Etiology

Although there appears to be an "autoimmune" attack against myelin and myelin-forming cells in the brain and spinal cord, multiple sclerosis is not a typical autoimmune disease. There is no proven target antigen, including the myelin proteins that generate experimental allergic encephalomyelitis (EAE), an antigen-induced animal model. Multiple sclerosis is an exclusively human disease. It does not arise spontaneously in wild mice or any other animals. Some HLA types are associated with multiple sclerosis, but the mechanism for the connection is unclear. The immune disruption of multiple sclerosis may begin with an inciting event in the periphery or in the central nervous system, followed by expanding, often intermittent, immune attacks against the brain and spinal cord. There are surprisingly few links to other autoimmune diseases, except inflammatory bowel disease (both Crohn disease and ulcerative colitis) and possibly thyroid disease. Down syndrome and systemic lupus erythematosus (SLE) are underrepresented in the multiple sclerosis populations and are linked to excessive responses to type I interferons. Conversely, in multiple sclerosis, IFN-alpha/beta levels and responses to these type I IFNs are low (143; 93).

Specific antigenic targets and possible biomarkers for inflammation in multiple sclerosis. Brain proteins could trigger antigen-specific responses or modify immune reactivity over time. This "epitope spreading" to recognize different, but related, antigens is seen in experimental allergic encephalomyelitis. In contrast, after monophasic postinfectious encephalomyelitis, there is epitope contraction. Candidate central nervous system antigen targets and disease modifiers include:

Antibody responses to CNS antigens differ among patients. Anti-myelin basic protein responses are normal or slightly increased in multiple sclerosis, unlike the strong responses seen in animal models. However, proinflammatory high avidity human T cell clones that recognize myelin basic protein are detectable (32). Antibodies to myelin basic protein are low in early multiple sclerosis and increase over time (273), but detection is inconsistent between laboratories. Anti-proteolipid antibodies in CSF are more common in women than men, in patients with later onset of multiple sclerosis or no family history of multiple sclerosis, and with low levels of CSF immunoglobulin and few oligoclonal bands (335). Antibodies to myelin oligodendrocyte protein are debatably elevated in all forms of multiple sclerosis. However, they exist in other inflammatory brain diseases, and in normal subjects, who can have anti-MBP T cells and activated anti-MOG memory T cells that produce IFN-gamma and nerve growth factor. Newly-formed myelin does not express MOG, and is resistant to inflammation. Anti-glycan IgM Abs, possibly provoked by breakdown products of the CNS escaping through the BBB, B1 B cells, and the presence of some bacteria, and fungi all correlate with faster progression. Even if antibodies to brain antigens do not cause multiple sclerosis, they may be induced by inflammation and CNS damage and could modify disease course.

In contrast, post-vaccinal and post-infectious encephalomyelitis have defined target antigens. For instance, 1 in 220 people vaccinated with the Semple rabies vaccine--which contains central nervous system tissue--develop autoimmune encephalitis (similar to experimental allergic encephalomyelitis). Patients susceptible to this encephalitis, however, have a human leukocyte antigen (HLA) makeup that is distinct from that of multiple sclerosis (245).

The lack of a causative “multiple sclerosis antigen” suggests that fundamental control of immune responses may be abnormal. Activated lymphocytes and monocytes might enter the central nervous system because of nonspecific adhesion to endothelial cells, become activated within the central nervous system, stay longer during trafficking through the central nervous system, and resist the normal CNS suppression of the immune response. Putative antigen-specific responses are described below.

Non-antigen-specific immunity in multiple sclerosis inflammation. Etiologies that do not invoke specific target antigens are possible.

Viruses. Viruses could cause direct damage to oligodendroglia or could share peptides with myelin. T and B cells could then react against oligodendroglia (molecular mimicry). Viruses can enhance replication of other viruses, making linkage to a single virus difficult to determine.

Human endogenous retroviruses (HERV) make up 10% of the human genome but do not generate complete virions. Immune activation provokes production of incomplete virus nucleic acids or proteins. Expression of HERV nucleic acids and proteins correlates with a more progressive disease course, and serum levels rise with long-duration multiple sclerosis. Activated astrocytes produce retrovirus-encoded syncytin, which is toxic to oligodendrocytes. The HERV envelop protein inhibits differentiation of oligodendroglial precursor cells. IFN-beta therapy strongly reduces the load of HERV products in blood. There is a surprising trend for less multiple sclerosis in HIV-infected patients, perhaps due to immunosuppression from HIV or anti-retroviral therapy.

Multiple sclerosis susceptibility is linked to a history of infectious mononucleosis, but not to acute Epstein-Barr virus (EBV) infections. Onset of infection in young adults increases the risk of developing multiple sclerosis 32-fold (39). Circulating Epstein-Barr virus-infected B cells are rare (1/10,000), but could be a reservoir of viral antigens. Infected cells produce IL-6, which stimulates B cell proliferation, and B cells in multiple sclerosis produce higher than normal levels of IL-6. Antibodies to Epstein-Barr virus correlate with brain atrophy and are elevated early in the course of multiple sclerosis. This may simply reflect multiple sclerosis-characteristic high titers to many antigens and viruses. HLA-DR2 is over-represented in multiple sclerosis, and DR2-positive people have higher antibody titers to Epstein-Barr virus, measles, and rubella (59). Nonetheless, combination of HLA-DB1*15 positive and HLA-A*02 negative, with high antibody titers against Epstein-Barr virus nuclear antigen (EBNA) confers a 16-fold higher risk of developing multiple sclerosis than the opposite profile (127). Epstein-Barr virus-negative multiple sclerosis is very rare or even nonexistent.

In children, seropositivity to EBNA-1 protein increases the risk of multiple sclerosis 3.8-fold, and the disease is rare in seronegative children. Over 90% of children with multiple sclerosis are Epstein-Barr virus positive compared to 50% of control children. Shedding of virus appears in 50% of Epstein-Barr virus positive children with multiple sclerosis but in only 20% of Epstein-Barr virus positive healthy controls, suggesting impairment of immune control of EBV in multiple sclerosis (346). The EBNA-2 1.2 allele quintuples the risk of developing multiple sclerosis. Smokers have higher anti-EBNA titers, along with high titers to many antigens (below).

Anti-Epstein-Barr virus antibodies could arise from persistent infection of astrocytes or B cells, causing immune activation with costimulatory molecule expression and IL-6 secretion by B cells. Epstein-Barr virus-infected B cells could become resistant to apoptosis and immune control. Viral latent proteins appear in some meningeal germinal center-like areas in multiple sclerosis. Epstein-Barr virus-RNA in white matter lesions of multiple sclerosis is associated with interferon-alpha production by macrophages and microglia (321). Epstein-Barr virus nuclear antigen (EBNA-3) blocks the vitamin D receptor, and low vitamin D levels correlate with more Epstein-Barr virus shedding in saliva, linking two environmental factors important in multiple sclerosis.

Antibodies to cytomegalovirus, in contrast, correlate with better outcome and less risk of developing multiple sclerosis (39). Past cytomegalovirus infection confers a 0.27-fold lower risk of multiple sclerosis in children (336). Cytomegalovirus exhausts the immune system throughout the human lifetime, possibly inhibiting inflammation in multiple sclerosis.

Reports of varicella-zoster virus particles in multiple sclerosis brains have not been confirmed (47). Varicella-zoster virus DNA increases briefly in mononuclear cells during relapses, but varicella-zoster virus is generally assumed not to increase the risk of multiple sclerosis. However, Han Chinese patients who developed zoster in Taiwan were 4-fold more likely to develop multiple sclerosis in the next year than zoster-free patients (148).

Bacteria and chlamydia. Bacteria and chlamydia in posterior nasal sinuses and submucosa could trigger attacks through cross-reactive antigens, superantigen activation of pathogenic T cells, responses to induced heat shock proteins (all trigger cytokine release), and release of bacterial toxins (112). Conversely, parasite infestation is protective.

Diet. Diet affects immunity through oral tolerance and direct effects on the microbiome. Diet modifies macrophage and T cell function, membrane composition of immune cells, and prostaglandin synthesis. Fasting reduces monocyte number and inflammatory activity and improves chronic inflammation in animal models. Acinobacter and Akkermansia increase in untreated multiple sclerosis gut bacteria and, when transplanted, worsen experiment allergic encephalomyelitis in germ-free mice. These bacteria are also increased in the gut of untreated multiple sclerosis, but countered by interferon-beta therapy, which increases immune-suppressive short-chain fatty acid transporters.

Genetic. Predisposition is likely in responses to brain antigens, altered control of the immune response to brain antigens and viruses, lack of neurotrophic proteins, or poor ability to repair CNS damage.

Oligodendroglia. Oligodendroglial function and repair of demyelination is defective.

Sex. Multiple sclerosis is three times more common in women than men, and estrogens reduce attack rates in women.

Other mechanisms. Toxins, microchimerism of circulating blood cells, and interrelations among endocrine, catecholamine, stress, and immune systems have been proposed.

In the 1950s, it was theorized that CNS microvessels had poor blood flow in multiple sclerosis. Anticoagulants, however, failed to impact the course of multiple sclerosis.

Venous stenting to reverse putative cerebral venous outflow problems (CCSVI) has not been beneficial in controlled studies. The early studies that generated the hypothesis were carefully performed but have been difficult to replicate. Venous outflow is dynamic, and abnormalities can be reversed simply by drinking liquids. Many patients restrict fluid intake because of bladder urgency; others take diuretics for leg edema. In atrophic multiple sclerosis brains, metabolic needs diminish, and blood flow consequently may be reduced. Tens of millions of dollars in research money and medical costs, huge amounts of investigators’ intellectual energy, and misplaced hope by patients were directed at this therapy.

Pathogenesis

Multiple sclerosis is a demyelinating disease in which brain and meningeal inflammation also affect neuronal metabolism and survival. Demyelination and chronic cytokine exposure leads to brain atrophy, fatigue, cognitive loss, and neurologic abnormalities. The course of multiple sclerosis can be broken down into three phases:

Immunity underlying the CNS pathology in multiple sclerosis. The initiating event for the first exacerbation is unknown. Genetics and environment both play a role (233). Nearly 200 genes have weak links to multiple sclerosis; most are immune-related. Downstream, RNA expression in peripheral blood mononuclear cells is highly dysregulated, with 8800 abnormally expressed genes in untreated patients compared to healthy controls (92). Much of the signal is from immune genes. Cytokines, antigen affinity, and costimulation all have additive effects on immune responses. Interferon-beta, and likely other agents, markedly reduces the dysregulation.

Immune activation in multiple sclerosis is a multistep process, likely evolving over years. Cytokines, antigen affinity, and costimulation all have additive effects on immune responses. Medullary epithelial cells in the thymus express thousands of antigens that are also found in peripheral organs and create immune tolerance to the antigens. Type I interferons, dysregulated in multiple sclerosis, amplify antigen presentation. During development of the immune system or later, an autoimmune cascade may start with thymic presentation of alternately spliced golli-myelin basic protein or other self or viral proteins in the context of excessive expression of costimulatory molecules (194). Nonetheless, no “multiple sclerosis antigen” has been identified.

Following peripheral activation by unknown stimuli, circulating T cells adhere to post-capillary venules in the choroid plexus, brain, and spinal cord. The T cells pass directly through the endothelial cells or possibly breach the tight junctions of the endothelium. They then migrate into perivascular brain parenchyma and form plaques. Equivalent numbers of monocytes and T cells are present in plaques at early stages, but monocytes predominate in later multiple sclerosis lesions. Brain antigens and immune cells can emigrate to deep cervical lymph nodes and educate peripheral T cells. These T cells may then home back to the brain. In the plaque, the cellular infiltrate is associated with destruction of the inner myelin lamellae and dysfunction of oligodendroglia, and with neuronal loss and astrocytic scarring.

Inflammation, reflected by Gd-enhancing MRI lesions, largely resolves in 2 to 8 weeks. Unseen by MRI, some immune cells remain in plaques. They are poised for more activation and exhibit continued smoldering low-grade inflammation chronic active plaques, and slowly expanding lesions, causing chronic axonal loss and demyelination.

Immune activation and dysregulation. Immune activation in peripheral blood precedes neurologic problems and MRI activity. Several weeks before attacks, concanavalin A mitogen-stimulated IFN-gamma and TNF-alpha production increases (28), as do IFN-gamma levels in serum (79), IFN-gamma-induced [Ca++] influx in T cells, and secretion of prostaglandins by monocytes. Excessive numbers of cytokine-secreting cells appear early in multiple sclerosis and in acute optic neuritis. IFN-gamma, osteopontin, and IL-2 activate T cells and endothelial cells and induce costimulatory molecules that further enhance T cell proliferation and activation (248).

Th1 cell-mediated inflammation increases during active multiple sclerosis. There are excessive levels of the activating zeta chain of the T cell receptor on CD4 cells (156), activation proteins (HLA-DR and CD71), costimulatory molecules on B cells (CD80/B7-1) (114), and chemokine receptors (CCR5 and CXCR3) on Th1 cells (20). Inflammatory cytokines and messenger ribonucleic acid for IL-2, IL-15, IL-17, IL-23, and IFN-gamma are elevated in mononuclear cells (318; 184; 275; 48). IL-1, IL-6, IL-15, and TNF-alpha are present in the CSF (193; 161). These Th1-like cytokines and monokines amplify immune responses. IFN-gamma "therapy" and granulocyte colony-stimulating factor (G-CSF) infusions trigger attacks of multiple sclerosis. IFN-gamma, a pro-inflammatory cytokine, is toxic to actively remyelinating oligodendroglia, and it activates monocytes and microglia. However, elevated IFN-gamma receptors on activated proinflammatory Th17 cells in multiple sclerosis allow IFN-gamma to inhibit these cells through apoptosis (02) and protect mature oligodendroglia (179). Thus, cytokines affect timing, location, and degree of inflammation.

Control of inflammation is lost during attacks of multiple sclerosis. Concanavalin A-induced regulatory/suppressor T cell function drops (11). Low production of IL-10 removes another brake on Th1 cells. IL-15 levels rise in blood and, to a lesser extent, in CSF monocytes during attacks and progression. These changes could lead to delayed-type hypersensitivity (Th1-type) immune reactions, and enhanced CD8 T cell cytotoxicity.

However, the Th1/Th2 dichotomy is too simplistic:

(1) Th1 and Th2 cytokines both increase in white blood cells before attacks, known as a “cytokine storm” (180). Th1 and Th2 cytokines are both induced in CNS (51) and in peripheral immune cells following IFN-beta therapy (48; 334). Th1 and Th2 cytokine levels are balanced in healthy controls, but are dysregulated in multiple sclerosis. Balance returns towards normal with IFN-beta therapy (226).

(2) Therapy with anti-CD52 (alemtuzumab) depletes Th1 cells, but it does not stop progression in later multiple sclerosis.

(3) Th2 cytokines, if excessive, can also cause damage. A Th2-driven form of myelin-oligodendrocyte-glycoprotein-induced experimental allergic encephalomyelitis causes lethal demyelination.

(4) Monokines are increased in CSF (193). People with high IL-1/IL-1Ra (IL-1 receptor antagonist) ratios and high TNF-alpha/IL-10 ratios have a 6-fold higher risk of having a family member with multiple sclerosis (72).

(5) Microarrays of immune cell RNA from untreated multiple sclerosis patients show that the IFN-alpha/beta pathway is more dysregulated than the Th1 and Th2 pathway (345; 92).

