing MHC class II, CD68, CD86, and the chemokine receptor CCR7), are present both in the CSF and within the MS lesions.^[101] Together, these findings support a model in which the afferent limb of CNS-directed immune responses involves entry of dendritic cells that mature when they capture CNS antigens within MS lesions. The maturing dendritic cells then migrate via the CSF into the deep cervical lymph nodes, where they may present CNS antigens to T cells. The T cells turn into central memory (CCR7*) cells, which infiltrate and participate in the efferent limb of CNS immune responses. On subsequent restimulation by antigen within the CNS compartment, these cells downregulate CCR7 and become CCR7-effector memory T cells that are retained within the MS lesions where they contribute to the disease process.^{[101,120,129]} The chemokines CXCL12 (SDF-1) and CXCL13 (BCA-1) may be particularly relevant in regulating B-cell migration into, and possibly persistence within, the CNS.^[130]

Selective Expression of Matrix Proteinases in Multiple Sclerosis. The family of matrix proteinases (MPs) is comprised of tightly regulated proteolytic enzymes, including the MMPs, the related ADAM (a disintegrin and metal-loproteinase) molecules, and tissue inhibitors of matrix proteases (TIMPs). Collectively, these molecules contribute to diverse biological processes such as normal organ development, tissue remodeling, and immune responses. Activated immune cells are able to migrate across barriers such as the BBB and into the extracellular matrix of the CNS, in part, based on the local production of lytic enzymes (Fig. 3, Step 4). Dysregulation of MP molecules may contribute to abnormal tissue invasion by immune cells.^{[107,131-133]} Both MMPs and ADAMs are typically produced as proenzymes requiring proteolytic cleavage to become activated. The TIMPs function to downregulate the MP effects.^[134] Expression of MPs is also seen in CNS glial cells such as astrocytes and microglia.^[135-137] The MP family molecules may contribute to the MS process by: (1) facilitating infiltration of immune cells by disrupting the BBB basement membrane and extracellular matrix;^[134,135] (2) activating membrane-bound proinflammatory cytokines, such as tumor necrosis factor (TNF)α, by cleaving them off the cell surface;^[138,139] and (3) directly damaging CNS structures.^[140] It should be kept in mind that certain MPs may also be required during processes of repair and regeneration within the CNS.^{[107,141,142]} Increased activity of several CSF MMPs has been reported in MS patients compared with controls;^[143,144] gelatinase B (MMP-9) levels are abnormally elevated in both the serum and CSF of MS patients, particularly during acute relapses. A correlation exists between the elevated levels of MMP and the degree of BBB disruption assessed as the number of gadolinium-enhancing MRI lesions.^[145,146] Distinct expression profiles of MPs are found in T cells, B cells, and monocytes when purified cell subsets from patients with MS and from controls are examined.^[147,148] Of interest, monocytes express a broader range and often higher amounts of different proteolytic enzymes compared with T cells and B cells, which is consistent with the observation that invading monocytes/macrophages are major contributors to the MS inflammatory lesions.^[147] Microglia appear to produce even higher amounts of MPs, and this production may in turn regulate their effector cytokine expression.^[149]

It is important to keep in mind that the multistep molecular interactions involving adhesion molecules, chemokines, and MPs is fundamental to normal processes of immune surveillance and adaptive immunity. Dysregulated expression and/or function of these molecules may contribute to the pathology of MS. Efforts are aimed at elucidating how targeting these molecular interactions may therapeutically regulate trafficking of particular cell subsets, while minimizing the impact on normal immune functions.

