Multiple Sclerosis
A Disease of Miscommunication Between the Immune and Central Nervous Systems?
Monica J. Carson, Crystal S. Anglen, and Corinne Ploix
1. IMMUNE PRIVILEGE 1.1. The Problem
A functional central nervous system (CNS) is essential for mammalian survival; therefore, the CNS must be defended from insults and other pathogens. The molecules (e.g., free radicals, cytokines, proteases) produced in vast quantities by the activated immune system to combat pathogens have the demonstrated potential to disrupt CNS function (1–3). To balance these opposing needs, (sufficient defense of the CNS without loss of CNS function), the CNS and immune system have developed a unique relationship referred to as immune privilege. Disrup- tions in this unique relationship leading to disregulated CNS inflammation are now thought to contribute to the onset and progression of many diverse types of CNS pathology, including CNS autoimmune diseases such as multiple sclerosis (MS), Rasmussen’s encephalitis, and narcolepsy;
neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and stroke; and the secondary neurodegeneration associated with spinal cord injury (3–10).
Currently, the following three issues concerning the pathogenesis of CNS autoimmune and neurodegenerative diseases are under substantial debate:
1. Is the CNS really an immune-privileged site? Specifically, to what extent is the CNS passive in its inter- actions with the immune system, and to what extent is the CNS an active regulator of inflammatory responses?
2. Is all inflammation within the CNS maladaptive, or can CNS inflammation be beneficial for CNS function?
3. Do microglia, the resident macrophages of the brain, perform different functions from macrophages that acutely infiltrate the CNS in response to pathogenic signals?
In this chapter, we explore these three broad issues by focusing on the pathogenesis of a chronic demyelinating CNS inflammatory disease: MS. MS most frequently presents as a relapsing–remitting pathology, suggesting that its pathology may result from offsetting detrimental and protective/regen- erative interactions between the CNS and immune systems (11,12). Further, we examine the CNS mechanisms that contribute to maintaining immune privilege and promoting successful resolution of acute inflammatory responses. We specifically examine the potential for microglia to play key func- tions in MS pathogenesis because of their ability to regulate and be regulated by both the CNS and immune systems and to develop both neuroprotective and neurotoxic effector functions (13).
17
From: Current Clinical Neurology: Inflammatory Disorders of the Nervous System:
Pathogenesis, Immunology, and Clinical Management
Edited by: A. Minagar and J. S. Alexander © Humana Press Inc., Totowa, NJ
1.2. Immune Privilege Defined
More than 60 yr ago, Medawar illustrated in an elegant series of experiments that showed that the CNS was immunologically distinct from other peripheral tissues (14). When Medawar placed allografts (tissue grafts from the same species but expressing a different major histo- compatibility complex [MHC] haplotype) into peripheral tissue sites, the allografts were rapidly destroyed by the immune system. By contrast, allografts placed within the CNS were not immediately rejected and survived for substantially longer periods. Subsequent experiments from numerous groups confirmed the following two key tenets that define the current concept of CNS immune privilege:
1. The threshold for initiating T-cell responses against antigens found primarily within the CNS is much higher than for antigens found primarily outside the CNS (9,15).
2. The types of immune cells recruited to a tissue by inflammatory insults (e.g., lipopolysaccharide, viral infection, mechanical damage to stroma) and the kinetics of recruitment differ when the insult occurs in the CNS as opposed to other peripheral tissue sites. Within the CNS, the kinetics of inflammation are delayed, and there is a tendency for fewer granulocytes to be recruited than outside the CNS (9,16).
1.3. How Are Immune Responses Initiated in Nonimmune-Privileged Sites?
Before we can explore how immune privilege is maintained in the healthy CNS and overcome in MS, we need to define how T-cell responses are initiated outside of the CNS. In nonimmune- privileged sites, T-cell responses to pathology are shaped by the profile and progression of the innate immune response (e.g., macrophages, granulocytes, fibroblasts, stromal cells) (17,18). In brief, tissue macrophages/immature dendritic cells become activated by any of several non- specific danger signals (e.g., tumor necrosis factor [TNF]- α, cell debris) produced by dam- aged and distressed tissue cells and pathogen-associated molecular patterns (PAMPs) (e.g., endotoxin, peptidoglycans, guanine and cytosine-rich oliognucleotides/CpGs) recognized by pattern-recognition receptors (17,18). On activation, tissue macrophages/immature dendritic cells are stimulated to produce cytokines, chemokines, and free radicals, and to capture anti- gen from pathogens and cellular debris. Because T cells do not recognize whole proteins, macrophages/immature dendritic cells must proteolytically process the captured antigen by reducing the protein into antigenic peptides.
After antigen capture, tissue macrophages/tissue dendritic cells migrate to the draining lymph nodes, where they present antigen to T cells and initiate primary induction of T-cell-effector function (17,18). Acting as antigen-presenting cells (APCs), macrophages/dendritic cells activate CD4
+T cells by presenting antigen in the binding cleft of MHC class II and CD8
+T cells by presenting antigen within the cleft of MHC class I. Once activated by antigen presentation, T cells leave the lymph nodes and circulate throughout the body in search of their target antigen. After entering the target tissue, the target antigen must be re-presented to the infiltrating T cell by resident and infiltrat- ing macrophages/dendritic cells. In the absence of APCs, the T cell is blind to the presence of its target antigen, even when it is immersed in its target antigen. Without local antigen presentation, the T cell will leave the tissue to continue searching for its target.