Th17 cells amplify autoimmune CNS inflammation and are suggested to be important in multiple sclerosis. IL-6 plus transforming growth factor-beta generate IL-17-producing cells from naive CD4 cells. IL-23 maintains this population and also induces IL-17 protein in memory CD4 cells. IL-17 levels are elevated in secondary progressive multiple sclerosis and correlate with MRI lesion load and brain atrophy. The inflamed blood-brain barrier and dendritic cells help polarize naive T cells into Th17 cells (138). In contrast, IL-4, IL-27, IFN-gamma, and IFN-beta all inhibit IL-17 production.

IL-17-expressing cells increase during exacerbations and are higher in multiple sclerosis plaques and CSF than in serum (84). The IL-17 receptor is also elevated on brain endothelial cells. CD4 and CD8 cells, as well as oligodendrocytes in perivascular areas of active multiple sclerosis lesions produce IL-17 (320). Cells simultaneously secreting the Th1 cytokine and IFN-gamma plus IL-17 are also increased in multiple sclerosis and correlate with MRI T1 lesions. CSF levels of IL-17 and IL-8 correlate with the length of spinal cord lesions. Nonetheless, Th17 is difficult to detect in multiple sclerosis and is less important than in demyelinating Devic/NMO disease (140).

The aryl hydrocarbon receptor (AHR) binds dioxin, breakdown products of aromatic amino acids (eg, tryptophan), prostaglandins, compounds in cigarette smoke, products of ultraviolet light in the skin and flavonoids in vegetables, fruits, and tea (256). The AHR interacts with STAT transcription factors, retinoic and estrogen receptors, NF-kB, and the multiple sclerosis therapy, laquinimod. It induces inflammatory Th17 but also regulatory dendritic, NK (IL-10-secreting), Tr1 (IL-10 secreting), and T (Treg) cells. Culture conditions may explain published differences in Th17 function. Commonly-used RPMI culture media has low levels of AHR ligands, but Iscove’s media has high levels of aromatic amino acids and is much more conducive to Th17 cell induction (327). Further, modifying the aryl hydrocarbon receptor responses, the sodium concentrations of these media are RPMI: 8.8 g Na+ = 383 mM/L = 191 mEq/L, which is high compared to Iscove (IMDM) media: 7.6 g Na+= 330 mM/L = 165 mEq/L and X-VIVO 15 media: 7.5 g Na+ = 326 mM/L = 163 mEq/L. The high levels of sodium in RPMI and an additional 30 to 40 mEq/L of Na added to Iscove media induce Th17 (343).

Smoking products affect the AHR and inhibit NF-kB activation, reduce IFN-alpha and IFN-beta production, and increase virus infections (210). Because serum type I IFN levels and responses to IFN-beta are below normal in multiple sclerosis (94; 95), smoking is likely to amplify the IFN-beta deficit. Smoking triggers multiple sclerosis attacks and doubles the rate of brain atrophy (see Environment below); an effect through the AHR is possible. Effects on multiple sclerosis are likely to be complex. Nicotine inhibits immunity and experimental allergic encephalomyelitis (230). AHR ligands in germinal-center-like follicles in multiple sclerosis brain have not been studied, nor have effects of beauty salon vapors or diesel fumes. on the AHR in multiple sclerosis. These fumes suppress NF-kB and interferon-beta production.

Smoking also increases risk of onset and activity in lupus. It amplifies disease activity in rheumatoid arthritis 1.5-fold, but with variants of HLA and protein tyrosine phosphatase nonreceptor variant 22 (PTPN22), the risk of joint inflammation increases 20-fold. Smoking and inflammation induce homocitrulline, triggering antibodies to it that amplify severe rheumatoid arthritis. Aryl hydrocarbons reactivate Epstein-Barr virus and increase risk of Sjögren syndrome. Effects are possible on Devic disease, which is related to Sjögren syndrome.

CD2 is a costimulatory T cell molecule that binds CD58 (LFA-1). The conformation of CD2 may be altered in multiple sclerosis—there is a marked fall in avid RBC rosette-forming cells (CD2 on T cells binds CD58 on RBC), and some anti-CD2 antibodies do not bind CD2 in multiple sclerosis (266). Simulation through CD2 is reduced in progressive multiple sclerosis. A DNA allele of CD58 that increases CD58 mRNA is protective against multiple sclerosis (odds ratio = 0.82), and CD58 mRNA is elevated 1.2 times above normal in exacerbations and 1.7 times in remissions (71). There may be a reciprocal relationship between disease-activity-specific low CD2 function and CD58 expression. Activation through CD2 increases regulatory CD4 T cells number and function; effects of CD2 activation on CD8 cells are unknown.

Cytolytic CD8 cells and monocytes in plaques directly damage neurons and axons. Many of these CD8 cells are activated, react with EBV-infected B cells, and secrete toxic IL-17, suggesting they are mucosa-associated invariant (MAIT) T cells (320; 326). Although most evidence for a CD8 role is correlative, melanoma cell adhesion molecule-positive CD8 cells are elevated in multiple sclerosis CNS, produce IL-17, IFN-gamma, and GM-CSF, and kill oligodendroglia. Expanded CD8 clones appear in blood, CSF, and multiple sclerosis plaques, CD4 clones do not.

CD8+,CD28- regulatory/suppressor cell function is an important component of immune suppression in multiple sclerosis. Without control, inflammation can become self-sustaining. During exacerbations, the number of these CD8 cells drops and membrane CD8 protein diminishes, noted in the first study to quantitate membrane CD8 expression on lymphocytes (265). Importantly, concanavalin A mitogen-induced suppressor function drops during attacks of multiple sclerosis (11; 152; 64). Antel and colleagues showed that the T cells in multiple sclerosis that suppress immune reactions are predominantly CD8+CD28- (10; 68). CD8 cells have much more potent suppressor effects than CD4 cells in multiple sclerosis. CD8 suppressor cells form a 3-way bridge with monocytes to destroy pathogenic CD4 cells that express HLA-E (310; 64). CD8+,CD28-,FoxP3+ suppressor cells also induce tolerogenic ILT3 and ILT4 molecules on endothelial cells (195; 144) and on antigen-presenting cells. During exacerbations, high levels of IL-15 and IFN-gamma induce expression of the inhibitory NG2A protein on CD8 cells, further diminishing their suppressor function (64). In mice, similar CD8,CD122 regulatory cells produce IL-10 to inhibit proliferation and IFN-gamma production by CD8 cytotoxic cells. Four classes of multiple sclerosis therapy induce these suppressor cells (below).

Transfer of specific neuroantigen-reactive CD8 cells inhibits experimental allergic encephalomyelitis (347). In CD8 knockout mice, attacks resolve, but later relapses still occur. This suggests that regulatory CD8 cells do not terminate inflammation but do prevent recurrent attacks. Generalizations across species are suspect, however. The major suppressor cell subpopulation in mice consists of CD4+CD25+ T regulatory cells; in multiple sclerosis, the more potent subset is CD8+CCD28-.

The defect in mitogen-induced CD8 suppressor cell function in multiple sclerosis correlates highly with clinical activity (r = 0.79) (10), far better than enhancing MRI lesions correlate with clinical disease (r = 0.25) or serum cytokine levels (163). The CD8 suppressor defect in multiple sclerosis is corrected with IFN-beta, glatiramer acetate, fingolimod, ocrelizumab, beta2-adrenergic agonists, and Fc receptor ligands. Monitoring of suppressor cell function, or of expression of CD8, ILT3, or CD80, or specific Th1, Th2, and Th17 markers could predict impending attacks of multiple sclerosis, could differentiate multiple sclerosis attacks from transient fever-induced worsening, and could mirror therapeutic responses to drugs.

CD4+ CD25++ (CD39+) FoxP3+ T regulatory cells (Treg), CD56bright NK suppressor cells, and follicular regulatory T cells have reduced function in multiple sclerosis (308). CD4 Treg can function with or without contacting target cells and can create a tissue microenvironment that induces other immune suppressive populations, “infectious tolerance.”

Tr1 CD4 suppressor cells secrete 6-fold less inhibitory IL-10 in multiple sclerosis. Multiple sclerosis CD4 cells are also resistant to inhibition by IL-10 (200). Memory CD4 Treg return to normal levels in progressive disease (328). The environment in the eye is immunosuppressive; very small amounts of retinal antigens create CD4 CD25+ cells that inhibit immunity in mice. The immunologically privileged CNS is likely to behave similarly. CD4 Treg development requires IL-2, IL-7, vitamin A, TGF-beta, and indoleamine dioxygenase (induced by IFN-beta). These cells can also be induced by cAMP agonists and apoptotic cells, including antigens from dying brain cells draining to deep cervical lymph nodes.

Thymic export of new T cells is reduced in multiple sclerosis, so blood T cells have fewer T cell receptor recombinant DNA excision circles (Trec). The age of these cells can be quantitated; each time peripheral T cells divide, the number Trec per cell is halved. Recent thymic emigrants (RTE), including new naive T cells and Tregs, are reduced in relapsing-remitting multiple sclerosis (124). The Treg precursors are highly sensitive to endogenous signals that control development of mature peripheral Treg (234). Using RTE as a measure, the immune system in multiple sclerosis ages prematurely and is 30 years older than that of healthy controls (134). This indicates the need to understand the immune mechanisms of immune modulatory therapies in patients.

B cells have direct effects on immune regulation and brain destruction (208). B cells secrete antibodies -- generate oligoclonal bands, present antigen to T cells, secrete cytokines, form germinal centers and germinal center-like areas in the meninges, and can contain EBV and JC viruses. Potentially pathogenic inflammation- (IFN-gamma) and virus- (TLR9) triggered B cells (325), and microbiota-reactive IgA+ B cells, some producing IL-10, traffic into the CNS during exacerbations (252). B cells secrete pro-inflammatory IL-6, GM-CSF, TNF-alpha, and chemokines. IL-6 enhances generation of destructive Th17 cells. In contrast, B cells can produce anti-inflammatory IL-10 and IL-35, and also nerve growth factor and brain-derived neurotrophic factor, which may help repair the CNS. Anti-CD20 therapies will, therefore, have complex effects on B cell regulation of immunity.

B cells are activated in multiple sclerosis. Compared to healthy controls, naïve B cells secrete half as much inhibitory IL-10 after stimulation with anti-CD40 (a model of bystander T cell activation) or B cell receptor plus anti-CD40 (B cell plus T cell activation) (82), although the same laboratory later stated that Bregs may have increased function in multiple sclerosis. Circulating B cells in multiple sclerosis express high levels of costimulatory molecules (CD80/B7-1) pushing them above a signaling threshold to enhance inflammation. B cells are potent antigen-presenting cells because they are exquisitely focused against specific antigens (115). B cells are activated by B cell activating factor (BAFF), which is made by myeloid cells. CSF BAFF and CXCL13, a B cell attracting chemokine, increase during relapses and in secondary progressive multiple sclerosis (258). CSF BAFF correlates with IL-6 and IL-10 levels, suggesting that these factors amplify B cell function and CSF antibody production. CSF CXCL13 is elevated in all forms of multiple sclerosis and correlates with CSF white cell and B cell number, IgG synthesis, and MRI activity. The number of CSF B cells and plasmablasts correlates with active MRI lesions in the brain.

High CSF immunoglobulin synthesis and high CSF antibody titers to measles virus were reported in the 1950s. Using a sensitive immunoelectrophoresis technique, CSF IgG and oligoclonal bands are present in more than 95% of patients. Bands are a sensitive and specific diagnostic test that reflects CNS inflammation and cortical lesion load but do not correlate with prognosis. IgG kappa light chains in CSF are a more sensitive test but slightly less specific and do not require a paired serum sample. In clinically isolated syndromes, B cell clonal expansion is reflected by rearranged mRNA and certain heavy chains (VH4 or VH2) and is linked to development of multiple sclerosis, yet these antibodies do not predominantly react against myelin (29). In progressive multiple sclerosis, CSF and brain B cells clonally expand and are present in germinal center-like areas in the meninges. CSF and serum antibodies recognize unknown antigens, viruses, myelin proteins, axons (triose-phosphate isomerase), and DNA (ANA). Over 50% of brain plaques contain antibodies plus complement, although the antibodies do not cause demyelination (186). Some anti-brain antibodies enhance remyelination in mice, but not in human trials.

Chemokines attract immune cells. Monocytes in multiple sclerosis secrete excessive CXCL8 (IL-8) to attract other monocytes. IL-8 should also attract polymorphonuclear neutrophils, but these cells are rarely seen in multiple sclerosis CSF or brain. In Japanese optico-spinal multiple sclerosis, however, there is increased IL-8 and IL-17 as well as both Th1 (IFN-gamma) and Th2 (IL-4 and IL-5) cytokines. In a subset of patients with this Japanese NMO/Devic-like variant, IL-8 in CSF and neutrophils in lesions correlate with spinal cord lesion formation (140). IFN-beta decreases IL-8.

Myeloid suppressor cells are precursors of macrophages, dendritic cells, and granulocytes. They increase in number during exacerbations (140/million mononuclear cells) versus normal controls (5/million), fall during stable disease (15/million), and have potent function in multiple sclerosis (139). They promote function of regulatory B cells and CD4 T cells and suppress activated CD4 cells on contact.

Monocytes, and to a lesser extent, microglia present antigens and amplify immune responses. They communicate with cells hundreds of microns away through tunneling nanotubes that transmit calcium ions and antigens, and continuously modify neuronal networks. They over-express receptors for immunoglobulins and are activated by low levels of serum receptor for advanced glycation end-products (RAGE). Inhibitory molecules expressed by monocytes (HLA-G, ILT3) are reduced in multiple sclerosis but are upregulated by IFN-beta (213; 144) and by vitamin D, IL-10, and CD8 suppressor cells.

Microglia in the brain release nitric oxide, oxygen radicals, complement, proteases, and cytokines. CSF nitric oxide metabolites correlate with gadolinium-enhanced MRI lesions, clinical activity, and progression of multiple sclerosis. Nitric oxide also modifies brain proteins to form nitrotyrosine. This creates neo-antigens in the brain and generates antibodies to S-nitrosocysteine in the CNS (41). Myeloid dendritic cells in secondary progressive multiple sclerosis are activated and pro-inflammatory (154). Peripheral monocytes secrete excessive nitric oxide, which is neurotoxic and damages oligodendroglia, but also eliminates activated T cells and have other positive effects (See “Recovery from relapses,” below.).

Plasmacytoid dendritic cells are weak as antigen-presenting cells but are the major source of type I interferons. They are more frequent in early multiple sclerosis and relapses in some studies. However, these cells in multiple sclerosis produce less IL-10 and less IFN-alpha (305) and induce Th17 cells.

Inflammation, immunity, and trauma. Trauma and stress were proposed by some to cause multiple sclerosis and trigger exacerbations (206; 247; 46; 178). Stress and exacerbations are sometimes difficult to define. However, linkage of exacerbations to stress and trauma are nonexistent when stress, trauma, and concomitant clinical manifestations of multiple sclerosis are carefully analyzed (293; 294; 299). One group, however, finds a slight increase in new MRI lesions with stress (214) and transient reduction of new MRI lesions by stress management (215). Stress in the home and physical abuse during childhood appear to prevent multiple sclerosis. Gunshot wounds and SCUD missile attacks actually seem to protect against exacerbations (293; 228), although another war report suggests increased exacerbations (119). Local irradiation of the brain increases lesions of multiple sclerosis within the radiation field, possibly by disrupting or activating the blood-brain barrier (224).