Activation (and Reactivation) of Immune Cells within the Central Nervous System

CNS-reactive T cells may be activated (or reactivated) within the CNS itself (Fig. 3, Step 5). It is well accepted that under normal physiologic conditions, lymphocytes traffic into the CNS.^[150-152] Resident CNS microglial cells, both perivascular and parenchymal, have been identified as competent APCs.^{[7,49,153,154]} Antigen presentation could thus be accomplished by resident cells or by invading monocytes/macrophages and dendritic cells.^[155-158] Dendritic cells have emerged as important myeloid APCs in the context of CNS inflammation.^[159-163] The migration of monocytes across the BBB may be associated with dendritic cell differentiation, which may result in unique APC populations in a subendothelial distribution.^[164,165] B cells represent another potential source of APCs capable of presenting antigens to T cells in both the periphery and within the CNS. Studies of CSF and the meninges of MS patients have revealed chronically activated B cells/plasma cells that are organized in structures resembling germinal centers and express high levels of costimulatory molecules.^[166] The presence of inflammatory factors produced within the CNS, including cytokines, chemokines, and matrix proteases, can stimulate invading APCs, as well as primed T cells and B cells, in the process called “bystander activation.” Abnormal costimulatory molecule profiles may contribute to the abnormal activation of CNS autoreactive T cells, both within and outside of the CNS. Expression of CD80 (B7.1) is reported to be selectively upregulated in MS plaques,^[167,168] and CD80 but not CD86 (B7.2) was found upregulated on immune cells in CSF and peripheral blood of MS patients compared with controls.^[169] CD80 and CD86 may preferentially activate Th₁ and Th₂ responses, respectively, and blockade of CD80 in EAE inhibited disease induction.^[20,170,171] Tissue damage may further expose CNS components that can then become targets of subsequent immune attack (epitope spread), possibly contributing to clinical relapses.^[172]

Active immune responses could impact on the integrity of CNS myelin, oligodendrocytes (OLGs), and neural elements by several distinct mechanisms (Fig. 3, Step 6, and Table 1 and Table 2 ). CD4 and CD8 T cells can mediate antigen-specific injury to targets that they recognize. Autoreactive antibodies may contribute to antigen-specific injury through complement fixation or antibody-directed cytotoxicity (ADCC). In vitro studies suggest that CD4 and CD8 T cells may also impact the health of neurons by nonspecific cytotoxic mechanisms.^[173,174] Injury to neural elements may also be mediated indirectly, for example, efficient killing by NK cells of supporting astrocytes, resulting in secondary compromise to neurons.^[175] A variety of soluble factors, including proinflammatory cytokines, glutamate, matrix proteases, nitric oxide, oxygen radicals, and proapoptotic signals may also mediate injury in a non-antigen-dependent way.^{[49,75,88,176]}

Involvement of Antibody-Dependent and -Independent B-Cell Responses in Multiple Sclerosis. As members of the adaptive immune system, B cells, like T cells, have the capacity to recognize particular antigens through their surface B-cell receptors (BCRs). The typical sequence of normal B-cell activation involves encounter with the specific antigen that binds the BCR followed by T-cell help, which involves engagement of CD40 on the B-cell surface by CD40L expressed on the activated T cell. The B cells may then proliferate and differentiate into memory B cells as well as plasma cells that secrete antibodies (immunoglobulins [Igs]), and contribute to the humoral immune response against the stimulating antigen. The potential role of B cells in MS pathophysiology has generally been considered based on their potential to react to particular CNS antigens and subsequently produce CNS-directed auto-antibodies. The CSF of MS patients has long since been known to contain abnormally elevated levels of Ig, typically displaying a pattern of oligoclonal bands (OCBs) on an electrophoretic gel. The presence of deposited Ig and complement molecules is reportedly a common occurrence in MS lesions;^[177,178] in both MS and EAE, antibodies bound to fragments of myelin are found within phagocytic cells at sites of demyelination.^[179] Consistent with the oligoclonal Ig pattern, somatic hypermutation and clonal analysis of cells found within MS CSF and lesions indicate that particular B-cell clones are expanded in response to antigenic encounter within the CNS compartment. Together, these findings point to contribution of complement-mediated injury and ADCC, mediated by CNS-specific antibodies in MS.^[180,181] The antigenic specificity of the OCB in MS has been challenging to elucidate. A series of elegant molecular and cell-based approaches have shown that at least a portion of the CNS Ig appears to bind myelin targets, including MOG.^[182-185] Meaningful analysis of serum antibodies against MOG requires assays that use conformationally true epitopes.^[183-186] Serum antibodies against CNS myelin components such as MBP appear to be of relatively low affinity.^[187] It is not clear whether such antibodies directly contribute to pathogenesis; they may be generated as a consequence of tissue injury and as such, may provide a marker of how aggressive the inflammatory process is.^[188,189] It has also been suggested that certain CNS-directed antibodies may actually be beneficial, such as through the support of remyelination^[190,191] or by helping to remove debris— including growth inhibitory molecules such as Nogo^[192]—thereby providing a more permissive environment for axonal regeneration.