1.4. Is Immune Privilege Equivalent to Immune Isolation?
In the decades immediately following Medawar’s seminal experiments, the immune privilege
of the CNS was assumed to be caused by the immune system’s inability to detect antigens within
the CNS (19). The presence of the blood–brain barrier (BBB) and the absence of a lymphatic sys-
tem that can passively drain CNS antigens into lymph nodes coupled with the absence of a resi-
dent population of APCs capable of presenting antigen or migrating to the lymph nodes were
together presumed to sequester the CNS from the immune system. If the immune system became
aware of CNS antigens by a rupture in the BBB, the subsequent drainage of CNS antigens into the
cervical lymph nodes, and/or the intrusion of APCs into the CNS, the immediate result was pre- sumed to be the initiation of destructive CNS autoimmune inflammation. Conversely, anti-CNS immune responses could be initiated outside the CNS by viruses or other pathogens expressing an antigen target similar in molecular structure to an endogenous CNS antigen (a molecular mimic) (20,21). If a sufficiently strong anti-CNS immune response was initiated outside the CNS, the activated immune cells were presumed to breach the BBB, encounter their CNS antigen targets, and launch destructive CNS autoimmunity. In this paradigm, the CNS is a passive victim of the immune system; thus, the damage to CNS myelin and neurons observed in the histopathology of MS was previously viewed as the inevitable and sequential result of failing to prevent macrophages and T cells from infiltrating the CNS and becoming aware of CNS antigens.
Currently, a very different view of CNS–immune system interactions has begun to evolve that alters our view of the pathogenesis of CNS inflammatory diseases (3,22–24). First, immunocompe- tent T cells, macrophages and dendritic cells do readily cross the BBB and transverse the healthy CNS in the absence of neuropathology and clinical symptoms (5). Second, several classes of viral infection can be cleared from the CNS by an acute self-resolving CNS inflammation without evi- dence of any long-lasting bystander effects and without induction of chronic CNS autoimmunity (25). Third, brain injuries (e.g., stroke, blunt injury, axotomies) do not initiate destructive CNS autoimmunity (3,22–24). Yet, these forms of injury do cause the release of CNS antigens into circu- lation and are associated with infiltration of CNS auto-reactive T cells and MHC-expressing APCs into the sites of CNS injury. Fourth, microglia have demonstrated potential to act as a resident CNS- specific type of APC that serves to limit, redirect, and even terminate proinflammatory T-cell responses (26,27). Finally, new data indicate that in some situations, CNS-autoimmunity may serve to limit neurodegeneration and perhaps promote neuroprotection. In this paradigm, MS histopathology is likely a consequence not only of an inappropriately activated peripheral immune system but also of failures in the active immunosuppressive and neuroprotective mechanisms oper- ating within the CNS.
2. MULTIPLE SCLEROSIS: THE STEREOTYPIC DISEASE OF CNS INFLAMMATION
2.1. What is Multiple Sclerosis?
MS has been actively studied for more than a century, but much about MS, including its cause, remains a mystery. What is known? MS is one of the first chronic CNS neurodegenerative diseases recognized to have an inflammatory component driving pathogenesis and clinical disability (11,12).
Although MS-initiating agents/events remain a subject of speculation, pedigree and epidemiological studies indicate that MS susceptibility has both environmental and genetic components. On the genetic level, the MHC class II locus has been most consistently correlated with MS susceptibility, although many loci appear to have roles in modulating MS risk and pathogenesis. From these obser- vations, inappropriate activation of myelin-specific CD4
+T cells are suspected to have causative roles in MS pathogenesis. Interestingly, epidemiology studies also suggest that encounters with unknown environmental factors around puberty prime individuals to develop MS 10 to 20 yr later. Furthermore, researchers have speculated that MS susceptibility may result from early encounters with viruses or bacteria that prime the immune system to generate destructive antimyelin-T-cell responses at later ages in response to reactivating signals (e.g., pathogens, bacteria, viruses) (11,12,20).
Clinically, onset of MS symptoms most often occurs between 20 and 40 yr of age (11,28).
About 85% of persons experience a relapsing–remitting form of MS in which periods of full or partial symptom remission follow clinical episodes of disability. The frequency, severity, and types of clinical episodes vary widely between individuals. Initial symptoms can include transient bouts of extreme fatigue, vertigo, optic neuritis, weakness, and/or numbness in the extremities.
With time, symptoms become much more severe and debilitating and can include ataxia, paraparesis,
painful limb spasms, and cognitive impairment. After several years, the disease progression tends to convert from an episodic to a progressive neurological deterioration. Approximately 15% of persons experience a progressive deterioration from the initial onset of symptoms.
Because none of these symptoms are unique to MS, the diagnosis of MS is based on the occur- rence of two or more episodes of clinical disability coupled with finding two or more lesions within CNS-white-matter tracts disseminated in time and location (11,28). Furthermore, because a definitive laboratory test for MS does not exist, the diagnosis of MS can be made only if all other potential causes of these clinical signs have been excluded. For example, diseases with viral, bacterial, or even parasitic causes (e.g., acute disseminated encephalomyelitis, sarcoidosis, disseminated Lyme disease), can all initially present with symptoms similar to MS.