Autonomic control and damage in multiple sclerosis. The hypothalamus regulates autonomic functions, body temperature, sleep, and sexual activity. Hypothalamic corticotrophin releasing hormone (CRH) begins an endocrine cascade to adrenocorticotropic hormone and then to cortisol production. Serum cortisol feedback and exogenous steroids turn down corticotrophin secretion.

Hypothalamic plaques are common in multiple sclerosis and disrupt endocrine regulation (136). Surviving myelin bundles are next to HLA class II positive microglia. Neurons that secrete regulatory hormones are localized to periventricular and arcuate nuclei, and are perhaps more susceptible to CSF toxins. Inflammation in the hypothalamus may explain the high number of double-positive corticotrophin and arginine-vasopressin neurons that are unique to multiple sclerosis, especially in disease of long duration. Arginine-vasopressin potentiates the action of corticotrophin on adrenocorticotropic hormone (ACTH) release. The resultant elevation in cortisol could inhibit inflammation because high numbers of corticotrophin-releasing factor/arginine-vasopressin neurons correlate with low hypothalamic lesion load. Similarly, rats with high corticosterone are protected against experimental allergic encephalomyelitis.

The hypothalamic-pituitary-adrenal (HPA) axis is hyper-responsive to corticotrophin-releasing hormone, especially in primary progressive multiple sclerosis (311). Chronic HPA axis overactivity renders cells insensitive to glucocorticoids allowing them to escape from immune restraint. Levels of cortisol, adrenocorticotropic hormone, and dehydroepiandrosterone, and the number of cells secreting corticotropin releasing hormone are increased most in progressive and active forms of multiple sclerosis (349).

The steroid-regulated diurnal migration of B cells from bone marrow to blood is likely to be disrupted. Acute and chronic inflammation induces high serum cortisol levels that cause systemic and local steroid resistance. IL-1alpha, produced by activated macrophages (M1), inhibits glucocorticoid receptor translocation to the cell nucleus (239). High levels of tumor necrosis factor, IL-1, and IL-6 correlate with hypothalamic-pituitary-adrenal axis (HPA) activation and with fatigue. During active multiple sclerosis, the hypothalamic-pituitary-adrenal axis and immune cells are hyporesponsive to dexamethasone feedback (268). Conversely, cyclic adenosine monophosphate (cAMP) agonists (prostaglandins, beta-adrenergic agonists such as terbutaline, and some antidepressants) enhance steroid receptor translocation and could potentiate glucocorticoids and normalize the HPA axis. Weak response to steroids correlates with high CSF white blood cell counts and enhancing lesions on MRI (90). Mechanisms for resistance to steroids include (1) downregulation from chronic high cortisol (mildly increased in multiple sclerosis), possibly from adrenocorticotropic hormone released by immune cells (263; 269; 189); (2) mutations in steroid receptors; and (3) interaction with other signaling pathways.

During pregnancy, the exacerbation rate falls (34). Regulatory cytokines and hormones such as estriol act together to frustrate Th1 responses and cause immunosuppression. Estriol therapy reduces exacerbations in multiple sclerosis. CD4 and CD8 regulatory T cells tolerize against the fetus. They persist after delivery, perhaps correcting some of the regulatory defect in multiple sclerosis. In contrast, perhaps, mild maternal infections during mouse pregnancy lead to enrichment of persisting IL-17+ cells in the small intestine of the pups.

Recovery from relapses. As clinical symptoms wane, there is suppression of inflammation linked to a rise in inhibitory Th2 cytokines, immunoglobulins, and glucocorticoids (270), and rebound of subnormal suppressor T cell function (10). In addition to immune changes, axonal sodium channels redistribute in ravaged but surviving axons, and there is remyelination and rewiring of the brain (compensatory adaptation; functional reorganization of neurons and synapses).

Inflammation is turned off by apoptosis and suppression of activated immune cells. Apoptosis of Th1 cells is mediated by steroids (endogenous or therapeutic), IFN-gamma (109; 02), TNF-alpha, and nitric oxide. IFN-beta causes apoptosis of Th17 cells, which express high levels of the type I interferon receptor (84). Unfortunately, some of these regulatory compounds are also toxic to neurons and oligodendroglia (TNF-alpha, high levels of IFN-alpha, glutamate, and nitric oxide).

A subgroup of macrophages (M2) secrete neuroprotective compounds, suggesting there is a counterbalance between destruction and repair during recovery from inflammation. Macrophages produce platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-beta), insulin-like growth factor 1 (IGF-1), neural growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3). BDNF is expressed in lesions by T cells, macrophages, microglia, and astrocytes. Immune cells secrete more BDNF during relapse, but levels fall with progression. Other neurotrophic factors rise after relapses, including glial derived neurotrophic factor (GDNF), NT3, NT4, NGF, and possibly ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF). Foamy macrophages, after ingesting myelin, secrete anti-inflammatory IL-4, IL-10, and prostaglandin (PGE). Staph protein A-activated monocytes produce ten times more IL-10 (3000 pg/ml) than lymphocytes (125). IL-4, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in turn induce microglia and monocytes that have anti-inflammatory properties. In focal and traumatic brain injury, and perhaps in multiple sclerosis, activated microglia phagocytose CNS debris. The microglial triggering receptor expressed on myeloid cells (TREM2) binds phospholipids and pathological amyloid. It associates with transmembrane adaptor (DAP12) on microglia and activates SYK to help clear A-beta amyloid plaques. However, this microglial activation may be detrimental in multiple sclerosis lesions and reflects disease activity.

IFN-beta and IFN-gamma cause macrophages to produce indoleamine 2,3 dioxygenase, an anti-inflammatory compound that induces regulatory T cells. Glatiramer acetate activates type II monocytes (M2), which induce Th2 cells and regulatory CD4 T cells, and these inhibit experimental allergic encephalomyelitis (338).

Remyelination occurs in most lesions soon after lesion onset and also lasts for up to 6 months. It prevents axonal loss and slows progression. Remyelination is the normal default response to insults, reinforcing the need to reduce inflammation. Remyelination is quite extensive in 20% of patients and in more than 40% of MRI lesions. It occurs in both relapsing-remitting and primary progressive disease and in both early and late multiple sclerosis (241). It can be prominent in older patients, in disease of long duration, and in subcortical and deep white matter but is infrequent in periventricular plaques.

Evolution of the progressive clinical course: many possible causes. Repeated exacerbations and remissions change to a progressive clinical course approximately 10 years after disease onset. Clinical remissions, associated with T- and B-cell-mediated immunity, begin to wane, but constant low-grade immune activation smolders and monocyte-mediated damage begins to predominate. In progressive multiple sclerosis, cumulative loss of oligodendroglia and neurons, increasing demands on surviving, yet compromised, cells.

One theory for the evolution from relapsing-remitting to progressive multiple sclerosis is that early subclinical neurodegeneration simply becomes noticeable at some point. However, patients who continue to have frequent relapses after the first 2 years are less likely to become progressive (286), and superimposed relapses actually slow worsening in progressive from onset patients (135). This suggests that there is a distinctive transformation from relapsing-remitting to progressive multiple sclerosis. The mechanism for this failure of immune regulation and CNS repair is unknown (194; 77), but there are many hypotheses for the mechanism:

Changes that could provoke the transition from relapses to progression.

(1) Interferon signaling becomes continuously subnormal in mononuclear cells from patients with progressive multiple sclerosis—a transition from near-normal function in stable relapsing-remitting disease (94).

(2) Weak CD8 suppressor T cell function, seen intermittently during exacerbations, becomes continuous with progression (267; 11). Over time there is loss of peripheral immune tolerance and loss of many immune regulatory pathways (92).

(3) Naive CD4 T cells that express high levels of immune response genes (activation markers, pro-apoptotic markers, CD28,chemokines, vitamin D receptor, and TLRs 1,2,4,7) correlate with rapid transition from relapsing to progressive multiple sclerosis (350).

(4) T cell clones from patients with progressive multiple sclerosis express costimulatory CD80 and CD86 molecules and can present antigens. These cells become insensitive to steroids, which only weakly inhibit proliferation and cause little apoptosis, akin to a “pre-leukemic state” (63). They are also resistant to inhibition of growth by TGF-beta.

(5) Spontaneous and activation-induced apoptosis is impaired in T cells during clinically active multiple sclerosis (353; 291), so autoimmune cells are not eliminated.

(6) Shift from fluctuating dysregulated Th1 and Th2 immunity to chronic neurogenerative innate inflammation. Gene dysregulation in untreated relapsing/remitting multiple sclerosis with 8800 differentially-expressed genes versus healthy control mononuclear cells falls to less than 400 in secondary progressive disease (92).

(7) Adhesion molecules are shed from the lymphocyte surface in relapsing-remitting and secondary progressive disease but not primary progressive multiple sclerosis (83). This suggests that T cell-endothelial cell adhesion is most abnormal in relapsing disease.

(8) Germinal center-like areas appear in the meninges, often deep in sulci, in secondary progressive multiple sclerosis. They are less frequent in relapsing and primary progressive multiple sclerosis. Organized sites with chronic B cell activation suggests there is loss of CNS immune control. Antibodies to many targets generally increase more with progression. Other cytokine toxins may escape from germinal centers. A subarachnoid lymphatic-like membrane (SLYM) lines meningeal sinus endothelial cells, allowing exchange of small molecules between venous blood and CSF. Perhaps related, there is a high TSPO PET signal in bones at the temporal pole and base of skull, suggesting inflammation of the calvarium in multiple sclerosis.

(9) Gadolinium-enhancing MRI lesions decrease in frequency, possibly from a change in the makeup of inflammatory peripheral or CNS cells or in endothelial cell activation.

(10) Patients with primary progressive multiple sclerosis and high MRI T2-weighted lesion volume, nonetheless, have excessive IFN-gamma production and rapid T cell migration through endothelial cells (249).

(11) Monocyte and microglial evince excessive innate immunity, with a proinflammatory profile. Monocytes produce 5- to 10-fold more IL-12 and IL-18, and more IL-23, in progressive multiple sclerosis (21). These interleukins induce IFN-gamma and allow dendritic cells to activate pro-inflammatory Th1 cells (154). Macrophages (M1) and Th1 and Th17 cells activate astrocytes to secreteTIMP-1 and CXCL10 (IP-10), which are toxic to oligodendrocytes. In contrast, protective IL-10, PGE, and BDNF decrease. Low IL-10 is associated with more disability and more MRI lesions. Immune cells in secondary progressive multiple sclerosis also secrete high levels of kallikreins and destructive serine proteases; both are associated with more disability.

(12) Plaques contain more monocytes and fewer T cells in chronic disease. Perhaps reflecting a shift to innate immunity, anti-T cell therapy has no or minimal clinical benefit in progressive multiple sclerosis.

(13) Neurotrophic abilities are lost as disability progresses, both in the central nervous system and in peripheral blood cells. Nerve growth factor produced by endothelial cells drops with increasing disability (33) as does BDNF produced by T cells (125). Chronic defeat stress, and possibly the stress of ongoing brain inflammation, reduces BDNF levels through di-methylation of the BDNF gene, a DNA-repressive modification (319). N-acetylglucosamine (GlcNAc) serum levels are low in progressive disease, perhaps reducing N-glycan branching, leading to demyelination and inflammation.

(14) Inexorable damage. In animal models, noninflammatory damage from cuprizone, followed by completed remyelination, is clinically stable for some time. After 6 months, slow axonal degeneration crosses a threshold and mice lose clinical function (196). WNT repair pathway expression declines in mononuclear cells, coinciding with reduced repair capability (105). In multiple sclerosis, residua from old lesions, plus ongoing inflammation, combined with aging, may cause relentless loss of function and atrophy. In addition to an effect of age, leukocyte telomeres are shorter in secondary progressive disease, perhaps from environmental or endogenous stressors (126), and shortness correlates with rate of progression.

(15) In progressive multiple sclerosis, the deep gray matter becomes more abnormal on MRI (254; 103) and the cortex atrophies (232). Deep cortical invaginations are most affected and abut many germinal center-like inflammatory areas (171). In relapsing multiple sclerosis, brain atrophy is largely periventricular. At the transition to progressive multiple sclerosis, gray matter loss accelerates and is the main contributor to total brain atrophy (102), and cortical remyelination and plasticity fail. In a prospective study, cortical lesion load and, most significantly, cerebellar cortical and white matter volume predict the change in course (50). In contrast, white matter damage is obvious in relapsing/remitting multiple sclerosis but does not predict progression. Retinal nerve fibers and macular ganglion cells thin faster in progressive than in relapsing multiple sclerosis, and damage is not slowed by therapy in progressive disease (301).

(16) Cord atrophy and neuronal loss are present in all forms of multiple sclerosis. Atrophy correlates with progression and disability, especially in those with higher EDSS scores. Cord lesions are likely to damage sympathetic fibers (see 22).

(17) Axonal damage markers in CSF after a first attack predict conversion to definite multiple sclerosis (44). Tau and neurofilament proteins (40%) are slightly better than MRI (34%) as predictors. Both increase further during exacerbations and in secondary progressive multiple sclerosis, reflecting ongoing damage. Amplifying symptoms, axonal energy demand is increased in parallel with detached and uncompact myelin, plus unmasked potassium channels at the nodes of Ranvier, in normal-appearing white matter NAWM) (322).

(18) Destruction of oligodendrocytes continues at a faster pace in secondary progressive multiple sclerosis than in relapsing-remitting multiple sclerosis, and there is more myelin basic protein-like material in urine.

(19) Oligodendrocyte precursor cells are lost as plaques accumulate. Oligodendrocyte precursor cells are also not recruited to the lesions, fail to differentiate, and do not remyelinate, possibly from inhibitory factors in plaques such as chondroitin sulfate, hyaluronan, or from inhibitory molecules on demyelinated axons such as polysialylated-neural cell adhesion molecule (PSA-NCAM, NOGO, and RGM) (106).

(20) In chronic plaques, progenitor oligodendrocytes often extend processes to axons but do not wrap around axons. The axons that spurn the oligodendrocyte remain dystrophic, swollen, and vulnerable to insults (53).

(21) Activated astrocytes inhibit extension of oligodendrocyte processes. Normal astrocytes express beta2-adrenergic receptors, which reduce major histocompatibility complex class II and adhesion molecule expression, inhibit immune responses, and prompt secretion of trophic factors and lactate, an energy source for axons and oligodendrocytes. In multiple sclerosis and in Alzheimer disease, adrenergic receptors are absent from astrocytes (74); this is associated with more neuropathology. Loss of these receptors may overcome immune privilege (above) and allow overactive immune responses in the central nervous system.

(22) Disrupted sympathetic and other neural pathways that regulate immunity is from a gradual increase in cord plaque burden. A reflex arc of sensation monitors the gut with transmission to the CNS--with memory stored in the insular cortex. Motor efferent output affects the gut microbiome. Associated with monocytes and macrophages, nociceptor neurons sense bacterial invasion and secrete CGRP to inhibit invasion and also feed back into the nervous system. Sympathetic nerves innervate the gut and bone marrow and inhibit immunity. Disrupted sympathetic effects on bone marrow and T cell homing are also of interest because bone marrow has more polymorphonuclear and myeloid cells, and fewer B and NK cells in untreated multiple sclerosis (292).