B-cell contributions to MS have also been considered beyond their capacity to become antibody-producing cells. For example, B-cell subsets have been identified that can function as effective APCs to T cells,^[193] and normal human B cells are now recognized to actively contribute to the regulation of immune responses through secretion of distinct effector cytokine profiles in a context-dependent fashion.^[194-196] In patients with MS, the meninges and CSF are found to contain chronically activated B cells^[65] in structures reminiscent of germinal centers that may promote ongoing T-cell activation and propagation of the MS disease activity.^{[166,197,198]} A deficiency in the ability of MS patient B cells to produce the downregulatory cytokine IL-10 has been reported,^[199] implicating them directly in the dysregulated MS immune process. Promising results from early-phase MS studies of B-cell depletion with rituximab (anti-CD20 antibody) further support a role for B cells in disease pathogenesis,^[200,201] and the rapid onset of action of this strategy (without obvious effects on Ig levels) suggests that antibody-independent functions of B cells likely contribute to the observed therapeutic effect. In the context of B-cell-targeted therapy, it is noteworthy that the molecular interactions that underlie human B-cell entry into the CNS (involving adhesion molecules, chemokines, and MMPs) partially overlap with, but are not identical to, those employed by T cells.^[147,148] This becomes relevant when considering the impact of T-cell-directed therapies on B-cell physiology, as well as the optimal approach to the development of B-cell-directed therapies.

Contribution of Immune Responses to Remissions. Recovery from an MS relapse involves both immune and nonimmune mechanisms. Nonimmune mechanisms include redistribution of sodium channels on demyelinated nerve fibers,^[202] recruitment of myelinating cells, and remyelination,^[203] as well as axonal regeneration and reorganization of networks.^[204] Immune mechanisms that contribute to recovery include active resolution and/or suppression of the injurious inflammatory processes and promotion of immune responses that may support protection and repair. Resolving active inflammation requires clearance of infiltrating immune cells from the CNS through either emigration or through in situ apoptosis. This limits further injury and enables reversal of acute edema. The balance between proinflammatory and proretention factors and the profile of proapoptotic and antiapoptotic molecules together determine whether activated immune cells persist within MS lesions and whether they are available to impact on recovery.^[88]

As noted previously, active immune responses are not necessarily detrimental during CNS inflammation. Table 2 identifies several ways in which immune mediators may be beneficial in MS. Particular immune responses may suppress or downregulate others: Th₂ cytokines are known to inhibit Th₁ differentiation, and may thereby limit proinflammatory Th₁ responses through “bystander suppression.” Modulation of infiltrating monocytes/macrophages and a resident microglia toward a type-2 APC phenotype could promote antiinflammatory Th₂ responses within the CNS.^[158,205] Overly aggressive shifts toward Th₂ responses may be harmful,^[56,57] and, in some settings, Th₁ responses have been identified as potentially beneficial, pointing to a possible role for “protective autoimmunity.”^[89] Subsets of regulatory T cells (noted earlier)^[33-39,50] are thought to actively participate in dampening immune responses, thereby contributing to remission following a relapse. In addition to providing antiinflammatory or down-regulatory signals, immune responses may also be beneficial in the CNS of MS patients by actively contributing to survival and repair of neural elements. This can occur through release of neurotrophic factors,^[206,207] cytokines that promote OLG progenitor survival and proliferation (e.g., TNFα),^[208] and secretion of particular myelin-directed antibodies that may actually support remyelination.^{[190,192,209,210]} Immune responses are also required to create a more permissive CNS environment through the removal of molecules that otherwise inhibit axonal regeneration such as Nogo and myelin-associated glyco-protein (MAG).^[211] Certain immune mediators (e.g., IL-1β, TNFα, apoptotic molecules) may have both detrimental and beneficial effects during CNS inflammation ( Table 1 and Table 2 ). For example, IL-1β can contribute to tissue injury by enhancing the function of certain MMPs and by exacerbating glutamate cytotoxicity. However, IL-1β is also required for the production of ciliary neurotrophic factor (CNTF)^[212] and promotes remyelination by inducing production of insulinlike growth factor (IGF)-1.^[213] Similarly, TNFα, often considered a Th₁ proinflammatory cytokine, may be directly cytotoxic when acting via TNF receptor (R)1, but can support remyelination when acting via TNFR2. This poses particular therapeutic challenges and may explain why anti-TNFα therapies (which work well for patients with rheumatoid arthritis) have been unsuccessful and, indeed, detrimental in MS.