2.2. Does Multiple Sclerosis Have Variable Pathogenesis or a Collection of Related Syndromes?
The extreme heterogeneity of disease onset, progression, clinical symptoms, and histopathology has also raised the possibility that MS in not a single disease with a single cause but a collection of symptomatically related syndromes that may have multiple causes. For example, tropic spastic paraparesis (TSP) (also known as human T-cell lymphoma/leukemia virus [HTLV]-associated myelopathy) was originally defined as a form of MS (29); however, once TSP was identified as one of the many clinical consequences of HTLV-1 infection, its unique histopathology was distin- guished from other forms of MS (29,30). With continued epidemiology and research, some current subtypes of MS may eventually be recognized as distinct diseases with causes and pathologies that differ from those of MS.
Likewise, it should be cautioned that the heterogeneous nature of MS in itself is not a conclusive indication that MS is more than one disease. For example, immunization of susceptible rodent strains with myelin antigens in the presence of strong adjuvants triggers a T-cell-dependent demyelinating disease called experimentally induced autoimmune encephalomyelitis (EAE) (31);
however, the clinical symptoms, kinetics, severity, and histopathology of EAE are highly depen- dent on the type of adjuvant and the genetic background of the treated animal (32). For example, the same antigenic peptide from the myelin oligodendrocyte glycoprotein (MOG) (92–106) has been used to immunize SJL/J and A.SW mice, two different mouse strains expressing the same MHC class II haplotype (H-2
s) but with different propensities for generating CD4
+T-cell pheno- types (33). SJL/J mice lack natural killer (NK)1.1 T cells and are more prone to generate T-helper type 1 (Th1)-CD4
+T-cell responses (interleukin [IL]-2 and interferon [IFN; producing T cells]), whereas A.SW mice have a normal complement of NK1.1 T cells and are more prone to generate Th2 CD4
+T-cell responses (IL-4, IL-10 [producing T cells]). Immunizing SJL/J mice with MOG in the presence of complete Freud’s adjuvant (CFA; the oil and water immersion of killed mycobacterium tuberculosis) leads to a relapsing–remitting form of EAE. By contrast, immunizing A.SW mice with the same protocol leads to a primary progressive model of EAE. Addition of per- tussis toxin (PT) treatment to the immunization protocol failed to alter the long-term disease pro- gression observed in the SJL/J mice but did convert clinical disease from a primary progressive to a secondary progressive form in A.SW mice.
Histologically, the pathology induced by the identical causative agents also differed in the
two mouse strains (33). In SJL/J mice, only mild demyelination occurred and was accompa-
nied by T-cell infiltration. In the A.SW mice, large areas of demyelination developed accom-
panied by neutrophil infiltration and minimal T-cell infiltration. In immunized A.SW mice
without PT treatment, immunoglobin (Ig) deposition was detected in the CNS and high titers
of MOG antibodies were detected in the serum. These results raise the question of which
mouse model is the correct model of MS but also highlight that the apparent heterogeneity of
MS could also be largely a function of the polymorphic immunological genotypes of those
persons with this disease.
2.3. Multiple Sclerosis Pathogenesis
Early in the study of MS, the pathogenomonic feature of the disease was the appearance within the CNS of demyelinated lesions often infiltrated with lymphocytes and activated macrophages (11,28).
Although numerous studies have documented the extreme heterogeneity of lesion types, it remains unclear to what extent lesion heterogeneity is caused by the evolution of lesions with disease progression or whether it reflects distinct pathogenic processes.
2.3.1. Classification of Multiple Sclerosis Lesions
Recently, Lucchinetti et al. performed a large-scale analysis of MS lesions from biopsy and autopsy material (34). Although many of their observations confirmed those of previous investi- gators, they made a notable addition. All of the analyzed lesions could be grouped into four dis- tinct patterns, and all lesions from a single individual belonged to only one pattern. Owing to the small amount of tissue taken for biopsy analysis, there has been some discussion of whether the authors would have been able to detect lesion heterogeneity within a single individual. Despite this caveat, the primary implication from their study is that symptomatic MS results from at least four distinct types of pathogenic mechanisms.
Interestingly, the authors note that both type I and II MS lesions shared features with those detected in EAE. In both type I and II MS lesions, myelin loss was predominantly seen in perive- nous locations and the edges of the lesions were sharply demarcated. Areas of active demyelina- tion were associated with lymphocytic and myeloid inflammation. As noted by previous investigators, remyelination and preservation of oligodendrocytes were also readily apparent in these areas of active destruction. The primary difference between patterns I and II was the deposi- tion of Ig G and activated complement in pattern II lesions and the absence of such deposition in pattern I lesions. Of the four described lesion types, type I was the most frequent and was most associated with the relapsing–remitting form of MS.