Spinal cord atrophy predicts progressive disease in relapsing multiple sclerosis (36), and autonomic responses are frequently disrupted in progressive forms of multiple sclerosis. A "strategic hit" to central autonomic pathways may interfere with the immuno-inhibitory tone from spinal sympathetic adrenergic fibers that innervate the spleen (153), and bathe choline acetyl-transferase-containing T lymphocytes that secret acetylcholine and shift monocytes to an M2 anti-inflammatory phenotype. With autonomic loss, a form of denervation supersensitivity appears in immune cells, and beta2 adrenergic receptors are overexpressed on CD8 T cells in progressive multiple sclerosis. These cells have exaggerated cyclic AMP responses that can affect suppressor CD8 T cell function and innate immunity. Oral terbutaline, a beta2-adrenergic agonist, increases CD8 suppressor function and could be more effective in progressive disease.

(23) Adrenals increase in size, cortisol levels rise, and feedback inhibition of the hypothalamic-pituitary-adrenal axis by glucocorticoids is abnormal in progressive multiple sclerosis, perhaps from excessive production of AVP/CRH in the plaque-filled hypothalamus. High glucocorticoid levels, endogenous and therapeutic, are linked to hippocampal atrophy. Glucocorticoid therapy no longer suppresses multiple sclerosis symptoms. Hypercortisolemia may be corrected with antidepressants. The role of sympathetic input, enteric neuron damage, gut-innervating pain neurons, constipation, and effects of disease-modifying therapies and symptom treatments (opiates, antibiotics, gabapentin, steroids) versus the microbiome in multiple sclerosis need to be explored.

(24) Adrenals increase in size, cortisol levels rise, and feedback inhibition of the hypothalamic-pituitary-adrenal axis by glucocorticoids is abnormal in progressive multiple sclerosis, perhaps from excessive production of AVP/CRH in the plaque-filled hypothalamus. High glucocorticoid levels, endogenous and therapeutic, are linked to hippocampal atrophy. Glucocorticoid therapy no longer suppresses multiple sclerosis symptoms. Hypercortisolemia may be corrected with antidepressants.

(25) Therapies directed at T and B cells, which are effective in relapsing or transitional multiple sclerosis, lose efficacy in purely progressive multiple sclerosis. These include alemtuzumab, glatiramer, IFN-beta, mitoxantrone, fingolimod, natalizumab, chemotherapy, IVIG, and rituximab.

Clinical characteristics interact with the above factors. Progression and poor prognosis are more likely in those with poor recovery after attacks, in older patients and men, and in those with sphincter, motor, and multiple system dysfunction, and with comorbid illnesses that blossom with aging. There is more brain atrophy, most dramatic in the cortex, during progression independent of relapse activity (PIRA), paralleling accumulation of disability.

Pathology of the multiple sclerosis lesions. Multiple sclerosis plaques are found in both gray and white matter throughout the brain and spinal cord. Lesions appear random but have predilection for certain brain regions. Periventricular and periaqueductal sites are most likely to suffer, and optic nerves are almost always involved. Cord lesions are often subpial. Even “normal appearing white matter” is abnormal on magnetic resonance spectroscopy. On histology, there is significant axonal loss, especially near plaques.

A plaque is a well-demarcated area with myelin loss, inflammatory cells, gliosis, and often partial preservation of axons and neurons. Demyelination usually predominates, but in some cases, axonal loss is also severe (314). Mitochondria are increased in axons and astrocytes of active and inactive lesions, but can become dysfunctional during inflammation.

Swollen, hypertrophied astrocytes at plaque edges contain dense core particles and sometimes endocytosed oligodendroglia. Perivascular end-feet of astrocytes are damaged early in plaque formation, reminiscent of the pathology in neuromyelitis optica. Astrocytes are initially hypertrophic or “gemistocytic” with high levels of GFAP. When stimulated by IL-9, they produce CCL20, which attracts Th17+ CD4 and CD8 T cells. Astrocytes also produce potentially beneficial trophic factors, BDNF, TrK receptors, and VEGF (188). Months to years later, they become fibrillary and form fibrous scars (sclerosis; gliosis).

The periventricular location of plaques has been explained by multiple theories: damage from CSF toxins or cytokines, regional variation in microglia or capillary pericytes, and slow blood flow in post-capillary venules to facilitate T cell adhesion. Local toxins are unlikely because abluminal molecules diffuse throughout the brain within 6 minutes, facilitated by arterial pulses (274). Rapidly diffusing cytokines should activate pericytes or endothelial cells throughout the CNS, unless toxins are efficiently bound by nearby cells. MRI in multiple sclerosis suggests that a slow flow rate (less than 50% of normal) through periventricular veins allows immune cells more time to attach to endothelium (174). Slow flow occurs despite the reduced diameter of intra-lesional veins and increased diameter of extra-lesional veins (110). All plaques are perivenular on MRI (309), and areas with high vein density are most frequently affected. In radiologically-isolated syndromes without clinical activity, only those who have lesions surrounding a central vein develop multiple sclerosis (212; 255).

Choroid plexus cells are activated in multiple sclerosis, with HLA-DR and VCAM-1 expression in macrophages, dendritic cells, and epiplexus cells (329). It may be an important site for antigen presentation and for the earliest T, B, and NK cell entry into the CNS, leading to CSF-mediated surface-in or ependymal-in gradient of thalamic damage (89). The activated choroid plexus is enlarged in multiple sclerosis, but not in NMO, and this is linked to periventricular neuroinflammation, brain lesions, and atrophy. Thalamic neurodegeneration is more prominent than demyelination (192).

The blood-brain barrier consists of specialized endothelial cells connected by tight junctions in the lumen of blood vessels. On the abluminal surface are vascular smooth muscle cells and pericytes. Surrounding these cells, the inner layer of the basement membrane includes endothelial cells and pericytes. Astrocyte foot processes generate the outer layer. A few blood-brain barrier areas, such as the area postrema, lack tight junctions but have no increase in plaques. This discordance suggests that activated endothelial cells in the postcapillary venules actively attract immune cells into restricted areas.

The endothelium is abnormal in multiple sclerosis and in experimental allergic encephalomyelitis (219). In 1872, Rindfleisch described abnormal blood vessels in all multiple sclerosis lesions (160; 188). Active plaques appear on MRI because of leakage though the barrier and perhaps Gd uptake by activated endothelial cells. Lack of Gd+ lesions in progressive multiple sclerosis would suggest there is less breakdown of the blood-brain barrier, yet serum proteins extrude through the blood-brain barrier in progressive multiple sclerosis more than in other forms (176). The type of inflammation may differ, or tight junctions may be compromised, even with less apparent inflammation, because fibrin is increased in the perivascular space in chronic plaques (169; 58; 176). Many active demyelinating lesions are missed on MRI, suggesting there is a spectrum from profound inflammation in classical plaques, to moderate inflammation in slowly T cells expanding plaques, to mild or no inflammation in inactive plaques (171).

T cell adhesion molecules bind ligands on endothelial cells. Bound T cells then penetrate directly through the endothelial cells (emperipolesis), not necessarily through the tight junctions (12). The self-amplifying loop between activated T cells and activated endothelial cells is blocked by natalizumab and by interferon. After monocytes cross through endothelial cells of the blood-brain barrier, they pile up in the perivascular space. The glia limitans outside the basement membrane or basal lamina is then breeched by matrix metalloproteases. These are diminished by IFN-beta therapy. The combination of natalizumab and IFN-beta could synergistically decrease leukocyte traffic into the CNS.

The initial inflammatory lesion is a cuff of macrophages and CD4 T lymphocytes that surround vessels lined with endothelial cells expressing major histocompatibility complex class II proteins (317; 259), similar to the lesions of experimental allergic encephalomyelitis. Very early, the margin may be indistinct (259). Sensitive magnetization transfer ratio MRI scans can detect abnormalities weeks prior to Gd+ enhancement. The cellular infiltration is minimal in some acute plaques, suggesting a direct insult to oligodendrocytes that is similar to the Lucchinetti type III lesions described below (26). These authors argue that in some early cases, oligodendroglial damage precedes immune infiltration; then macrophages arrive, followed by T cells, suggesting that multiple sclerosis is a primary degenerative disorder (debated). In another series studying very early plaques, there were clusters of activated microglia, a few CD8 > CD4 cells, activated complement but not on myelin, and mild to moderate demyelination, all near Virchow-Robin spaces filled with CD4 and B cells (113).

The Virchow-Robin spaces are dilated in multiple sclerosis. These extravascular spaces drain into the CNS glymphatic system, then to leptomeninges and cervical lymph nodes where the antigens may provoke anti-brain immunity. In addition, dendritic cells from inflammatory CNS sites also migrate and aggregate with lymphatic endothelial cells in the olfactory cribriform plate where the immune cells secrete immunosuppressive cytokines.

Acute plaques typically spread out from the post-capillary venules. Clonally expanded (“oligoclonal”) CD8 cells begin to outnumber CD4 cells at the plaque margins (40; 14). These margins are often abrupt, suggesting a battle between the spread of inflammation and endogenous resistance to the intruders. “Immune privilege” in the brain may contribute to the immune cloaking of brain cells. Neurons express immune-inhibitory transforming growth factor-beta. Neuron-T cell contact converts encephalitogenic CD4 T cells to regulatory CD4 T cells that inhibit experimental allergic encephalomyelitis (182). Nonetheless, more-frequent cytolytic CD8 cells can damage neurites, axons, and oligodendroglia.

Plaques are of various ages in multiple sclerosis, unlike the monophasic lesions of postinfectious and postvaccinal encephalomyelitis. Some old multiple sclerosis plaques contain infiltrating macrophages that phagocytose compact myelin and show smoldering demyelination.

Gray matter contains myelinated fibers, and it is affected. There is neuronal and synaptic loss in the cerebral cortex and death in up to 35% of thalamic neurons (57). Although cortical T2 lesions are rare on conventional MRI, demyelination can be extensive. Cortical atrophy correlates with fatigue (frontal cortex), cognitive loss, and physical disability. A subset, myelocortical disease, affects cerebral cortex and spinal cord but largely spares white matter (316). Atrophic deep gray matter (thalamus, putamen, caudate) has reduced blood flow (See CCSVI). Extensive neuronal loss in the hypothalamus is common (136) and may explain disruption of circadian rhythm, alterations in cortisol regulation, sexual function, depression, weight control, and even poor sleep (143). Forty-percent of cortical neurons show synaptic loss after 30 years of disease. Spinal cords are even more affected, with synaptic loss in 60% to 95% and anterior horn cell loss in 50% (244).

Cortical lesions can be contiguous with expanding subcortical lesions (cortical type I), can be confined to small perivascular areas of the cortex (type II), or can extend from pia to cortical layer 3 or 4, usually in chronic, progressive disease (type III) (243). These multiple sclerosis-specific, subpial lesions mainly appear in cerebellum, hippocampus, and deep invaginations--in the cortex of the insula, cingulate, and deep occiput and fronto-and temporal-basal areas (171). Cortical plaques compromise 60% of all brain lesions but are easily missed on histopathology when a long-duration Luxol Fast Blue destaining step is used to detect centrum semiovale white matter demyelination. The type I cortical lesions (later multiple sclerosis) are linked to cognitive loss and white matter loss, perhaps from disruption of U fibers and intracortical communication. Type II cortical lesions (early multiple sclerosis) correlate with physical disability; type III subpial lesions suggest ”outside in” damage under the inflamed meninges due to soluble factors (227).

Cortical lesions are much less inflammatory than white matter lesions. There are 10 to 40 times fewer T cells and six times fewer macrophages and microglia, less complement activation, less blood-brain barrier breakdown, and minimal edema compared to adjacent white matter plaques. This parallels lack of MRI enhancement. Despite fewer invading immune cells, activated microglia begin to ensheathe cortical neurites and apoptotic neurons. Neuronal gray matter breakdown components are higher in CSF from first attacks than in later stages of multiple sclerosis (288), suggesting very early damage of neurons.

Lymphoid germinal center-like B cell clusters in the meninges, over the surface of the brain, are associated with cortical lesions. Supernatant from multiple sclerosis B cell cultures is toxic to oligodendrocytes, and the toxin is not immunoglobulin. Type 1 cortical lesions have less GAP43 protein, suggesting that atrophic cortex has lost neurons (approximately 10% fewer) and glia (36%). Repair is more efficient in cortical than white matter lesions (52). Myelin sheaths reappear in 71% of subpial lesions, astrocytosis is decreased, and there are normal numbers of oligodendrocytes progenitor cells.

Oligodendrocytes are the major target in multiple sclerosis. One oligodendrocyte maintains myelin on 50 axons and contributes to neuron and axon survival. This has an extraordinary metabolic demand. They are easily damaged, yet contain plentiful protective mechanisms and can remyelinate. In cultures of stressed adult, but not fetal, oligodendroglia, the Antel lab finds myelin changes precede cell body death, including degeneration of the inner cytoplasmic tongue and bleb formation (69). The mRNA binding proteins, TDP-43 and PTB, are mislocalized in oligodendrocytes, likely affecting alternative splicing of RNA and altering production of multiple myelin proteins (201). The myelin-axon interaction is impaired in early multiple sclerosis plaques, and later, oligodendrocytes are often apoptotic. This is followed by demyelination, gliosis, and microglial activation. Axonal damage depends on the type of inflammation, size of lesion, and ability to repair. Oligodendrocyte progenitor cells (OPC) are numerous, widely distributed, and can differentiate to remyelinate naked axons, along with surviving mature oligodendrocytes. Possibly related, the myelin sheath is stochastically built by homotypic fusion of myelin membranes at the front of an oligodendrocyte process (306).

Monocytes and macrophages destroy neurons and oligodendroglia; their proportion in lesions increases in later stages of multiple sclerosis. Oligodendrocyte loss correlates with the number of macrophages but not with T cells or plasma cells (186). CD8 T cells, NK cells, and gamma/delta T cells also damage oligodendrocytes through the NKG2D protein and other targets. Gamma/delta T cells in multiple sclerosis CSF secrete slightly more IL-17 than nonmultiple sclerosis cells. CD4 cells do not directly damage oligodendrocytes but do secrete cytokines that activate CD8 cells and macrophages.

Monocytes and microglia activate NOS and also cause lipid peroxidation, tyrosine nitrosylation, and DNA strand breaks (351). Macrophages and microglia release IL-1, which then induces glutamate to cause synaptic hyperexcitability and death of neurons and a fall in neuronal N-acetyl-aspartate (NAA). Glutamate is also toxic to oligodendroglia. Within active lesions next to dystrophic axons, macrophages express high levels of glutaminase involved in glutamate synthesis (340). CSF IL-1beta levels correlate with number of MRI lesions and with poor prognosis. Potentially preventing damage, IFN-beta blocks secretion of anti-inflammatory IL-10 in activated macrophages and possibly in microglia, contrary to its effect in the periphery where it increases T cell IL-10 (97). This suggests therapy, perhaps combination therapy, could be tailored to the inflammatory makeup of the brain in different forms of multiple sclerosis.

Partial remyelination during recovery from relapse is common in early multiple sclerosis but also occurs in progressive disease (241). Repaired fibers are thinly myelinated within acute plaques or at the edge of chronic plaques during and after active myelin breakdown. Remyelination is more extensive in the cortex than in the white matter (03). Moderate remyelination by hyperplastic oligodendroglia sometimes forms a "shadow plaque" with severe demyelination in the center but thin myelin sheaths in the shadowy periphery of the plaque (251).

Importantly, few MRI studies measure the density of demyelination, “shades of gray,” in T1 black holes.