Immune Mechanisms of Disease Progression. Whether and how inflammation contributes to the progressive accumulation of neurological disability in the absence of relapsing/remitting biology remains to be fully elucidated. Failure of the resolution phase of acute relapses due to persistence of proretention and prosurvival cues ( Table 1 ) may play a role in prolonging the injurious impact of infiltrating immune cells. Chronic activation of resident CNS glial cells, such as microglia and astrocytes, as well as persisting immune cells, provides the basis for generation and maintenance of progressive pathologic responses, even in the absence of acute waves of infiltrating inflammatory cells from the periphery. CNS or invading cells may become activated through TLRs or by scavenging the CNS debris itself. Germinal centerlike structures developing within the CNS may provide further nidi for chronic activation.^{[166,197-199]} Collectively, the activated cells residing within the CNS, whether from peripheral or central origin, are likely sources of effector molecules contributing to smoldering tissue injury. This reflects an important CNS-compartmentalized component of MS inflammation that would be relatively resilient to peripheral immune intervention and may be particularly dominant later in the disease process (Fig. 2).

Space precludes detailed discussion of immuneneural interactions and the neurobiology involved in the gradual loss of neural elements and progressive neurological dysfunction over the course of MS. A “multihit” model is likely relevant with initial sublethal injury that renders OLGs and neurons more vulnerable to accumulating insults. For example, in culture, human OLGs chronically exposed to inflammatory cytokines do not die, but do upregulate intracellular p53 that in turn results in upregulation of death receptors, making the cell more susceptible to Fas-ligand and TRAIL (TNF-related apoptosis inducing ligand)-mediated apoptosis.^[214] Injury of OLG progenitor cells early in the disease course may lead to their eventual depletion and progressively limit the capacity of CNS remyelination.^[215] Axons rely on support from OLG and astrocytes; in their absence, they become more vulnerable to insults that would otherwise have a lesser impact. Combinations of sublethal insults likely contribute to the chronic loss of CNS elements,^[216] quite possibly starting in the earliest stages of the MS process. It is not known whether the mechanisms underlying chronic progression in patients with the primary progressive form of MS are identical to those that account for the progression seen later in the course of patients with a relapsing/remitting onset.

Table 1. Immune Mechanisms that Could Contribute to Central Nervous System Injury

Table 2. Immune Mechanisms that Could Contribute to Central Nervous System Recovery, Protection, and Repair

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Acknowledgements

I wish to thank Claudine McDuff for assistance in preparing the manuscript. This article is dedicated to Mom and Dad—how they shared their worlds, and their world with others.

Funding Information

The author is supported by the Canadian Institutes of Health Research (CIHR), the Multiple Sclerosis Society of Canada (MSSC), and the Immune Tolerance Network (ITN). He is recipient of the Don Paty Career Scientist Award of the MSSC and the FRSQ Chercheur Boursier-Clinicien, and he is awarded a McGill William Dawson Chair.

Reprint Address

Amit Bar-Or, M.D., F.R.C.P.C., Neuroimmunology Unit, Montreal Neurological Institute, 3801 University Street, Room #111, Montreal, Quebec, Canada H3A 2B4. E-mail: amit.bar-or@mcgill.ca .

Amit Bar-Or, M.D., F.R.C.P.C., Experimental Therapeutics Program, Montreal Neurological Institute, Montreal, Quebec, Canada.

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