Denise I. Campagnolo, MD, MS, Director Clinical Multiple Sclerosis Research, Barrow Neurological Institute, Phoenix,
Arizona; Timothy L.Vollmer, MD, VanDenburgh Fellow, Director BNI Neuroimmunology Program, Barrow Neurological
Institute, Phoenix, Arizona

Key Words:

Antigens – Any substance that provokes an immune response. Ideally, the antigen is a foreign substance, like a protein made by a bacterium. Sometimes T cells inappropriately react with “self-antigens” ( i.e., substances made by the body). When this happens, an autoimmune disease can develop. MS is thought to be, at least in part, an autoimmune disease.

Antigen Presenting Cells (APCs) – Cells that take up antigens from the body and insert them into their membranes for “presentation” to T cells. If there is a perfect match between proteins on the T cell and proteins on the APC, including the antigen, the T cell will become activated.

Cytokines – Signaling molecules of the immune system. They have many functions and the types of cytokines made by immune cells can determine whether the immune system mounts an inflammatory response. Cytokines can call other immune cells to a lesion site, instruct other cells to release cytokines, and directly damage other tissues and cells.

Enzymes – Molecules that perform actions on other molecules, which can include cutting them up.

Macrophages – Immune cells whose primary job is to clean up cell debris by engulfing and then breaking down the material. Macrophages can also act as APCs.

Microglia – The only immune cell native to the central nervous system (CNS). Microglia are made by cells of the bone marrow, but they enter the immune system during the animal’s development. They are believed to act mostly like macrophages, cleaning up dead cells or other debris in the CNS.

Myelin –A fatty substance that is wrapped around nerve fibers and provides the electrical insulation needed to transmit nerve signals quickly over long distances. Myelin is damaged in MS lesions.

Oligodendrocytes – The myelin-making cells of the CNS.

T cells – Infection-fighting immune cells. Once activated, they can take on specific roles and characteristics. In MS,T helper 1 (Th1) cells promote inflammation, and T helper 2 (Th2) cells suppress it.

T cell activation – Activation requires a specific set of molecular circumstances. T cells express on their surface a T Cell Receptor (TCR) that is unique to that T cell and, along with the TCR, they express “costimulatory proteins.” APCs express a protein called the Major Histocompatibility Complex (MHC), which provides a required context for any antigen presented to T cells.That is,T cells will not respond to an antigen unless the MHC is the correct match for that T cell. APCs also express “co-stimulatory proteins.” When all the proteins match, that is, when the TCR and the Tcell’s co-stimulatory proteins match the shape of the MHC and antigen and the APCs co-stimulatory proteins, the T cell becomes activated to fight the antigen. If co-stimulatory proteins on the T cell are missing, the T cell may be driven into an inactive state that prevents its response to the antigen.

Introduction
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). At the time of diagnosis, most people with MS have the relapsing-remitting form of the disease (RRMS). This form of MS, which often lasts many years, is characterized by intermittent, sudden loss or impairment of a neurological system. Examples of symptoms experienced during typical relapses are loss of vision known as optic neuritis, or dizziness with a lesion in the deep parts of the brain, called the brain stem. These events, called “attacks,” “relapses,” or “exacerbations,” are caused by a new localized area of active demyelination/inflammation or reactivation of an old one somewhere in the brain or spinal cord. These active lesions disrupt outgoing nerve signals from the brain to the affected part of the body and also incoming signals in the opposite direction. Clinically, a relapse is defined as new or reappearance of a resolved neurological symptom lasting at least 24 to 48 hours, that is accompanied by a change in neurological examination that cannot be explained by infection or other illness. Typically, exacerbations resolve over a period of days to weeks, and, early in the disease, full function is often regained. Some attacks, however, do leave a permanent problem. That is why limiting the numbers of attacks people have with disease-modifying agents, or quieting an attack as soon as possible is considered optimal. In most cases, RRMS eventually changes into secondary progressive multiple sclerosis (SPMS), which is characterized by few or no exacerbations but steadily worsening disabilities. The other feature article in this issue of MSQR discusses some practical aspects of the progressive form of this disease (see page 12).