By contrast, the pathologies of pattern III and IV lesions were noted to be reminiscent of viral- or toxin-induced models of oligodendroctye dystrophy. Most notably, type III lesions, unlike type I and II lesions, were not centered around veins and lesion borders were diffuse and irregular. In addition, oligodendrocyte death was quite prominent in both type III and IV lesions. The apparent method of death differed between the two lesions. In type III lesions, oligodendrocytes displayed nuclear condensation and fragmentation characteristic of apoptotic death, while those in type IV lesions displayed features suggesting necrotic death. Deposition of IgG and activated complement were not detected in either type III or IV lesions, although both lesion types contained lymphocytes and activated myeloid cells.
2.3.2. Axonal Damage Underlies Progressive and Permanent Multiple Sclerosis-Associated Disability
Although generally described as a disease of demyelination, MS is also a disease of axonal damage and neuronal dysfunction (35–39). Indeed, magnetic resonance imaging, magnetic reso- nance spectroscopy, and positron emission tomography examinations of MS patients during dis- ease progression indicate that accumulating permanent clinical dysfunction correlates more closely with axonal damage than with infiltration of activated immune cells. Histological analysis has confirmed that neurons are damaged during the earliest stages of MS. Axonal damage and transection have even been detected in neurons with apparently intact healthy myelin sheaths and in brain regions without overt inflammation.
Within lesions, progressive irreversible axonal damage is likely the consequence of sustained
demyelination and exposure to neurotoxic products produced during the inflammatory process. The
cause of neuronal damage in apparently healthy brain regions distant from inflammation is less clear
(40). It is likely that this latter form of neuronal damage is owing in part to Wallerian degeneration of
neurons damaged within inflamed lesions. Of unknown significance is the microglia clustering and
global expression of microglial-activation markers in cells with an unactivated ramified morphology observed coincident with neuronal damage in otherwise normal-appearing brain regions (40–42).
Are these merely microglial responses to neuronal pathology with little consequence for the progres- sion of disease, or do these microglial responses prime these healthy brain regions for autoimmune attack? Alternatively, as some have suggested, are these activated microglia initially attempting to save stressed neurons, perhaps by the production of growth factors (43,44)?
Ultimately, attempting to interrupt MS pathogenesis requires identifying sites of intervention prior to the accumulation of substantial irreversible axonal damage. Thus, the key question is:
which comes first? Does dysfunction of microglia and/or astrocytes inappropriately recruit lym- phocytes, macrophages and dendritic cells across the BBB? Does microglial activation increase the ability of microglia to present antigen to T cells and promote detrimental T-cell responses? Do CNS-infiltrating T cells and macrophages directly and indirectly activate microglia and astroyctes?
Conversely, does neuronal pathology and/or oligodendrocyte dysfunction lead to inappropriate activation of microglia and astrocytes?
3. AUTOIMMUNITY
3.1. How Is Autoimmunity Initiated?
Before we can address how the immune-privileged status of the CNS is breached in MS, we need to understand how autoimmunity is initiated against nonimmune-privileged targets.
3.1.1. How Is Autoimmunity Initiated Against Nonimmune-Privileged Organs?
Peripheral organs are largely protected from autoimmune attack because T cells with high affinity for antigens expressed by peripheral organs (e.g., pancreas, liver, kidney) are deleted from the T-cell population during development (18,45,46). Despite the removal of these self-reactive T cells, T cells autoreactive for peripheral organ antigens (and CNS antigens) are readily detected in all adult mammalian species. Furthermore, simply transferring high-affinity autoreactive T cells specific for peripheral organ antigens is by itself insufficient to trigger autoimmunity. For example, transfer of T cells specific for the influenza hemagglutinin (HA) into mice expressing the HA antigen in their pancreatic islets (Ins-HA mice) is insufficient to cause pancreatic autoim- mune diabetes (45,46). This is not because the HA-T cells fail to detect and respond to their target antigen. After transfer into Ins-HA mice, the transferred T cells proliferate and a few T cells even accumulate around the islets. These results indicate that induction of destructive proin- flammatory T-cell-effector function requires more than just antigen presentation. For instance, to induce autoimmune diabetes in Ins-HA mice, HA-specific T cells must be primed by home- ostatic or adjuvant signals in addition to antigenic signals (47).
3.1.2. What Are Homeostatic and Adjuvant Signals, and Why Do They Promote Autoimmunity?
The numbers of T cells in circulation are maintained at a species-specific and strain-specific home-
ostatic set point. T cells sense the total numbers of T cells in circulation by competing for survival fac-
tors (CCL21 for CD4
+T cells and IL-15 for CD8
+T cells) (47,48). When T cell numbers are low
(because of partial depletion of T cells by genetic or environmental factors), high concentrations of
these survival factors become available, and T cells begin to proliferate and acquire a semiactivated
phenotype (capable of crossing the BBB) in the absence of antigen presentation (47). Homeostatic
proliferation/activation by itself is insufficient to drive the differentiation of T-cell-effector function
(47,48). However, when homeostatic proliferation is coupled with antigen presentation, all aspects
of T-cell activation are dramatically accelerated and augmented (47). Similarly, strong adjuvants
such as CFA, the oil–water immersion homogenate of killed mycobacterium tuberculosis, or PT
have many effects on T cells, APCs, and stromal cells (17). The net result of these adjuvants is to
bypass and/or amplify the receptor pathways triggered by these homeostatic survival factors, leading
to accelerated and augmented T-cell responses to antigenic signals.