Cytokines and gliotrophic factors secreted by lymphocytes and some macrophages enhance remyelination. Low levels of inflammatory cytokines can trigger protective oligodendroglial genes, such as HIF-1alpha and HSP70; oligodendrocytes also produce growth factors, NGF, IGF-1, and TGF-beta (351). N-acetyl-aspartate (NAA), synthesized in neuronal mitochondria, is reduced in lesions. As the brain recovers, N-acetyl-aspartate in plaques increases in patients with better clinical outcome (225; 56), suggesting that formerly compromised neurons are now healthy.

Oligodendroglia precursor cells (OPC), expressing the anti-apoptotic protein Bcl-2, arise in the plaque or migrate out from the subventricular zone into demyelinated lesions. In white matter plaque subtypes I and II (187), oligo precursors are preserved and remyelinate axons. Macrophage products, plus IL-11, chemokine CXCL2, platelet-derived growth factor (PDGF), and fibroblast growth factor-2 (FGF-2), increase the number of oligodendroglia precursor cells. Clemastine (clinically intolerable) and ketoconazole modify cholesterol synthesis and increase sterols, which stimulate oligodendroglia precursor cell differentiation and remyelination. Opposing repair, fibroblast growth factor enhances oligodendroglia precursor cell recruitment but inhibits differentiation. Myelin basic protein inhibits oligodendroglia precursor cell differentiation; IFN-gamma inhibits remyelination. (See “Recovery from relapses,” above.)

Perineuronal oligodendrocytes are nonmyelinating progeny of oligodendroglia precursor cells with a unique membrane and RNA profile (307). These cortical cells support neurons and protect themselves and neurons from apoptosis. They are unexplored in multiple sclerosis but do respond to oligo-preserving therapies used in multiple sclerosis and amyotrophic lateral sclerosis.

There are few mature oligodendrocytes and surprisingly little remyelination of adjacent bare axons despite the presence of premyelinating oligodendrocyte precursors in chronic plaques (53). Loss of axonal receptivity for remyelination and lack of remyelination could be from multiple factors:

With each CNS insult, and continuously during chronic multiple sclerosis, oligodendrocyte precursors and mature cells die, and remyelination falters. With chronic progressive disease, normal-appearing white matter shows diffuse loss of myelin, infiltration of T cells, and microglial activation (171). Reactive oxygen species, glutamate, proteases, viruses, and immune cell products (nitric oxide, IL-1-beta, and tumor necrosis factor-alpha) damage myelin. IFN-gamma protects mature oligos against oxidative stress but damages immature oligos (19). Loss of trophic support from compromised oligodendroglia and myelin damages axons, even in the absence of inflammation. Nonetheless, some patients remyelinate, trumping the effects of inflammation and age. Some shadow plaques show extensive remyelination, including two elderly patients with long-standing disease (241).

Axons are damaged in multiple sclerosis. During the earliest stages, there are abundant axonal “ovoids,” ends of transected axons ballooning from ongoing anterograde transport (Charcot 1850; 314). There are 10,000 axonal spheroids/mm3 in multiple sclerosis plaques (transected days or years earlier), but only 2/mm3 in healthy controls (314). Early active lesions have 10% to 20% axonal loss. Even in normal-appearing white matter, axons are half as numerous as in control brains. In chronic progressive multiple sclerosis, one half of the axons in many long tracts and the corpus callosum disappear (38). Unexpectedly, there is little correlation between plaque load and axonal loss, suggesting there are different types of repair and inflammation. The atrophy rate on MRI in multiple sclerosis is approximately 1% per year--higher than in healthy controls (0.12%) but less than in Alzheimer disease (3%).

Axonal damage predominates in the optic nerve, corpus callosum, and spinal cord. In the cervical cord, up to 65% of the axons can be lost. In the corpus callosum, transcallosal bands of Wallerian degeneration predict poor prognosis. Axonal loss correlates with clinical disability and with central nervous system atrophy (314). The damage predominates in small-sized axons, in corticospinal axons at all levels, and in sensory axons in the cervical spinal cord (76). Axonal loss can be severe enough to cause elevated CSF neurofilament light chain protein, Wallerian degeneration and low magnetization transfer ratio on MRI, and decreased N-acetyl aspartate (NAA) on MR spectroscopy.

Acute axonal damage is worst in the first year of disease activity. Damage in plaques correlates with the number of CD8 T cells, monocytes, and activated microglia (164). Cytotoxic CD8 cells attach to dendrites and axons and transect them by releasing perforin. Monocytes release nitric oxide and glutamate--also toxic to neurons. Myelin-specific CD8 cells are more frequent in relapsing than in progressive multiple sclerosis. T cells proliferate to GM3 and GQ1b in primary progressive disease. Amyloid precursor protein, reflecting acute damage to neurons days to weeks after the insult, predominates in acute early multiple sclerosis and in edges of chronic active plaques in secondary progressive disease (37). Amyloid precursor protein levels correlate with the presence of monocytes and CD8 cells but not with CD4 cells. Some CD8 cells secrete anti-inflammatory cytokines, but this type of CD8 cell is reduced in patients with high MRI T1 lesion load (157).

Circulating antiganglioside antibodies in progressive multiple sclerosis reflect axonal damage. Antibodies generally bind to axons but not to myelin. They are present in 50% of primary or secondary progressive patients compared to only 3% of relapsing-remitting patients (278). Demyelination and toxic cytokines increase the energy demand of impulse conduction. As inflammation subsides, axonal sodium channels redistribute out from the nodes of Ranvier into demyelinated regions. This greater Na+ influx is accompanied by a greater Ca++ influx (one tenth of the Na+ flux), so compromised axons must contend with sequestration of potentially toxic Ca++ (277). Axonal mitochondria are dysfunctional, also leading to Ca++-mediated axonal degeneration. Low axonal ATP is akin to a state of chronic hypoxia with mitochondrial dysfunction, and high Na+ and Ca++ influx (315). A patient said her brain and immune system were both “tired.”

Altered ion channels affect neuronal function. Ten subtypes of sodium channels control timing and duration of axon potentials. A channelopathy can arise when new types of sodium channels appear at high density in demyelinated axons (337). There is also robust expression of sodium channels on activated microglia and monocytes. Blockade of Na channels with phenytoin decreases inflammation (66), and phenytoin and flecainide inhibit experimental allergic encephalomyelitis. However, abrupt withdrawal of phenytoin or carbamazepine provokes exacerbation of experimental allergic encephalomyelitis, although this is not reported in multiple sclerosis. Lamotrigine, a Na channel blocker directed at immune cells and naked axons, slightly enhanced walking speed in multiple sclerosis but was linked to more brain atrophy (151).

The central nervous system is normally hostile to immune activation (“immune privilege”). Reflecting this brain tumors that produce inhibitory cytokines are more aggressive (341). The blood-brain barrier prevents access of white blood cells and cytokines to the brain because of tight junctions, few lymphocyte adhesion molecules on endothelial cells, less endothelial transcytosis, and potent extrinsic pumps (PgP) and active transporters. Glia secrete transforming growth factor-beta, platelet-derived growth factor, and prostaglandin E that inhibit lymphocyte proliferation (271).

Many mechanisms subvert immune privilege of the brain. Myelin basic protein, tau, neurofilaments, and 14-3-3 proteins in CSF reflect neuronal and glial damage. CNS antigens drain into brain lymphatics (250), exiting not through the nasal cribriform plate but through arachnoid granulations, emptying into dural sinuses and then into the deep cervical lymphatics (261). Brain antigens in cervical lymph nodes promote B cell maturation, provoke strong antibody responses, and educate Th1 cells, but also enhance regulatory Th2 immunity. Immune privilege can be subverted in multiple sclerosis lesions, perhaps by cells educated in the cervical lymph nodes without normal regulatory control. These educated T cells can rapidly return to the CNS.

The Virchow-Robin spaces in the brain contain extracellular matrix proteins, facilitating migration of MHC-expressing macrophages that are well-placed to interact with T and B cells. In chronic plaques, astrocytes hypertrophy and express costimulatory B7-1 and B7-2 molecules, possibly allowing them to present antigens. B cells also act as antigen-presenting cells, co-stimulate T cells, and secrete cytokines (115; 67).

Antibodies are produced within the CNS itself. B cells in the CNS undergo local clonal expansion, activation, “receptor editing,” and hypermutation and so develop an activated memory cell phenotype (218). Immunoglobulin from these few clones forms an oligoclonal band pattern. Editing of surface immunoglobulin in response to antigens improves affinity but ordinarily reduces proclivity to autoimmune disease. B cells recognize their cognate target in its native form; T cells recognize degraded antigen. Nonetheless, a disease-inciting antigen has not been identified in multiple sclerosis.

Germinal center-like areas (resembling those in the spleen) and lymph node medulla-like areas appear in the perivascular spaces of some old multiple sclerosis plaques (250) and in the meninges, largely restricted to secondary progressive multiple sclerosis. Similar structures are seen in affected tissues in rheumatoid arthritis, Sjögren syndrome, Crohn disease, and Hashimoto thyroiditis and in female dogs with granulomatous meningoencephalomyelitis. This organized inflammation appears in the meninges, often near sulci, in 40% to 54% of autopsy cases. Germinal center-like areas in the meninges contain follicular dendritic cells, plasma cells, and proliferating B cells (290). B cells and myeloid cells produce cytokines that drive growth of these tertiary lymphoid tissues. For instance, TNF-alpha induces B cells to proliferate and form germinal center-like areas. Lymphotoxin-alpha, essential in formation of tertiary lymphoid structures, is elevated in multiple sclerosis CSF. These ectopic B cell follicles can be sites of Epstein-Barr virus persistence, possibly driving antibody production detectable in CSF (289). An alternate explanation for these germinal center-like areas is that CNS injury itself or unknown antigens trigger systemic autoimmunity and local B cell activation (09). T cells, which control B cell function, are present in spinal meninges and near activated microglia in the normal-appearing white matter of the spinal cord. The B cell follicle-like areas are associated with subpial microglial activation, demyelination, and cortical atrophy. Axonal density in the cervical cord of patients with progressive multiple sclerosis is 25% less than in controls, and T cells in the meninges correlate with the atrophy (08). Parenchymal and meningeal sites are difficult to reach with some multiple sclerosis therapies.

As the plaque ages, its inflammation and edema partially resolve, and the immune cell composition changes. The relative number of B cells, CD8 cells, and monocytes increases, compared to CD4 cells (40; 173). The margins of chronic inactive plaques have fewer inflammatory cells and CD8 cells are 10 times more numerous than CD4 T cells. There are also myelin-laden macrophages, occasional plasma cells and mast cells, a glial scar, and damaged and demyelinated axons. In late chronic plaques, the minimal inflammation is comparable to other neurologic disease controls. Antiviral CD8 cytolytic cells persist in the CNS long after the virus disappears. The inciting agent may be missing in adult plaques, but its legacy remains.

Mast cells are typically missed by usual histological stains, but they are visible in chronic active lesions with specific stains (137). Mast cells may be a consequence of any kind of chronic inflammation and are not specific to multiple sclerosis. Mast cells could encourage immune cell migration through the blood-brain barrier, activate Th1 cells, and release destructive molecules. Opposing these effects, mast cells could secrete neuroprotective molecules and induce IL-10+ regulatory B cells; histamine also induces Th2 cells and myeloid suppressor cells. On microarrays of mast cells, there is high expression of mRNA for “allergic” molecules, such as prostaglandin D synthase, histamine receptors, immunoglobulin Fc-epsilon receptor, tryptase, and chemokine CCL5, stem cell factor (242; 65).

RNA profiles differ between multiple sclerosis plaques and normal white matter. The signature is of neuroprotection, anti-oxidative stress (inflammatory and anti-inflammatory), and mitochondrial deactivation, with nearby glial and astrocytic activation. Proteomic analysis shows activation of tissue factor and other coagulation molecules. Conclusions are difficult because of heterogeneity of lesion activity, varying damage, a mix of cells, and varied age of subjects.

Brain pathology in multiple sclerosis is not stereotypical. The MRI ranges from small lesions in the white matter to huge plaques that are sometimes mistaken for gliomas. Large solitary demyelinating lesions in the centrum semiovale are often biopsied. These large lesions, even if associated with multiple plaques, sometimes have good prognosis (155). Brain pathology differs between primary progressive and relapsing-remitting multiple sclerosis. In primary progressive disease, spinal cord lesions predominate and cause gradual paraparesis. Less inflammation is reflected by fewer Gd-enhancing lesions in white matter, but brain myelin is pale (“dirty” MRI) in the white matter. Cortical atrophy and demyelinated plaques in deep gyri of the cerebral cortex, insula, cingulate, limbic circuit, and cerebellar cortex are much more severe than in relapsing-remitting multiple sclerosis (172). The damaged area of cortex is 4-fold greater in secondary and primary progressive than in relapsing/remitting multiple sclerosis. N-acetyl aspartate levels are low in the cortical gray matter (285). Subpial germinal center-like areas may contribute to cortical damage. In Alzheimer disease, beta-amyloid deposition increases near sulci, suggesting CSF flow plus deep germinal center-like areas may affect the cortex. Urine myelin basic protein-like material is lower than in secondary progressive multiple sclerosis, suggesting a slower rate of destruction in primary progression. Generation of new oligodendrocytes aids brain plasticity and learning, and oligodendrogenesis is promoted by neuronal activity. Extensive hippocampal demyelination in chronic multiple sclerosis interferes with cognition (116).

Distinct patterns in different brains, but similar within a given brain, appear in biopsies of large lesions and at autopsy (187). Pathological subtypes depend on the degree of inflammation, myelin destruction, and oligodendroglial preservation. In each case, macrophages outnumber T cells by 10-fold and T cells are 10-fold more numerous than B cells.

Four pathological subtypes are described by Lucchinetti, Lassmann, and Bruck.

Patterns I and II are seen in acute, early multiple sclerosis. The intense perivenous immune reaction causes a sharply demarcated area of demyelination and destruction of oligodendroglia, astrocytes, and axons. Oligodendrocytes are preserved and there is significant remyelination (shadow plaques), without (pattern I) or with deposition of activated complement and IgG (pattern II). Many myelin proteins are decreased, but myelin-associated glycoprotein is not lost. Oligodendrocytes die at the expanding plaque edge, but they reappear in the remyelinating plaque center.

Patterns I and II show ring enhancement with a hypointense rim on T2 MRI. They resemble the lesions of experimental allergic encephalomyelitis in which there is an autoimmune attack against myelin. Pattern I resembles destruction of myelin by macrophage products (TNF-alpha and reactive oxygen species). Pattern II is similar to experimental allergic encephalitis induced by myelin oligodendrocyte glycoprotein (MOG), mediated by T cells interacting with anti-MOG antibodies.

Antibody and complement-facilitated pattern II is the most common. Most antibodies in plaques are “nonsense” antibodies to unknown determinants, and their relevance is unknown. They usually do not react with myelin antigens. Some, especially IgM, may stimulate remyelination (however, clinical trials failed). Others are probably pathogenic, ie, antibodies to gangliosides (above) and IgM against myelin and S-nitrosocysteine (from nitric oxide reactants, some directed against myelin-associated glycoprotein on oligodendrocytes) (41). Complement binding to antibodies increases the destruction. Nonetheless, myelin damage in pattern II appears to be macrophage-mediated, and some authors argue that immunoglobulin and complement deposition in multiple sclerosis tissue is a nonspecific feature (25). However, plasma exchange has benefit in pattern II but not in patterns I and III.