Although exacerbations can cause severe neurological problems, most are not lifethreatening. Rather, they can be highly disruptive, debilitating, and painful. Depending on the part of the CNS affected, an exacerbation can have negative implications on the patient’s quality of life, including employment. Therefore, the cost of exacerbations is monetary as well as personal, with the financial loss being in medical treatment, patient care, and lost wages. The more quickly patients can recover from exacerbations the better, both medically and financially.

Pseudoexacerbation is a term used to describe episodes of worsening of old symptoms, lasting less than 24 hours. These do not represent new damage and can be triggered by things like stress or heat. They are not treated with steroids, they are managed by removing or lessening the trigger (i.e., cooling off in air conditioning, relaxing after a stressful situation, etc.).

Treatments for Exacerbations
Active MS lesions show inflammation––an immune response. Therefore, therapies for relapses have largely focused on suppressing the immune system. The current treatment of choice for MS exacerbations is methylprednisolone (MP), a synthetic steroid hormone that is related to the hormones released by the adrenal gland as part of the “fight or flight” mechanism. Other steroids such as intravenous dexamethasone can also be used. Steroid hormones have long been known to suppress the immune system, including the activity of immune cells, and have been used with success to hasten patient recovery from MS exacerbations. Before MP came into standard use, patients with MS were often given ACTH (adrenocorticotropic hormone), a substance naturally produced by the brain that stimulates the production of stress-response steroid hormones, like cortisone. ACTH, given as intramuscular injections for 14 days, does shorten the duration of a relapse. It is used very little now, however, because the synthetic hormone MP is just as effective, well tolerated by patients, and can be given for only 3 to 5 days intravenously (IV), or orally in tablet form.

There is currently considerable controversy over whether steroids are best given orally or intravenously. A recent study indicates that oral MP can be equally well absorbed by the body and reach the same concentrations in the blood. It requires taking many pills a day, however, which can be irritating to the stomach. Oral treatment with MP is relatively inexpensive and non-invasive, which makes it attractive. A large-scale study of oral versus IV MP treatment will be needed to determine if oral treatment is as safe and effective as IV treatment (see Morrow et al., 2004 and associated commentaries).

Despite MP’s effectiveness, it has some deleterious side effects (Table 1), particularly with long-term use, and its benefits are shortlived (in the order of weeks). Furthermore, neither the most effective dose of MP for relapses nor the best length of treatment has been established conclusively. Generally, 1,000 mg given by IV each day for three to five days (depending on the severity of the relapse) is sufficient. Another question is timing. Not every relapse requires treatment, but if it is severe enough to require MP, then the sooner the better. A clear timetable has not been established.

Life Cycle of an MS Lesion
To understand how steroids limit the inflammation of MS lesions, it is helpful to know more about how inflammatory lesions occur and what kind of damage is caused. Inflammation can only occur in the brain or spinal cord when a number of protective mechanisms have failed.

First, immune cells cannot attack the CNS without migrating into it from elsewhere in the body. They gain access to the CNS by way of blood vessels. Normally, immune cells migrate into tissues from the bloodstream by attaching to the blood vessel walls through “adhesion” proteins. Adhesion proteins expressed on the immune cell bind to their counterparts on the blood vessel wall, permitting the cells to cross into the tissue on the other side. Unlike other tissues, the CNS is protected from the rest of the body, including the immune system, by specialized blood-vessel walls called the “blood-brain barrier” (BBB) that prevents unwanted cells and many molecules from crossing into the brain. The cells of the BBB vessel wall, unlike the equivalent cells elsewhere in the body, form tight attachments to one another, blocking unwanted substances from squeezing between them. In addition, they do not make the adhesion molecules that immune cells need to attach to and cross the blood vessel wall. Finally, on the other side of the BBB is another barrier, called the basement membrane. This is a matrix of proteins that traps cells and molecules that manage to cross the vessel wall. For an MS lesion to form, this security system must be circumvented.