3.1.3. Do Homeostatic and Adjuvant Signals Have a Role in Spontaneous Autoimmunity?
During development, the number of T cells maintained in circulation is primarily determined by thymic production of T cells. With increasing age, thymic output declines, and homeostatic mechanisms begin to significantly contribute to the number of T cells maintained in circulation (49,50). Strikingly, two separate studies have suggested that thymic output of T cells is dramati- cally decreased in patients with either MS or autoimmune rheumatoid arthritis (RA) (49,50). In both studies, the thymic T-cell output of rheumatoid arthritis and MS patients was reduced to that observed in healthy individuals who were 30 yr older. Thus, it is likely that in these MS patients, circulating T cells were subject to strong homeostatic (autoimmune-priming) signals.
Increased homeostatic proliferation is not only a risk factor for lymphopenic (T-cell deficient) individuals but also may be a risk factor in those with normal levels of T cells. Abnormal increases in the production of survival factors would also inappropriately induce homeostatic T- cell proliferation. For example, transgenic overexpression of CCL21 outside the CNS induced homeostatic CD4
+T-cell proliferation in non-lymphopenic mice (47). Without other amplifying factors, CCL21 overexpression was sufficient to prime autoreactive T cells to inititiate destructive autoimmunity against peripheral organs. Suggestively, CCL21 expression is induced in the CNS and pancreas during the preclinical stages of rodent EAE and autoimmune diabetes, respectively (51,52). Taken together, these data strongly imply that homeostatic signals can have a role in priming the immune system for destructive autoimmune responses.
Although MS patients are quite unlikely to have been subjected to CFA or PT treatments prior to the onset or relapse of symptoms, several groups have correlated the impact of systemic infections with the onset and progression of MS. Although this remains an area of substantial debate, one epidemiological study found that the risk of MS relapse was nearly threefold higher following systemic infections, typically upper respiratory tract infections (53). Data derived from transgenic mouse models provide some support for systemic infections acting as nonspe- cific adjuvants able promote CNS inflammation. For example, mice in which nearly the entire circulating T-cell population has been transgenically engineered to be specific for myelin basic protein (MBP) do not develop CNS autoimmunity if maintained in a clean, specific pathogen- free animal colony (54); however, when these transgenic mice were maintained in a dirty colony and presumably became infected with common murine pathogens, the mice developed EAE symptoms and pathology.
3.2. Are Homeostatic and Adjuvant Signals Sufficient to Enable Destructive CNS Autoimmunity?
Our discussion of homeostatic and adjuvant signals has focused primarily on the priming of the immune system—and not the CNS—in autoimmunity. Because homeostatic signals do not alter the CNS, it is unlikely that homeostatic signals by themselves are sufficient to induce CNS autoimmunity.
It is highly likely that the effectiveness of adjuvants at promoting CNS autoimmunity is dependent
on their ability to regulate both the CNS and immune system. At least four types of observations
support this conclusion. First, autoreactive T cells are readily detected in the bloodstream of healthy
people and rodents (55,56). Yet, systemic infection does not automatically result in CNS autoim-
mune destruction. Second, treating mice with CFA alone and not with immunizing antigen leads to a
rapid and robust accumulation of T cells within the CNS (57–59). Yet, these cells are not retained
within the CNS, nor do clinical and/or histopathological signs develop. Third, even when CCL21
and other strong T-cell chemoattractants are transgenically overexpressed within the murine CNS, T
cells fail to accumulate within the CNS, and CNS autoimmune destruction does not occur (60)
(Ploix and Carson, unpublished results). Finally, humans with autoimmune diabetes develop robust
proinflammatory T-cell responses against a variety of antigens expressed in both the pancreas
and the CNS (such as glutamic acid decarboxylase [GAD65], glial fibrillary acidic protein, IA-2
antigen) (61–64). Despite the presence of activated T cells autoreactive for CNS antigens, diabetic
individuals do not have a higher incidence of encephalitis than the population at large.
The existence of a much higher threshold for initiating autoreactive immune responses against the CNS (as compared to other tissues) is reaffirmed by a close comparison of just the immune responses to GAD65 between individuals with autoimmune diabetes and stiff-man’s syndrome, a form of CNS autoimmune-induced ataxia (63). GAD is the biosynthesizing enzyme of the inhibitory neurotransmitter γ-aminobutyric acid and is highly expressed in pancreatic islet cells and a subpopulation of CNS neurons. Analysis of first-degree “at-risk” relatives of patients with autoimmune diabetes reveals that antibody and T-cell responses against GAD65 not only are strong predictors of incipient autoimmune diabetes but also that these responses can be detected years before the onset of clinical autoimmune diabetes. Although 60 to 80% of persons with autoimmune diabetes have strong detectable GAD65 responses, the risk of their developing stiff- man’s syndrome is rare (62). Stiff man’s syndrome’s incidence in persons with autoimmune diabetes is less than 0.001%. Surprisingly, the reverse is not true. Approximately 30% of individ- uals with stiff-man’s syndrome develop a mild form of diabetes in adulthood. Individuals with stiff man’s syndrome also have anti-GAD antibodies with a higher titer and increased epitope recognition as compared with those detected in individuals with autoimmune diabetes (62). These data imply that the initiation of CNS autoimmune disease requires a stronger immune response than that required to initiate autoimmune destruction.