Patterns III and IV exhibit oligodendroglial dysfunction, ensuing demyelination, and death. Pattern III consists of an inflammatory infiltrate of macrophages, microglia, and T cells but no immunoglobulin, with ill-defined, nonperivenous areas of demyelination (preservation of oligodendroglia near venules) and only limited remyelination. Concentric rings of demyelination are reminiscent of Balo concentric sclerosis, with “dying back” destruction and apoptosis of oligodendrocytes. In distal periaxonal oligodendrocyte processes, there is a marked and predominant fall in myelin-associated glycoprotein, which is needed for myelin attachment to axons and possibly for remyelination (53). This pattern resembles acute white matter hypoxia and suggests a virus or toxin, such as nitric oxide, that interferes with mitochondrial energy production. On third of multiple sclerosis brains show actual cortical microvascular hypoxia on near-infrared spectroscopy.

Pattern IV consists of an inflammatory perivenous plaque with a sharp border of destruction and apoptotic loss of oligodendroglia with little remyelination. This rare pattern is seen only in primary progressive multiple sclerosis. It may reflect an underlying dysfunction in oligodendroglia (oligo-opathy) (123).

Patterns I, II, and III are seen in acute, relapsing-remitting, and secondary progressive multiple sclerosis. Patterns I, II, and IV are seen in progressive multiple sclerosis.

Active plaques throughout a given brain exhibit a similar histopathological subtype and similar pattern of MRI lesions, suggesting a consistent immune and brain response at the time of biopsy (187). Differences in pathology between patients suggest heterogeneity in disease pathogenesis, in the mechanism and targets of demyelination, and probably in therapeutic responses. Theoretically, agents that modify cellular immunity (eg, interferons) are theoretically best for subtype I. Plasmapheresis or intravenous immunoglobulin might be of benefit in antibody-mediated subgroup II. Growth factors for oligodendroglial progenitors or actual transplants are potential therapies for types III and IV. Nonetheless, there are no obvious subtype-specific responses to any multiple sclerosis therapy.

In a smaller series, there were combinations of different categories in the same brain, such as pattern IV in some plaques, but other plaques had remyelination (ie, patterns I or II) (26). They suggest that oligodendroglial apoptosis may precede inflammation. In contrast, in late “established” multiple sclerosis, all lesions show complement and antibodies associated with macrophages in areas of active demyelination (43), suggesting that heterogeneity disappears over time.

MRI versus histopathological subtypes, clinical symptoms, and therapies. MRI determines extent of brain and cord lesions, presence of new lesions, atrophy, and certain responses to therapy (171). MRI T2 signal is from edema; T1 hypointensity is from axonal loss, myelin loss, edema, and widening of the extracellular space.

Endovascular and perivascular inflammation causes gadolinium-enhancing lesions. Small nodular lesions enhance from the center outward; ring-shaped lesions enhance centripetally over 30 minutes (111). Ring-enhancing lesions are areas of new inflammation, consisting largely of a sharp border of macrophages that secrete TNF-alpha, some T cells, oligodendroglia with DNA fragmentation, and axonal loss. This ring surrounds older lesions and is characterized by protein leakage (blood-brain barrier breakdown), isointense T1, and hyperintense T2 MRI (45). Ring-enhancing lesions correspond to pattern I and II lesions described above. T2 activity persists even after contrast enhancement resolves, reflecting degeneration and repair. Small T2 lesions are disproportionately more damaging than large ones (207). Demyelinated or remyelinating lesions have less inflammation and modest blood-brain barrier breakdown. They are hypointense on T1 (less so with remyelination) and hyperintense on T2 and are variably enhancing (45).

A central vein is visible in the center of 90% of new and old T2 lesions on susceptibility-based MRI, illustrating the attachment then egress of immune cells through endothelial cells of postcapillary venules followed over weeks by tissue remodeling of the vessel wall, enlargement of the lumen, and perivenular collagen-I deposition (01). These lesions are more common in multiple sclerosis than in other inflammatory brain diseases. Smoldering areas of inflammation and iron deposition surround the plaques and form a paramagnetic ring on susceptibility-based MRI. This rim is due to iron-laden macrophages/microglia, still active after the acute lesion subsides. At least one paramagnetic rim lesion appears in 52% of multiple sclerosis cases but only in 7% of other inflammatory brain diseases (191). These lesions are detectable during treatment, and they correlate with worse and accelerated disability. They likely reflect a different form of immunity, coinciding with reduced repair capability (105).

Although MRI is a dramatic way to demonstrate CNS lesions to patients, there are caveats for using MRI as a biological marker for multiple sclerosis. The T2 edema signal alone can’t differentiate between demyelinated and partially myelinated lesions. Two of nine T2 MRI lesions show no demyelination on postmortem analysis (24). Lesions in many parts of the brain are clinically silent. Correlation between T2 lesions and clinical symptoms is poor (r = 0.2 to 0.3; less than 6% of the variance) (“clinical-tomographic or clinical-MRI dissociation”). In 1354 placebo-treated relapsing-remitting patients from 45 clinical trials and natural history databases, T2 total lesion load did not predict change in disability from baseline to trials’ end (70). There was a small predictive effect of total lesion load on disability in secondary progressive multiple sclerosis (r = 0.21). Gd+ lesions did not predict clinical relapses. In contrast, T1 black holes measure lost axons, and correlate well with spinal cord atrophy and with clinical deterioration in secondary progressive multiple sclerosis (r = 0.8) (23), but not in relapsing-remitting multiple sclerosis (r = 0.3) (297). Eighty percent of T1 black holes are demyelinated, but in “T2 only” lesions, merely 20% of plaques are demyelinated (217). Glatiramer acetate and IFN-beta reduce the chance that black holes will become permanent (101; 100), and both therapies prevent black holes ab initio.

Gd+ lesions are most common early in the course of multiple sclerosis and in relapsing compared to primary progressive disease. Multiple new and reactivated Gd+ lesions appear in concert during disease activity. Occasional T2 lesions arise without enhancement, especially in periventricular areas (175). Long-duration Gd+ lesions are most likely to evolve into a hypointense. T1 MRI lesion. Two or more Gd+ lesions strongly predict the development of multiple sclerosis (96%) after an isolated clinical attack (Group CHAMPS 2002). However, Gd-enhancing lesions are only modest predictors of a worse clinical course. Changes in therapy must be made in the context of clinical patterns and not simply based on Gd+ lesions. In a cohort of untreated patients with Gd-positive scans, the number of contrast-enhancing lesions falls at 3 months by 4%, at 6 months by 29%, and at 9 months by 48% (352).

Brain regions differ in Gd enhancement. Cortical gray matter lesions are difficult to see on T2-weighted MRI; immune responses and edema are reduced from less myelin, little water, and 10 times fewer inflammatory cells than in white matter lesions (243). This may explain why plaques enhance in the white matter, but do not extend into the gray matter on T1 MRI, forming an “open ring.” This is highly specific for a demyelinating lesion. It reflects the different ability of gray and white matter to form T2 and FLAIR lesions. Important in MRI diagnosis of multiple sclerosis, subcortical U fibers are seldom bright on T2 or FLAIR MRI, perhaps because of higher iron content. Cortical lesions are rare on T2 MRI, but are sometimes seen with FLAIR (17). Double inversion recovery MRI is more sensitive and detects cortical lesions in over 80% of primary progressive multiple sclerosis brains (49). MRI with an 8 Tesla magnet easily demonstrates gray matter plaques (18). Even with minimal inflammation, cortical neurons are injured, contributing to motor, sensory, and cognitive losses and, possibly, fatigue.

Lesion location sometimes determines clinical symptoms, although patients with “benign multiple sclerosis” and those with severe disability can have similar brain atrophy and N-acetyl aspartate content. Disabled patients often have significant atrophy at the second cervical cord segment (42). Some patients with primary progressive multiple sclerosis exclusively have diffuse MRI abnormalities in brain and cord (“dirty white matter”) (354). This represents ongoing inflammation and significant axonal pathology.

Functional MRI measures blood flow to areas of brain involved in various tasks. Myelin speeds axonal impulses, but myelin is more than simple insulation. Myelin controls timing of information flow and organizes network connections. Abnormal activation of wide areas of primary cortex and supplementary motor cortex appears even in very early multiple sclerosis, indicating disruption of neural networks. The enlarged cortical area on fMRI is presumably less efficient because plaques and demyelinated axons have disrupted normal connections, forcing cortical reorganization or unmasking of less efficient latent pathways.

The rate of brain atrophy is increased up to 10-fold in progressive multiple sclerosis. In early relapsing-remitting multiple sclerosis, gray matter atrophies on MRI at twice the rate of normal controls (312). Atrophy is caused by loss of neurons and axons, with some contribution from damaged oligodendroglia and myelin. Measures are variable and difficult to apply to individual patients. Dehydration for 16 hours reduces brain volume by 0.55% and can confound measurement of atrophy. Studies do not account for possible diuretic effects of interferons. Gd+ lesions often do not predict brain atrophy (282) but are more predictive of future atrophy when they are ring-enhancing with central contrast pallor (177) and when they are present at onset of multiple sclerosis (297).

Gray matter atrophy includes cortex (especially in deep sulci), thalamus, and hippocampus (CA1 and subiculum). Of note, in Alzheimer disease, cortical amyloid deposits are high near deep sulci. Hippocampal volume loss is associated with high cortisol and depression in multiple sclerosis. Deep gray atrophy correlates with slowed cognition. The intercaudate nucleus distance correlates with loss of clinical function (brain atrophy versus disability, r = 0.67; versus cognitive function, r = -0.42). T2 lesions do not predict cord atrophy (31). Cord atrophy correlates best with clinical disability and poor walking. Cigarette smoking correlates with lower brain volume and with faster progression (85). Serum cholesterol increases by 4.4% for each gadolinium-enhancing MRI lesion, demonstrating the role of comorbidities (238). Nonetheless, atrophy is slowed by many multiple sclerosis therapies.

Magnetic resonance spectroscopy detects constituents of neurons and glial cells. N-acetylaspartate is part of an osmoregulatory molecular water pump (27) that reflects neuronal and axonal function but not necessarily neuronal loss. N-acetylaspartate is also present in oligodendroglia. Concentrations are very high in mast cells, which could confuse magnetic resonance spectroscopy readings of presumed neuron and axon integrity. Early in multiple sclerosis, N-acetylaspartate is decreased in “normal-appearing white matter” and thalamic and cortical gray matter (54; 99). Reduced N-acetylaspartate correlates with the number of clinical relapses over the preceding 2 years (240), suggesting that it forecasts prognosis even before T2 lesions and atrophy are visible. Loss of N-acetyl aspartate strongly correlates with fatigue, lateralized cognitive dysfunction, abnormal visual evoked potentials, and disability (38). Low cortical N-acetyl aspartate levels correlate with disability in primary progressive multiple sclerosis (285). Periventricular N-acetylaspartate is lowest in secondary progressive multiple sclerosis (203). Levels continue to fall in untreated patients but rise back toward normal after 6 months of IFN-beta therapy (225), indicating that metabolic disturbances in oligodendroglia, neurons, axons, and perhaps mast cells may be reversible.

MRI using ultra-small particles of iron oxide (USPIO) can trace macrophage activity and also label oligodendrocyte progenitor and hematopoietic stem cells. Benzodiazepine receptor-labeled microglia on PET scans (with PK1195) show lesions that are not detectable on regular MRI (22) and which are excessive in thalami and normal-appearing white matter in secondary progressive disease.

Cytokines, chemokines, autoantibodies, and Th1/Th2/monocyte ratios vary between patients and over time, possibly explaining some of the differences in disease course, pathology, or MRI lesions (132). Correlation of immune function, urine myelin basic protein (342), and MRI subtypes versus clinical responses in drug trials could enhance determination of prognosis or the best drug therapy.

Epidemiology

The prevalence of multiple sclerosis in the United States was estimated at 250,000 to 350,000 30 years ago (07), revised to 400,000 in 2007 to account for population growth, and revised again to 730,000 by the U.S. National Multiple Sclerosis Society in 2019 based on datasets from 2010 (333). The prevalence is now 309 cases per 100,000 people, or 1 in 323. World prevalence was estimated at 1.25 million (75) and revised to 3 million, again rising over the past 5 decades. The incidence was 3.2 per 100,000 cases a year in the United States in the 1990s (142), 4.2 in the U.S. in 2007, and 7.5 in Olmsted County, Minnesota, the nidus of the Mayo Clinic (205). Higher prevalence is, thus, from longer lifespan for patients and is highest in those 55 to 64 years old.

The total number of cases in other locales is clearly increasing, although it appears to have plateaued in some high-prevalence areas (205). In Olmsted County, the prevalence quintupled and the incidence quadrupled in the past 70 years (344). In Canada, the increase is largely in females (231). Over the past 4 decades, the ratio of females to males with multiple sclerosis increased from 2 to 1 to 3.5 to 1. The prevalence of multiple sclerosis has increased in regions of Scotland, Finland, Norway, Lower Saxony, Sardinia, Italy, Sicily, and the French West Indies (166). Allergy, Crohn disease, and type I diabetes show similar geographical distribution and increasing incidence. The increase has been attributed to altered immune regulation as exposure to infectious diseases has diminished (15).

Geographical variation in the prevalence of multiple sclerosis is striking. Multiple sclerosis is rare in equatorial countries and becomes more common with distance from the equator in either hemisphere. Differences in prevalence are partially due to Northern European, especially Scandinavian, ancestry in affected populations, but there is also an environmental influence (233). Incidence is high (greater than 30 in 100,000) in northern Europe from Iceland to Russia, and in Canada, New Zealand, and southern Australia. Incidence is moderate (5 to 29 in 100,000) in the Mediterranean basin, the southern United States, and southern South America. Incidence is low (less than 5 in 100,000) in East Asia, India, Africa, the Caribbean, Central America, Mexico (especially in Indians and mestizos), and northern South America (165; 168; 253). In the United States, early studies showed northern areas had a prevalence of over 100 per 100,000 whereas it was only 20 per 100,000 in southern states. This gradient attenuated over time.

Is the cause of multiple sclerosis genetic or environmental? Migration, ethnic, and twin studies indicate that genes and environment both influence the development of multiple sclerosis. Northern European and Scandinavian ancestry is strongly correlated with multiple sclerosis risk (Pearson product-moment correlation = 0.5). English ancestry is negatively correlated in the United States (-0.5) (233). The prevalence in England is 42 to 80 per 100,000 (165). Israeli Jews have a prevalence of up to 62 per 100,000, but rates are lower in Christians (35 per 100,000), Moslem Arabs (15), Druze (11), and Bedouins (17) (04). Genetically similar immigrants have half the rate of native-born Jews, suggesting an environmental factor. There is a low incidence of multiple sclerosis in Gypsies, Asians, and native Black Africans. Black Americans of African ancestry (often racially mixed) born anywhere in the United States have a relatively high risk compared to native Africans. Decades ago, Blacks had half the rate of whites in the United States (167), but in 2010 in Southern California, the incidence of newly diagnosed multiple sclerosis was 10 per 100,00 person-years in Blacks, 6.9 in whites, 2.9 in Hispanics, and 1.4 in Asians (170). The main effect was from the high rate in Black women. The average age of onset was later, 42 years, suggesting this cohort differs from many others. Blacks are more likely to have optico-spinal symptoms (and possible misdiagnosis of neuromyelitis optica), larger MRI lesion volumes, and faster disease progression than whites. In contrast, people of Japanese ancestry in the United States have low rates of multiple sclerosis (78), but much or all of the association disappears when covariates such as socioeconomic status are excluded (198).