It takes a specially-equipped burglar to avoid these high-tech security devices, and the cellular equivalent is the activated T cell (see keywords, pg. 6). These cells can release a variety of chemicals that alter their environment. When activated T cells encounter the vessel wall of the BBB, they secrete chemicals called cytokines that instruct the BBB cells to make adhesion molecules, thus allowing the T cells to cross the blood vessel wall. Once across the BBB, the activated T cells are faced with the basement membrane. For this, they make an enzyme called a gelatinase, which chews through the basement membrane proteins. Gelatinases, in turn, can cause other cytokines to be released, including one called Tumor Necrosis Factor alpha (TNF-alpha), which makes the blood vessel wall leaky and activates other immune cells. Other cytokines act as chemical lures, attracting large numbers of immune cells to the lesion site.

The major target of immune cells in an MS lesion is myelin, a substance that ensheathes nerve fibers and supports the transmission of nerve signals. Once the BBB is damaged, blood cells can readily cross into the CNS, and large numbers of immune cells invade the lesioned area, attacking the myelin. The chemicals released by the immune cells add to the tissue damage. In the melee of the immune reaction, oligodendrocytes, the cells that make myelin, are attacked and killed, myelin is destroyed, remyelination fails, there is local swelling of the CNS tissue, and nerve fibers are severed. Inflamed lesions like this are visible as a bright area on magnetic resonance imaging (MRIs) that are taken after the patient is injected with an enhancing agent called gadolinium. These active lesions are called “Gad enhancing lesions.”

Eventually, as the immune system destroys the proteins that triggered the inflammation, the immune attack subsides, the attacking cells die (as they are programmed to do at the end of an attack), the breach in the BBB is repaired, and the CNS tries to fix whatever damage it can––mainly through remyelination. Once the inflammation resolves, lesions no longer appear bright on gadolinium-enhanced MRI images. Their scars will continue to appear as white spots on the T2 or FLAIR (Fluid Attenuation Inversion Recovery) images without gadolinium enhancement.

How Steroids Work in MS
Steroid hormones are used to treat many different kinds of immune reactions, from poison ivy rashes to MS exacerbations. One reason these drugs are so effective for a wide-range of conditions is that they have multi-pronged effects, affecting the immune response at many different points (Sloka and Stefanelli, 2005). For example, MP reduces the activation of the T cells that enter the CNS in the first place. It does this in part by altering the co-stimulatory molecules expressed on Antigen Presenting Cells (APCs) and T cells (see sidebar), thus biasing the immune system against T cell activation.

MP can also intervene in lesion formation by reducing the ability of T cells to cross the BBB wall. Apparently, MP lowers the amounts of adhesion proteins expressed by activated T-cells and the cells of the BBB wall, making T cell entry more difficult. This reduction in adhesion molecules may be involved in the ability of MP to reduce the formation of new lesions.

MP also helps repair the BBB after it has been breached. Studies have shown that lesions that look bright on gadolinium-enhanced MRI images before treatment of an exacerbation, quickly become dim or completely dark with MP treatment, indicating that the damage to the BBB has been repaired. In some cases, when scientistscould monitor inflammatory lesions in the parts of the brain that corresponded with a patient’s neurological symptoms, the dimming of the lesion on MRIs was accompanied by improved neurological function. This suggested that MP was indeed suppressing lesion activity, and that the suppression was reflected in the repair of the BBB.

A hint about how MP might affect the BBB comes from a recent paper that examined the kinds of immune cells and immune-related chemicals that were in the blood of patients with MS during a relapse, before and after 7 days of treatment with intravenous MP (Mirowska-Guzel et al., 2006). One of the chemicals studied was a cytokine called interleukin-8 (IL-8), which tends to be elevated in the blood of people with MS. Because IL-8 is known to attract other immune cells to sites of inflammation and to damage tissues directly, it might contribute to the breakdown of the BBB in MS. The authors of the IL-8 study reported that MP treatment significantly lowered the level of IL-8 in the blood of the patients with MS. Therefore, MP may work in part by preventing the damage caused by high levels of IL-8.

Once immune cells have invaded the CNS, the kinds of cytokines (or chemical messengers) released by the cells can determine the response of the immune system. In MS lesions, the T cells that attack myelin proteins are typically of the T helper 1 (Th1) type, which drive an inflammatory immune response. One of the most damaging of the cytokines is TNF-alpha, which is secreted by the microglia, the native immune cells of the CNS, and by macrophages, which clean up cell debris. TNF-alpha damages myelin directly and can kill oligodendrocytes. The resulting cell debris attracts more macrophages to the lesion. TNF-alpha also instructs other immune cells, including T cells, to release inflammatory cytokines. When this happens, a feed-forward cycle can ensue, in which inflammatory cytokines instruct the increasing numbers of cells invading the lesion to release even more inflammatory cytokines, thereby attracting even more cells.