4. CAN THE HEALTHY CNS SUPPORT ANTIGEN-SPECIFIC T-CELL RESPONSES?
The previous discussion demonstrates that chemokine induction, homeostatic signals, and/or peripheral immune responses are sufficient to initiate destructive autoimmunity against peripheral tissues; however, these same signals are by themselves insufficient to initiate CNS autoimmunity.
Consequently, it has been proposed that the CNS lacks an effective resident APC population capa- ble of effectively recruiting, retaining, or activating T-cell responses. From this viewpoint, initia- tion of CNS autoimmunity (and MS) would be dependent on one of two events: the infiltration of sufficient numbers of peripheral APCs (macrophages and dendritic cells) into the CNS or the induction of inappropriate APC function by CNS cells.
4.1. Microglia Are the Resident Tissue Macrophages of the CNS
Of all the cells in the CNS, only microglia are hemopoetically derived and readily able to express sufficient levels of MHC class I, MHC class II, and the costimulatory molecules required to fully activate both CD4
+and CD8
+T cells (13,16,26,65,66). Microglia comprise between 5 and 15%
of the cells in the CNS. Found in all regions of the CNS, microglia are strategically situated to control the onset and progression of autoreactive T-cell responses; however, under nonpathological conditions, microglia in the CNS of healthy rodents and humans are largely MHC class I- and II-negative. In their inactivated state, microglial expression of costimulatory molecules B7.2 and CD40 is low to negligible. Therefore, in the absence of other signals, microglia are incapable of acting as APCs and thus of retaining or locally activating T cells within the CNS.
Microglia do appear poised to transform rapidly into APCs (13,16,26,65,66). In response to a wide array of signals (e.g., age, mechanical injury, bacterial or viral exposure), microglia rapidly express MHC. Exposure to proinflammatory cytokines produced by activated T cells and/or dam- aged cells, such as interferon (IFN)- γ and TNF-α, dramatically increases microglial expression of intercellular adhesion molecule (ICAM)-1, CD40, B7.2, and B7.1. Direct interactions between microglia and T cells can also amplify the ability of microglia to become APCs. The outcome of these interactions is in part dependent on T-cell activation state.
Activated T cells express CD154, the ligand for CD40, a receptor expressed on activated
microglia and other potential APCs. Recently, several studies have demonstrated that CD40-
triggered microglia express higher levels of MHC class II, CD40, B7.2, and B7.1 and begin to pro-
duce IL-12 and IL-23 (67–69). The production of IL-12 and IL-23 in turn increases T-cell production
of IFN- γ. CD40-triggered microglia are also primed to respond more robustly to other environmental cues. For example, CD40 ligation dramatically increases the β-amyloid and IFN-γ- induced production of TNF- α by microglia (67–69). T cells can even selectively cross-regulate microglial expression of costimulatory molecules. For example, in organotypic cultures, Th1 (IFN- γ-, TNF-α producing) T cells were able to selectively increase microglial expression of B7.1, while selectively decreasing expression of B7.2. Th2 (IL-10-, IL-4-producing) cells could only decrease B7.2 without increasing B7.1 expression (70). By contrast, Th2 cells, but not Th1 cells, decreased microglial expression of ICAM-1 and thus decreased the efficiency of antigen-presen- tation by microglia. In summary, as one of their most universal responses to pathology in general and to exposure of activated cytokine-producing T cells in particular, microglia acquire the poten- tial to interact with T cells in an antigen-specific manner. Microglial ability to function as an APC is in turn modified by antigen-activated T cells.
CNS astrocytes have also occasionally been postulated to have a role in antigen-presentation to CD4
+T cells. Although astrocytes can be induced to express MHC class II in vitro, several studies have failed to detect astrocytic expression of MHC class II in either rodent models of demyelination or in biopsy/autopsy CNS tissue from patients with MS (71,72). Taken together, these data suggest that the potential for astrocytes to act as APCs to CD4
+T cells does exist;
however, in all pathologies currently characterized, microglia are likely to be the only CNS resi- dent cell able to act as a potent APC to CD4
+T cells in vivo. By contrast, nearly all cells in the CNS have been detected to express MHC class I in at least some forms of CNS pathology (71,72). Thus, all cells in the CNS have the potential to present antigen to CD8
+T cells and, in turn, to be harmed by the induced cytotoxic functions of activated CD8
+T cells. For the remain- der of this chapter, we focus primarily on antigen-presentation to CD4
+T cells because linkage studies have consistently implicated an MHC class II locus as a MS susceptibility factor, CD4
+T-cell responses are required for antibody mediated responses, and CD4
+T cells have pivotal roles in CD8
+T-cell-induced demyelination.