Genetic influences. A genetic component to multiple sclerosis is clear from twin studies. The monozygotic twin concordance rate is 31% (200 times background), the dizygotic rate is 5% after 7.5 years of observation (279), and the sibling risk is 3.5%. First-degree relatives have a 25-fold, and monozygotic twins a 300-fold, increased risk of developing multiple sclerosis compared to the general population (133). Another family member has multiple sclerosis 20% of the time. When both parents are affected, 9% of the children develop multiple sclerosis. In theory, the highest-risk monozygotic twin has an affected parent and a twin sister with multiple sclerosis onset before 21 years of age. Mothers and fathers are equally likely to transmit the disease, with no evidence of a Carter effect--where the parent who is less likely to be affected is more likely to transmit the disease (131). However, a high-risk mother (white) married to a low-risk husband (aboriginal) is more likely to transmit multiple sclerosis to a daughter than a low-risk mother plus a high-risk father, suggesting a strong effect of maternal environment (260). Gender, age at onset, disease course, and severity are more similar than expected among patients in a family (149; 130), but others believe phenotypes are not concordant (87). Children of patients (108), 10% of first-degree relatives, and unaffected twins often have abnormal MRIs (220), but their T cell responses to myelin basic protein are normal (199; 257). Other investigators find excessive responses to myelin antigens in normal family members. The large number of unaffected monozygotic twins (70%) is a strong argument for a significant environmental contribution.

No single Mendelian locus causes multiple sclerosis. However, a limited number of interacting genes might affect susceptibility (279). Linkage to DR2 (HLA-DRB1*1501, possibly with DQB1*0602) is strongest in Northern Europeans. Other HLA-DR subtypes are linked to multiple sclerosis in the Middle East, Turkey, and Sardinia. Western forms of multiple sclerosis are linked to DR2 (HLA-DRB1*1501) in Japan (158) and in Southern Han Chinese. DRB1*0901 is protective against the Western form of multiple sclerosis and is frequent in Orientals (30%) compared to Occidentals (1%) (348).

In Japanese and Chinese patients, with and without HLA DPB1*1501, multiple sclerosis often resembles Devic disease (eye and spinal cord involvement, infrequent CSF oligoclonal bands), and typical Western multiple sclerosis is much less likely. In Caucasians, Caribbeans, and Brazilians, DRB1*03 increases risk for Devic disease.

In Black Americans, African HLA ancestry with DRB1*1503 and DRB1*0405, correlates with disability. In Europeans, DR2 correlates with the presence of oligoclonal bands in the CSF but not with MRI lesions (300). DRB1*15 positive patients have greater gray and white matter damage and more T2 lesions than DRB1*15 negative patients. DR4 is linked to a primary progressive course (149). DR2 and DR4 links suggest two different HLA-linked mechanisms for central nervous system lesions. Resistance genes are HLA-Bw4, DRB5 (less progression and severity), DRB1*01, CDR1*14, B*4402, HLA-A*0201, and HLA-C*05. HLA-B12 has been linked to multiple sclerosis and, as fate would have it, to vitamin B12 deficiency and myelopathy.

Non-HLA genes detected with genome-wide association studies (GWAS) of thousands of patients show subtle links to the onset or course multiple sclerosis. These include T cell receptors, immunoglobulin allotypes, POU2AF1 (transcriptional coactivator that regulates immunoglobulin expression), complement factors (C6, C7, properdin), the IL-2 receptor beta chain, IL-7 receptor alpha chain, intercellular adhesion molecule-1 (K469E), tumor necrosis factor alleles, the CD45 tyrosine phosphatase, CD24 (a heat stable antigen that enhances T cell persistence in the brain), synapsin III, and Tyk2 and 2,5-oligoadenylate synthase (OAS1) in the interferon response pathway, and possibly mitochondrial DNA. Other candidate genes code for myelin basic protein, transketolase, IL-10, chemokines, p53, estrogen and vitamin A receptors, Jagged1 (oligodendrocyte differentiation), and proteolytic enzymes such as calpain. Many of these correlation are highly significant, but biologically weak, with odds ratios of only 1.2.

Single nucleotide polymorphisms (SNPs) linked to multiple sclerosis appear are predominantly immune-related. They include the T cell receptor-related SH2D2A, GM21* immunoglobulin haplotype, IL-1 and IL-2 receptor, IL-7 receptor alpha chain, CD6 (CD4 T cell proliferation), CD24 (costimulatory and antigen presentation), CD58 adhesion molecule, EOMES, LMP2 (proteosome MBP antigen processing), MLANA, THADA, IFN-gamma, IFN-gamma receptor (debated), STAT3, interferon regulatory factor-5 (IRF-5), MxA, OAS1, RAGE, TNFRSF1A (type I TNF receptor), chemokines (CCL3, CCL15, and others). Nonimmune SNPs include brain-derived neurotrophic factor (BDNF), P2X7 (purinergic receptor; small study), tissue plasminogen activator, GPC5, KIF1B (axonal transport of mitochondria and synaptic vesicles), mitochondrial complex I, free radical scavengers (paraoxonase I), anti-glycation (glyoxalase I), and CYP27B1 (vitamin D synthesis and degradation). Vitamin D response elements are present in HLA-DRB1, CD40, CXCR4, and CXCR5 genes.

The IL-7 receptor alpha link is weak. However, altered IL-7Ra function could be important in a subset of patients. IL-7 enhances immunity and affects thymic emigration, and the IL-7 receptor chain is upregulated by steroids, tumor necrosis factor, and type I interferons.

Some genes modify the course of multiple sclerosis but not susceptibility to the disease. These genes affect immune regulation and glial or neuronal vulnerability. Three percent of Europeans have a homozygous deletion of ciliary neurotrophic factor (CNTF), a growth factor for neurons (181). In this group, multiple sclerosis is more severe and onset is earlier (118). Mice lacking CNTF or leukemia inhibitory factor (LIF) have worse experimental allergic encephalomyelitis. ApoE4 may be more common in progressive forms of multiple sclerosis and augers cognitive impairment, a faster rate of disability progression, and more MRI destruction and deep gray matter atrophy, although some studies and a large meta-analysis find no link. APOE helps phagocytes clear toxic cholesterol ingested from damaged myelin. ApoB is linked to new T2 MRI lesions. Chemokine receptor-5 positive monocytes accumulate in multiple sclerosis lesions; CCR5+ T cells correlate with MRI lesions. A mutation of the receptor, CCR5-delta 32, (homozygous in 1% and heterozygous in 13% of Caucasians) protects against HIV infection by preventing attachment to monocytes, and protects against severe rheumatoid arthritis (197) and West Nile virus. This mutation is associated with multiple sclerosis (91) but slows progression (150). Other putative or unconfirmed genetic links to the course of multiple sclerosis include the IL-1beta receptor and IL-1 receptor antagonist, transforming growth factor-beta, immunoglobulin Fc receptors, CD24, CTLA-4, and phenylethanolamine N-methyl transferase (converts norepinephrine to epinephrine). TOB1 is linked to exacerbations, and alphaB-crystallin is linked to progression. Multiple sclerosis is linked to ulcerative colitis, but not to lupus or autoimmune and neurodegenerative diseases. Overall, there is an excessive inflammatory response to multiple antigens, coupled with a decreased rate of virus infections and perhaps less cancer. Strong antipathogen responses without regulatory tempering may predispose to CNS damage. Some of these immune-linked genes arose in Neanderthals, and 8000 years ago, Yamnaya cattle herders, perhaps related to stronger responses to pathogens (80).

Genetic risk scores have little meaning for an individual who is questioning her multiple sclerosis risk, however. Heritability is additive, from many common variants, each with weak effect.

Gene expression is highly dysregulated in untreated multiple sclerosis, in contrast to the subtle variation seen with genome-wide association studies. Expression of 8000 genes is abnormal in blood mononuclear cells of untested patients versus healthy controls (92), replicated in a second cohort of patients (105). These differentially-expressed genes reflect a defect in immune regulation, leading to excessive inflammation, as well as dysregulation in the interferon system and neuroprotective pathways.

Response to putative target antigens. Antibody response is increased against certain viruses, particularly measles and Epstein-Barr virus. Antiviral antibodies are not specific for a single inciting agent, as they vary among plaques and among patients (204).

Excessive antibody responses may be part of the immune dysregulation that characterizes multiple sclerosis, and a nonspecific rise in all titers would make it easier to detect specific antibodies. However, nonspecific activation of B cells through immune dysregulation or exposure to viruses and CNS antigens are potential driving factors.

There is an increase of autoantibodies to 2,’3’ cyclic nucleotide 3’ phosphodiesterase (IgM), alphaB- and alphaA-crystallin (anti-inflammatory), cardiolipin, chlamydia (debated), contactin-2/TAG-1 or contactin/TIP30 of the juxtaparanodal domain (rats), DNA, galactocerebroside, gangliosides (GM1, asilao-GM1, GD1a; plus GM3, which is highest in secondary progressive multiple sclerosis and primary progressive multiple sclerosis), glial fibrillary acidic protein (GFAP; in secondary progressive multiple sclerosis, strong correlation with clinical deficits), glycans, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, linked to fatigue), glycopeptides, heat shock proteins (60 and 90), myelin proteins (CNP, MAG, MBP, MOG, OSP, phosphatidylcholine, and PLP), neurofilament light chains (axons), neutrophil cytoplasmic antigen, NG-2 (AN-2), Nogo (debated), nuclear antigens, proteasomes, transaldolase, thyroid microsomal antigens, smooth muscle, and thyroglobulin (272). Autoantibodies are most detectable during exacerbations and in progressive forms of multiple sclerosis (302). IFN-beta does not induce autoantibodies, but interferon therapy on a background of autoantibodies is more likely to lead to neutralizing antibodies.

Excessive T cell reactions to brain antigens approach the threshold of statistical significance. This might be expected in a chronic inflammatory disease of the central nervous system and does not prove causation. Myelin basic protein-reactive T cells are more common than in controls, especially when high avidity cells are detected with low, physiologically relevant levels of MBP (32). Importantly, more frequent MBP-reactive cells are equivalent between patients and their normal family members (107), maybe reflecting familial HLA-regulated responses.

Cytokine production in both innate and adaptive immunity is hereditary and predicts the type of multiple sclerosis. Th1 responses are strongly linked in families, with 0.8 to 0.9 hereditability. In healthy family members of patients, lipopolysaccharide-stimulated IL-10 is reduced by 12%, and TNF-alpha is increased by 10% compared to multiple sclerosis-free families. Low IL-10 plus high TNF-alpha in a family predicts a 4-fold increased risk of developing multiple sclerosis and an 8-fold increase of relapsing-remitting over primary progressive multiple sclerosis (73).

Environmental influences. The “exposome” strongly affects the risk of developing multiple sclerosis (133). Migrants to a low incidence area have a smaller risk than if they had remained in situ (Ebers and 279). Asians and Latinos maintain their low risk after migration (78; Ebers and 279). People who migrate from a low incidence area to a high incidence area before the age of 15 years have a high risk, but after the age of 15, migration does not affect the risk of developing multiple sclerosis.

The ratio of “Asian” (prominent optic nerve and spinal cord demyelination) to “Western” clinical phenotypes has changed in Japan from 2:1 in patients who were born in the 1920s to 1:4 in patients born in the 1970s. This suggests the form of multiple sclerosis is greatly modified by environmental alterations, likely hygiene and exposure to bacteria and viruses (159). This evolution of the clinical character of multiple sclerosis could alter responses to multiple sclerosis therapies.

Canine distemper virus, related to measles, or other viruses carried by small house pets were once implicated in the development of multiple sclerosis, but the association was likely from recall bias during epidemiologic surveys, and many patients had not been exposed. Nonetheless, anti-measles antibodies in multiple sclerosis CSF might be linked to the measles infection-induced profound short-term immunosuppression and long-term loss of 50% of memory T and B cells (211). Epstein-Barr virus titers are also elevated, but could simply reflect the excess antibody responses in the disease. Army recruits who become positive, however, have a 32-fold increased risk of developing multiple sclerosis (39). In the Faroe Islands, four epidemics of multiple sclerosis appeared after British troops occupied the islands in 1940 through 1944. Multiple sclerosis onset was attributed to a virus carried by the British. The putative virus required prolonged exposure (2 years) in people 11 to 45 years of age, and was theorized to cause multiple sclerosis 5 to 8 years after exposure (168). It is also possible that contact with the multiple sclerosis agent at an early age (0 to 3 years old) is protective, as Faroese born between 1941 and 1945 do not have multiple sclerosis (60).

Multiple sclerosis is not transmitted vertically (breast milk), through transfusions, or conjugally. Doctors, nurses, and spouses of patients do not have an increased incidence of multiple sclerosis. Lack of transmission argues against known viral or retroviral infections in adults.

Environment changes the course of multiple sclerosis. One third of upper respiratory infections will trigger an exacerbation (296; 237; 62) and new MRI lesions during the “at risk” period, especially in early multiple sclerosis (295). Picornaviruses, and perhaps all rhinoviruses, may be the most potent triggers. Nonetheless, virus infections decrease by 20% to 50% in multiple sclerosis (296), especially when the disease becomes rapidly progressive (295). Bacterial infections increase exacerbations by 3-fold (262; 62). Excessive inflammation and altered interferon regulation may protect against infections (94).

Interferon therapy does not reduce virus infection rates, but it prevents virus infections from triggering exacerbations (236). There seems to be no effect of interferon-neutralizing antibodies on infections.

Smokers induce multiple sclerosis in themselves and their children and also enflame ongoing multiple sclerosis. The relative risk from smoking is 1.5, but combined with HLA-DRB*15+ and “HLA-A*02-, this rises to 14-fold more risk (127). Forty one percent of new cases in Sweden with the latter profile are in smokers. Smokers have a 60% increase in exacerbations, double the rate of progression, more conversion to progression, more new MRI lesions, and double the rate of brain atrophy. During the BENEFIT study of IFN-beta-1b after first demyelination, there was no effect of smoking on occurrence of the next attacks, progression, or MRI lesions (222). IFN-beta therapy prevents disease and MRI activity and also enhances brain repair, perhaps obscuring detriments of smoking in this study. Oral tobacco containing nicotine reduces the risk of multiple sclerosis (127). This suggests that lung irritation by smoking, environmental particulate matter, and volatile organics, perhaps through the aryl hydrocarbon receptor (above), activates pulmonary and systemic immunity. Smoking also increases anti-Epstein-Barr virus antibody titers and the formation of neutralizing antibodies to natalizumab and interferon-beta therapies.

Physical activity and body morphology affect the incidence and severity of multiple sclerosis. Obesity in teenagers increases the chance they will develop multiple sclerosis by 50% (129; 128). Obesity paired with HLA-DRB*15+ and “HLA-A*02- increases risk of developing multiple sclerosis to 14-fold. Obesity also lowers serum vitamin D levels. High-fat, high-salt diets and multiple proinflammatory products of adipose tissue affect immune regulation, especially during the critical teen years. Adipocytes and high-fat diets inhibit B cell lymphopoiesis and function and increase infections, but also activate microglia. High cholesterol increases lipid raft mobility in CD8 cell membranes and, thereby, enhances immune activation. In contrast, high-density lipoprotein (HDL) attenuates Th1 and Th17 function and cytokine production, potentially beneficial in multiple sclerosis. High serum omega-3 polyunsaturated fatty acids (PUFA) reduce MRI T2 lesions. Four cups of coffee per day may prevent development of multiple sclerosis.