MP treatment causes cells in the blood of patients with MS to make lower levels of TNFalpha, which argues that MP probably also reduces TNF-alpha levels in MS lesions. Similar evidence suggests that MP reduces the levels of other inflammatory cytokines. MP treatment also results in lower blood levels of proteins that are associated with oligodendrocyte death. Together, this evidence suggests that MPprobably intervenes directly in the manufacture of chemicals that sustain the immune response. Furthermore, MP promotes the production and release of cytokines that are anti-inflammatory, which probably further damp down the inflammation in the lesion.

Nitric oxide (NO) is another destructive, but non-cytokine, molecule whose level in MS lesions is increased by TNF-alpha and another pro-inflammatory cytokine, IFN-gamma. NO can instruct microglia to kill oligodendrocytes and it can directly damage myelin. The evidence that MP reduces NO in MS lesions is thin. Since MP does reduce TNF-alpha, however, it is reasonable to believe that it may thereby lower the levels of NO.

As the inflammation winds down, it is natural for the immune cells to die. This is a programmed cell death, called “apoptosis.” This apoptotic cell death helps contain the inflammatory response, and it is triggered when a protein (FasL) instructs the immune cells to die by binding to another protein (Fas) on the immune cell surface. In patients with MS, the amount of Fas-triggered apoptosis is lower than in control subjects. This delayed cell suicide may contribute to the damage caused by inflammation in MS lesions. MP can increase the production of Fas on the immune cells, likely making them more susceptible to suicide command. But, there is contradictory evidence for the role of MP in cell death. Some studies suggest that steroids block Fas-triggered cell death. Others suggest that MP promotes immune cell death, but not through the Fas-FasL mechanism. More research is needed to understand whether MP helps or hinders this process in MS lesions.

Plasmapheresis
One method for reducing the strength of the immune response is to remove antibodies from the blood stream, using a technique called plasmapheresis (also called plasma exchange). Unlike immune cells, antibodies are small proteins that can be relatively easily removed from blood plasma (the liquid part of the blood that does not contain cells). Although there is some evidence that plasmapheresis may improve patients’ recovery from relapses (see Weinshenker et al., 1999), its usefulness is limited by the fact that it requires a dedicated large bore IV that often needs to be placed under local anesthetic by a surgeon or interventional radiologist. In some centers an inpatient hospitalization is required to perform plasmapheresis. Plasmapheresis (aimed at the antibodies) in combination with therapies aimed at the immune cells (such as MP) are very effective at relieving the neurological symptom.

Implications for the Future
As the details of the initiation and natural history of MS lesions become clearer, and as the roles of steroid hormones in resolving inflammation are better understood, it should become possible to design specific therapies that effectively block lesion development and/or inflammation. When successful, such therapies should target known vulnerabilities in the development of MS lesions while avoiding injurious side-effects.

Recommended Reading
Mirowska-Guzel, D., Kurowska, K., Skierski, J., Koronkiewicz, M. Wicha, W., Kruszewska, J., et al. (2006). High dose of intravenously given glucocorticosteroids decrease IL-8 production by monocytes in multiple sclerosis patients treated during relapse. Journal of Neuroimmunology, 176, 134-140.

Morrow, S. A., Stoian, C. A., Dmitrovic, J., Chan, S. C., & Metz, L. M. (2004). The bioavailability of IV methylprednisolone and oral prednisone in multiple sclerosis. Neurology, 63, 1079-1080; Commentary, p. 945.

Sloka, J. S. & Stefanelli, M. (2005). The mechanism of action of methylprednisolone in the treatment of multiple sclerosis. Multiple Sclerosis 11, 425-432.

Weinshenker, B. G., O’Brien, P. C., Petterson, T. M., Noseworthy, J. H., Lucchinetti, C. F., Dodick, D. W., et al. (1999). A randomized trial of plasma exchange in acute central nervous system inflammatory demyelinating disease. Annals of Neurology 46, 878-886.

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