4.2. Are Microglia or Infiltrating Immune Cells Acting as Antigen-Presenting Cells in Multiple Sclerosis Lesions?
The identities of the myeloid cells within MS lesions are uncertain. Frequently, the characteri- zation of lesion-associated myeloid cells as either activated microglia or CNS-infiltrating macrophages has been based primarily on their morphology. Cells with ramified morphologies have generally been labeled as microglia, and those with amoeboid morphologies are labeled macrophages. The imprecision of morphologically based criteria to determine cell type has been experimentally demonstrated. When fluorescently labeled rodent microglia were placed on cul- tured rodent brain slices, they developed both ramified and amoeboid morphologies that appeared dependent on brain region and type of tissue damage (73). Conversely, ramified morphologies do not necessarily represent microglia. When fluorescently labeled myeloid dendritic cells are injected into the CNS parenchyma, they retain a ramified morphology even after migration into the CNS (74). Similarly, in autoimmune responses outside of the CNS, both macrophages and dendritic cells can display ramified rather than amoeboid morphologies (47).
4.3. Is It Relevant to Distinguish Microglia From Central Nervous System-Infiltrating Macrophages?
At present, there are no definitive markers that distinguish activated microglia from
macrophages in histological sections. Activated microglia express all of the common macrophage
markers, including Fc receptor, CD11b, F4/80, CD14, and CD45 (8,26,75,76). Therefore, from
histological sections alone, neither the identity of the myeloid cells in the MS lesions nor their rel-
ative functions can be determined. However, this overlap in markers and morphology does not
necessarily indicate that microglia and macrophages have identical functions.
For example, several studies have indicated that microglia initially seed the CNS at least as early as embryonic day 15 in rodents. Under most conditions, the parenchymal microglial population is self-renewing, receiving little contribution from the bone marrow (77–79). This is in contrast to what occurs in all other tissues. In nonimmune-privileged tissues, tissue macrophage populations are continually replaced by bone marrow stem cells throughout the life of the individual. The rate and extent that the bone marrow replaces tissue macrophage populations has been determined using irradiation bone marrow chimeric rodents. In these animals, the bone marrow of a recipient animal is killed by lethal irradiation and replaced by donor bone marrow expressing markers such as green fluorescent protein. In peripheral tissues, the replacement half-life ranges from days (liver) to months (skin, heart), varying in a tissue-specific manner. Irradiation bone marrow chimeric mice have also revealed that parenchymal microglia are phenotypically distinct from the macrophage populations located in nonparenchymal CNS sites (77–79). Although often referred to as microglia, macrophages located in the meninges, subarachnoid spaces, choroids plexus, and perivascular regions are replaced within weeks by donor bone marrow and are thus phenotypically distinct from parenchymal microglia.
These phenotypic differences between parenchymal microglia and nonparenchymal macrophages have largely been confirmed in humans, most notably, in bone marrow transplant studies (1,80). On occasion, bone marrow donor cells that are well matched for histocompatibility antigens are mis- matched by gender. For instance, when male donor cells are transplanted into female recipients, donor cells can be identified by in situ hybridization analysis using Y-chromosome-specific probes.
When cerebral spinal fluid (CSF) and brain tissue were examined by these methods after donor- cell reconstitution of the peripheral immune system, parenchymal microglia were found to be of the recipient genotype, whereas cells in nonparencyhmal sites and in the CSF were found to be of the donor genotype.
Flow cytometric analysis and quantitative real-time polymerase chain reaction analysis of rodent material have further revealed that parenchymal microglia differ by their relative expres- sion of CD45 from other tissue macrophages or macrophages that acutely infiltrate the CNS in response to pathogenic signals (65,76,81). In comparison to the other macrophage populations, parenchymal microglia express very low levels of CD45 (also called leukocyte common antigen), a protein tyrosine phosphatase expressed by all nucleated cells of hemopoetic lineage. All other differentiated hemopoetically derived cells in the mature adult animal (lymphocytes, macrophages, granulocytes) express high levels of CD45. In response to chronic pathology or robust in vivo inflammatory signals, the CD45 levels on both microglia and other macrophages increase; however, even after activation, microglial levels of CD45 tend to remain at an interme- diate level between that of unactivated microglia and mature macrophages (65,76,81). Although these fine gradations in expression can be measured in flow cytometric analysis of cell suspen- sions, differentiation of activated microglia and macrophages by differential expression of CD45 in histological sections is less certain.
The characteristically lower level of CD45 expression in microglia as compared to all other dif- ferentiated hemopoetically derived cells is not merely a useful biomarker for differential isolation of microglia. Although the function of CD45 within microglia is as yet ill-defined, in peripheral macrophages, CD45 has been shown to function as both an activating signal and as a Janus kinase tyrosine phosphatase that negatively regulates cytokine-receptor signaling involved in the differen- tiation, proliferation, and antiviral immunity of haematopoietic cells (82). The stable lower expres- sion observed in microglia indicates that the set-point or homeostatic balance of intracellular signaling are fundamentally different in microglia as compared to other macrophage populations.
Using bone marrow chimeric animals and/or flow cytometric measures of CD45, several
recent studies have begun to suggest that microglia and macrophages may be differentially recruited
during CNS pathogenesis. For example, in response to transient middle cerebral artery occlusion
(MCAO), both activated microglia and macrophages were recruited to the infarct site; however,
microglia dominated the early response to the infarct (days 1–4 post-MCAO) (79). Hematogenously derived macrophages were observed only in the CNS in significant numbers at days 4 and 7 post- MCAO, and even then, were the minority of the activated myeloid cells.