Triggers of exacerbation include several months postpartum and cranial irradiation (330). Exposure to interferon-gamma, altered peptide ligands, or anti-TNF antibodies causes exacerbations. A relationship with stress and head trauma is unlikely but strongly debated in court. Menopause accelerates progression and disability; perimenopausal estriol supplements temporarily reverse this. Shift-work and lack of sleep, especially in the teen years, increases risk of developing multiple sclerosis by 50%.

Environmental antigens and age shape immune responses. A dirty environment (ie, viral and bacterial exposure) allows development of experimental allergic encephalomyelitis in transgenic mice with V-beta-8.2, V-alpha-2.3, and myelin basic protein-specific T cell antigen receptor genes. However, no lesions develop in identical transgenic mice raised in a clean, specific pathogen-free facility (121). It is very difficult to induce experimental allergic encephalomyelitis in wild mice, perhaps because of broad microbial exposure. In humans, all environments contain pathogens. Type and timing of exposure could affect immunity and tolerance. Parasites and a rich microbiome balance the evolutionary selection of interleukin polymorphisms that are effective against viruses and bacteria. Exposure to infant siblings during the first 6 years of life decreases the incidence of multiple sclerosis by up to 8-fold (246). Regions with a high incidence of multiple sclerosis have a low incidence of hepatitis B and schistosomes. Perhaps reducing pathogen exposure, multiple sclerosis patients have more education and higher socioeconomic status than average. Sanitation may be better, and childhood infections occur later in these patients than in the general population (05). "Clean" environments and late-in-childhood infections seem predispose or trigger an autoimmune or bystander reaction in multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease--the “hygiene hypothesis.”

Dental caries correlate with higher incidence of multiple sclerosis. Periodontal disease bacteria increase the severity of experimental allergic encephalomyelitis and drive Th17 responses and arthritis. Oral bacteria also activate latent HIV-1; effects on endogenous retroviruses have not been studied.

Intestinal microflora create an enormous and diverse ecosystem. Most of their bacterial RNA sequences are from novel, often uncultivated microorganisms. Likely to shape immune responses in multiple sclerosis, the gut bacteria activate immune cells, guiding immune tolerance to food antigens and perhaps self-antigens. Immune privileged sites, such as the brain, however, could be insulated from this mechanism. Complex gut flora strengthen the blood-brain barrier. Importantly, dietary antigens directly shape the gut immune system and can induce peripheral regulatory T cells.

Skin, mouth, airways, and breast milk contain unique flora, as does the vagina, where the microbiota vary with race and change after menopause. Maternal diet during gestation and lactation influence immunity in offspring, a “lard legacy.” Caesarean section prevents contact with vaginal bacteria during delivery, increases the risk of immune-mediated diseases. Compared to bottled milk, breast milk induces robust populations of memory and Th17 T cells compared to bottled milk, and maternal cells transfer immune knowledge to the child, “maternal educational immunity.” Bacteroides fragilis and Lactobacillus spp. in the gut microbiome help maintain blood-brain barrier permeability, increase regulatory B and T cells, reduce Th17 cells, and also control fat storage and obesity, change behavior, and regulate some virus infections.

A Western diet of high sugar, animal fat, and protein is associated with specific bacterial enterotypes. A high-sugar, high-fat diet disrupts bacteria that control immunosuppressive lipid absorption in the intestine. Free fatty acids and lipopolysaccharide stimulate TLR4 and amplify inflammatory bowel disease and possibly other autoimmune conditions; other metabolites can stimulate the aryl hydrocarbon receptor and maintain intestinal immunity. In contrast, dietary fiber induces beneficial bacterial families that break down fiber to produce immunosuppressive acetate and propionate. Some microbiota (eg, Bacteroides fragilis) induce IL-10-secreting regulatory T cells; others induce Th17 or Th1 proinflammatory cells (141). The intestine in multiple sclerosis contains high numbers of Clostridium perfringens which produce epsilon toxin (190). This toxin binds to an endothelial cell protein, allowing breach of the blood-brain barrier and also targets immune cells (264).

Antibiotics can have a prolonged effect on some gut microbiota taxa. For instance, antibiotic treatment of H. pylori prevents peptic ulcers and gastric cancer but increases risk of gastro-esophageal reflux disease (GERD) and esophageal cancer. Women with multiple sclerosis have lower rates of H. pylori infections compared to controls, and H. pylori-negative women have less progression than those who are positive. Antibiotics reduce complexity of the microbiome and shift immunity from a mix of Th17 and T-regulatory cells to one of Th17 predominance in mice. A more extreme effect appears in the intensive care unit; combinations of stress, critical illness, antibiotics, proton pump inhibitors, H2 blockers, opiates, and ischemia will increase virulence of gut flora and allow emergence of pathogenic, ultra-low diversity bacterial communities (06). They induce IL-17 and tumor necrosis factor, cytokines that enhance resistance to some intestinal pathogens but that could also trigger autoimmunity. Antibiotic use actually correlates with increased risk of multiple sclerosis, from 1.08 to 1.83, depending on the type of antibiotic (229). The risk could be related to the pretreatment infections or to a change in microbiota. Multiple sclerosis therapies change the microbiota; the relevance is under exploration.

Some bacteria, “probiotics,” as well as parasitic infestation with helminths induce Th2 responses and reduce the severity of experimental colitis and human ulcerative colitis and Crohn disease. Lactobacillus (in yogurt) increases IFN-alpha and IFN-beta levels. Other probiotics increase Th1 or Treg responses (55). Controlled studies are essential in evaluating effects on multiple sclerosis. Infestation with parasites may prevent multiple sclerosis from developing (104). In patients with relapsing/remitting disease, parasites induce eosinophilia--but also more regulatory macrophages and T and B cells plus secretion of anti-inflammatory IL-4, IL-10, and transforming growth factor-beta, but less pro-inflammatory IFN-gamma and IL-12. Parasites also reduce new MRI lesions, clinical progression, and attack frequency 20-fold (61). Antiparasite treatment reverses the effect and causes clinical and MRI multiple sclerosis activity. A trial of ova from Trichuris suis, the pork whipworm (not the human form, Trichuris trichiura), had no benefit, however (104). Although oral tolerance with myelin basic protein does not affect the course of multiple sclerosis, the richer multiantigenic repertoire of parasites and probiotics could have benefit.

Vitamin D affects the onset and the course of multiple sclerosis (298). Some correlations with serum vitamin D levels could be incomplete because low sun exposure and vitamin D intake are independent predictors of first demyelinating events (185). Sunlight and ultraviolet radiation are strong inducers of IL-10, beta-defensins, and Th2 and Treg responses in skin. Illustrating this immune suppression, sunburn blocks immune responses to vaccination and various antigens. It is unknown whether sun-induced fluctuations in IL-10 and other cytokines affect the course of multiple sclerosis.

Vitamin D is immunosuppressive. It induces cAMP, IL-4, and transforming growth factor-beta, and it inhibits production of IL-2, IL-12, IFN-gamma, and TNF-alpha. It slows proliferation of CD4 T cells, B cells, and plasma cells and inhibits function of Th1 and type I dendritic cells. Vitamin D causes a shift to Th2 cytokine production and enhances interferon-beta signaling, suggesting a synergistic therapeutic effect (96). CD8 cells express 2- to 3-fold more vitamin D receptors than CD4 cells, and receptors increase on activated T cells. Vitamin D inhibits onset (more in females) and relapses of experimental allergic encephalomyelitis via an IL-10 pathway. It also induces regulatory T cells (117). Regulatory T cells migrate from the mother into fetal lymph nodes (216), affecting the fetus (below).

Provitamin 7-dehydrocholesterol is synthesized in the skin. Ultraviolet sunlight converts it to vitamin D3 (cholecalciferol), which is further modified in liver and then in renal mitochondria to active calcitriol (1,25(OH)2D3). Foods high in vitamin D are fatty seafood, liver, egg yolks (D3), and chanterelle and portabella mushrooms, especially if grown with UV light (ergocalciferol, vitamin D2). Vitamin D intake is inadequate in many seemingly healthy people.

Children of mothers with serum vitamin D levels in the lowest quintile are twice as likely to develop multiple sclerosis (221). Mothers with high milk or vitamin D intake are less likely to have children who develop multiple sclerosis. Serum vitamin D levels fluctuate with the seasons, possibly linked to the May/November birth month ratio of 1.43 for development of multiple sclerosis (280). More people with multiple sclerosis are born in the springtime than in the fall, suggesting vitamin D affects the fetus. Total births also increase in spring, paralleling more frequent multiple sclerosis. Even after correcting for birthrates, multiple sclerosis increases in April births (1.24) but decreases in November births (0.84) (276), but others disagree (98). Suggesting a relationship between low vitamin D and less autoimmunity, low serum vitamin D levels in May births are associated with more thymic output of new T cells (RTE; Trecs) (81), which are preferential precursors of peripheral CD4 regulatory T cells.

Seasonal variation in multiple sclerosis activity differs in various locales (120), perhaps related to vitamin D intake, sunlight, or exposure to viruses and parasites. MRI activity peaks in late springtime (13), correlating with low serum vitamin D levels. Relapses are most likely 1 month after the serum vitamin D seasonal trough, and the gap before exacerbations shrinks with distance from the equator (303). Vitamin D enhances interferon-beta signaling and shifts cytokines to Th2 immunity (96). Supplementation enhances the therapeutic effect of interferon-beta (below).

Tasmanian children exposed to large amounts of sunlight, especially in winter, are one third as likely to develop multiple sclerosis later in life (324). Consumption of fatty fish, outdoor work, and rural life prevent development of multiple sclerosis and reduce mortality from multiple sclerosis. Nurses taking vitamin D supplements (greater than or equal to 400 IU daily) are 40% less likely to develop multiple sclerosis (223; 246), though their lifestyle could differ from those without vitamin D supplements. Similar correlations are seen in Norway (147). Subjects with darker buttock skin (a measure of genetic background, presumably not exposed to sunlight) were less likely to develop multiple sclerosis. Whites have higher vitamin D levels than Blacks; levels in whites correlate with resistance to development of multiple sclerosis. In France, the regional multiple sclerosis prevalence matches the amount of cloud cover. Women shrouded for religious reasons develop osteoporosis, especially in Northern European countries; this could increase their risk of multiple sclerosis. In contrast to these data, multiple sclerosis appears to have been much less common during the industrial revolution, when the sun was obscured by industrial smoke. Different pathogen exposure and early mortality may explain this discrepancy.

Vitamin D increases intestinal calcium uptake and promotes bone mineralization. Multiple sclerosis patients can have low vitamin D levels and demineralized bone due to a combination of fear of the sun’s heat, treatment of pain with antiepileptic drugs, immobility, disrupted sympathetic innervation of bone (depression and sympathetic hyperactivity is linked to osteoporosis), and possibly to subnormal interferon responses in untreated multiple sclerosis (because interferon therapy enhances bone formation) (94). Vitamin D3 induces interferon-beta production by osteoclast progenitors, and the interferon inhibits osteoclast formation (283). Even at the onset of multiple sclerosis, bone mineral density and vitamin D levels are low. Surprisingly, vitamin D metabolic pathway genes do not correlate with multiple sclerosis risk.

The effects of melatonin are complex. This indoleamine antioxidant prevents carcinogenesis but is antiapoptotic in noncancer cells. Melatonin induces neuroprotective Nrf (fumarate therapy does too). It also stimulates Th1 pathways (inhibits cAMP and induces IL-1, 2, 6, 12, and 18). Serum melatonin levels are inversely correlated with multiple sclerosis activity, but sleep is disturbed in multiple sclerosis, so correlations may not indicate causation. Melatonin is elevated by IFN-beta therapy.

Other environmental factors that may increase multiple sclerosis activity include in vitro fertilization (LHRH agonists), exposure to wool or sheep, and consumption of smoked sausage or fresh cow milk (the milk protein, butyrophilin, shares antigens with myelin oligodendrocyte glycoprotein). A high socioeconomic status confounds some of these factors (30). Regular smoking doubles the risk of having multiple sclerosis; men are more susceptible than women, and adolescence may be a critical period of susceptibility (below). Salt consumption has been proposed as fomenting Th17 cell responses, amplifying experimental allergic encephalomyelitis, exaggerating the pro-inflammatory effects of aldosterone, and inducing salt-sensing serum glucocorticoid kinase (SGK1) (323). However, there is no epidemiologic evidence for salt induction of autoimmune diseases or multiple sclerosis.

Obesity in adolescents and young adults doubles the risk of developing multiple sclerosis. Leptin and adiponectin, made by adipocytes, are proinflammatory. Leptin (Greek “thin,” small) promotes weight loss, hair growth, wound healing, atherosclerosis, breast cancer, and autoimmunity and induces inflammatory Th17 cells. Fasting mice have lower leptin levels, inducing Treg expansion and less immune infiltration into the CNS. Serum leptin is increased in multiple sclerosis (202). Adiponectin also activates dendritic cells to induce Th1 and Th17 cells. IL-17 feedback inhibits expression of these adipocyte genes and causes weight loss.

A diet low in saturated fats (the Swank diet) or rich in antioxidants (T Wahls diet) or treatment with evening primrose oil (rich in linoleic and gamma-linolenic acids) reduce fatigue and improve quality of life (332) (no placebo group) and may modestly lower the rate of exacerbations (86; 331), but this has been disputed (313). Diet and supplements may add to the benefit of multiple sclerosis therapies.

Alcohol consumption has a strong dose-dependent inverse relationship with the onset of multiple sclerosis (128) and also in multiple autoimmune diseases. It also attenuates the negative effects of cigarette smoking.

Association with autoimmune diseases. Few autoimmune diseases are associated with multiple sclerosis (267; 344). There are scattered reports of multiple sclerosis coexisting with ulcerative colitis or Crohn disease and possibly with myasthenia gravis, type I diabetes, narcolepsy (also HLA-DR2-linked), and thyroid disease. Other associations are lacking or actually decreased in multiple sclerosis. This suggests that the etiology of multiple sclerosis differs from other autoimmune diseases. Therapy with alemtuzumab (anti-CD52, Campath-1H) induces antithyroid antibodies, presumably by altering immune regulation. Importantly, the demyelinating mimic of multiple sclerosis, Devic disease/neuromyelitis optica, is highly associated with autoimmune diseases (10-fold increase). Epidemiological mixing of multiple sclerosis with this variant leads to spurious associations of “multiple sclerosis” with autoimmune diseases such as lupus and Sjögren syndrome.

Some diseases are infrequent in multiple sclerosis. Asthma and allergies are half as common as in the general population. Cancer is reduced by two thirds or three fourths in multiple sclerosis compared to controls (281; 162; 16). During trials of subcutaneous IFN-beta-1a, the reported to expected ratio of cancer was 1:11, with 50% more cases in the placebo groups than in the interferon groups (284; 235). Multiple sclerosis patients have low uric acid levels and rarely develop gout. Uric acid ameliorates experimental allergic encephalomyelitis but not multiple sclerosis.

In multiple sclerosis serum type I, interferon levels are low, immune cell responses to it are subnormal, and interferon therapy is beneficial (143; 95). Likely related, interferon levels affect some disease correlations. There is a strong negative association with Down syndrome, possibly because chromosome 21 codes for type I interferon receptors, increasing the set point of responses to interferon (339). Perhaps related, lupus is rare in multiple sclerosis. Lupus immune cells produce excessive interferon and are hyperresponsive to it; interferon therapy causes lupus exacerbations.