Similarly, in response to traumatic spinal cord injury, microglia and hematogenously derived macrophages were each recruited to different portions of the developing lesion, with hematogenous macrophage accumulation following microglial activation (8,83). Hematogenously derived (CNS- infiltrating) macrophages accumulated within the regions with the most severe neurodegeneration and subsequent necrosis. In contrast, activated microglia were localized to the spared white-matter regions. Depletion of peripheral macrophages by C12MDP-liposome treatment dramatically decreased the numbers of hematogenously derived macrophages recruited to the injured spinal cord and dramat- ically decreased the area of spinal cord necrosis. In both the MCAO and spinal cord injury models, the putative cell-type specific morphologies (ramified and amoeboid) and cell-type specific markers (ED1,CD14) were displayed by both microglia and hematogenously derived macrophages.
Both of these studies clearly demonstrate the potential for microglia and macrophages to have sig- nificantly different roles in neurodegenerative disease. Since microglia and hematogenously derived macrophages and dendritic cells are likely to provide different types of targets for therapeutic inter- vention in MS, it is crucial to directly examine their differential ability to act as APCs in vivo.
4.4. Microglia Are a Central Nervous System-Specific Form of Antigen-Presenting Cell
Careful examinations of the potential for microglia to initiate and sustain antigen-specific T-cell responses suggest that their potential is dependent on their prior history and the status of their local environment (see Fig. 1). Microglia isolated from healthy adult rodent CNS were found to be relatively ineffective as compared to splenic or CNS-infiltrating macrophages at promoting CD4
+T-cell proliferation when tested ex vivo, even when MHC expression was induced by IFN- γ treatment (65,76,84). However, if microglia were activated by in vivo pathology (during EAE or by transgenic CNS overexpression of IL-3) to become not only MHC class II-positive but also CD45
intermediate, a slightly different picture was observed (27,81,85). These microglia were still rel- atively poor at stimulating T-cell proliferation but were found to be very potent at promoting T-cell production of proinflammatory Th1 cytokines. Indeed, microglia were found to be much more potent at driving Th1 T-cell-effector function than the other macrophage populations examined in these studies because fewer T cells were producing greater levels of IFN- γ in the absence of antigen- induced T-cell proliferation (27).
4.4.1. Microglial Antigen-Presenting Cell Function Is Regulated by Environmental Signals and Cellular Interactions
In the rodent models just described, microglia limit T-cell proliferation by at least two mecha- nisms: production of nitric oxide (NO) and prostaglandins (27,85). Both NO and prostaglandin production not only directly inhibit T-cell proliferation but also these substances repress the ability of microglia, macrophages, and dendritic cells to present antigen by suppressing their expression of MHC class II and costimulatory molecules (86,87). Therefore, the net effect of microglia antigen- presentation in vivo would be twofold: a strong brief burst of Th1 cytokine production coupled with the inhibition of antigen-presenting function of surrounding microglia, CNS-infiltrating macrophages, and/or dendritic cells. In an acute self-resolving form of CNS inflammation, this response would provide a robust defense against pathogens while simultaneously limiting expo- sure of CNS cells to activated immune cells. Consistent with this hypothesis, inhibition of NO production is sufficient to transform acute self-resolving EAE into a relapsing–remitting form of the disease (86).
Analysis of cultured microglia further illustrates how strongly environmental factors can modify
microglial-effector function. Cultured microglia, even if maintained in the presence of other CNS
cells and in the absence of known stimulatory factors, already display a quasi-activated phenotype (27,81,85). They express intermediate levels of CD45, and dependent on culture conditions, may be weakly positive for costimulatory factors and MHC class I and II. Using these cells, one set of studies has suggested that weakly activated microglia induce T-cell anergy or unresponsiveness, despite low-level expression of costimulatory molecules, CD40 and B7.2 (88). If these microglia were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN- γ, molecules found in abundance in inflammatory infiltrates, these cells rapidly transformed into potent APCs (88). Similar culture conditions also cause microglia to express high levels of the dendritic cell markers, CD11c and Dec-205 (88). Even in the absence of stimulatory factors, a small percentage of cultured microglia spontaneously express the dendritic cell marker, Dec-205 (27). This developmental plasticity is not only a feature of microglia derived from neonatal glial cultures but microglia isolated from adult murine CNS cultured with astrocytes and GM-CSF also begin to express dendritic cell antigens (89).
4.4.2. Central Nervous System Viral Infections Cause Dramatic Changes in Microglial Antigen-Presenting Cell Function
Perhaps of greater pertinence to MS, Theiler’s virus infection of the murine CNS results in a demyelinating encephalomyelitis (TMEV-IDD). TMEV-IDD is characterized by the activation of myelin-specific autoreactive CD4
+T cells and mimics many of the clinical and histopatho- logical features of the chronic progressive form of MS. In TMEV-IDD, microglia and macrophages
Fig. 1. Microglia are a heterogeneous class of resident central nervous system (CNS) macrophages.Capable of developing a broad array of effector functions and summating signals from a broad array of CNS and immune system cells, microglial phenotype can be precisely tailored to address the specific needs of each neuronal population. These same properties predispose microglia to amplify irrelevant or dysfunctional signals and thus inappropriately amplify or promote neurotoxic inflammation.
