Multiple Sclerosis: Immune Cell Access to the Central Nervous System

First Published in R&D Systems' 2006 Catalog

Contents

Multiple sclerosis (MS) is a chronic disease characterized by the destruction of central nervous system (CNS) myelin and often the underlying axons.1 Its pathology has hallmarks of an autoimmune disorder including an inflammatory response accompanied by leukocyte infiltration into the nervous system. This requires an array of physiological changes in the blood vessel endothelia, resident central nervous system cells, and cells of the immune system. For instance, activated blood vessel endothelial cells and immune cells upregulate adhesion molecules that provide the traction necessary for extravasation (or diapedesis). Members of the chemokine family are expressed by numerous cell types and may regulate adhesiveness or act as molecular chemoattractants in MS lesions. In addition, proteases target components of the blood-brain barrier (BBB), providing pathways for leukocyte migration into the CNS parenchyma. Many studies have examined how changes in the levels or activities of molecules implicated in leukocyte trafficking are involved in MS. This mini review will focus on several of the primary mechanisms thought to be involved in leukocyte transmigration by highlighting evidence obtained from MS studies, supplemented with information gleaned from the animal model, experimental autoimmune encephalomyelitis (EAE).

Multiple Sclerosis

MS progression and severity may vary significantly between individuals. The most prevalent form, relapsing and remitting MS (RRMS), is characterized by exacerbations followed by periods of inactivity. RRMS may develop into secondary progressive MS (SPMS), characterized by a lack of remissions. MS may also take on a primary progressive form (PPMS), characterized by a continued progression of the disease from its onset. Approximately 70% of RRMS patients are female.2, 3, 4 The age of onset of RRMS is highly variable (10 to 60 years), but peaks at approximately 28 years, making it the most common CNS disorder of young people.5 Myelin plays a critical role in insulation of nerve fibers and regulates the speed of neurotransmission. Accompanying myelin destruction and associated axonal degradation are among any number of neurological symptoms that vary widely between individuals. These range from minor tingling sensations and numbness to cognitive dysfunction, breathing difficulties, and blindness.

Etiology

The etiology of MS is complex and is largely unknown. It is likely that MS has multiple causal factors that differ between individuals. Both genetics and the environment are likely to play roles. Monozygotic twins exhibit a concordance rate of approximately 25% as compared to 3% with their non-twin siblings.6 The genetic susceptibility appears to be greatest for Histocompatibility Leukocyte Antigen (HLA) class II genes (HLA-DQ, HLA-DR).7, 8 The relatively low concordance rate in identical twins suggests that some environmental influences also exist. Diet, infections, and differences in exposure to sunlight have all been implicated in MS etiology.9, 10, 11, 12 People living in the Western Hemisphere, especially Canada, the Northern United States, and Northern Europe, exhibit an increased susceptibility to MS compared to those living in other geographic regions.12, 13, 14, 15 Furthermore, the susceptibility to MS is higher in people who migrate from low risk geographical areas to high-risk areas, and those who relocate from high-risk areas before the age of 15 years lose their increased susceptibility.13 The prevalence of MS in females also suggests that hormonal influences may play a part.16

Pathology

The predominant view is that MS is an autoimmune disease. However, it has yet to be definitively shown that the immune response is the cause and not a consequence of some as yet unidentified process.17 Indeed, MS exhibits many of the hallmarks of an inflammatory autoimmune disorder including breakdown of the BBB and the recruitment of lymphocytes, microglia, and macrophages to lesion sites. Cytotoxic factors including pro-inflammatory cytokines, proteases, and reactive oxygen species (ROS) accumulate and may contribute to myelin destruction. Although there appears to be marked variability between studies and individuals, several potential autoantigens have been described. Some are associated with CNS myelin and include Myelin Basic Protein (MBP), Proteolipid Protein (PLP), and Myelin Oligodendrocyte Glycoprotein (MOG).18, 19 EAE, an animal model that shares some pathological characteristics with MS, can be induced by these myelin-associated proteins.20, 21, 22 Some other putative autoantigens include aB Crystallin, S100B, and Transaldolase H.23, 24, 25

There is a widely held view that MS is a CD4+ Th1-mediated autoimmune disorder. Several lines of evidence support this notion. For instance, similar to other CD4+ T cell-mediated autoimmune diseases, genetic susceptibility has been tied to HLA class II genes.7, 8 Myelin-specific T cell clones and pro-inflammatory Th1 cytokines including IL-2, IFN-gamma, and TNF-alpha have been described in MS lesions.26, 27, 28 Th1 cells may stimulate macrophages and microglia and enhance their release of pro-inflammatory cytokines, proteases, excitoxic neurotransmitters, and reactive oxygen species (ROS).29, 30, 31 EAE is induced by the transfer of myelin-specific CD4+ T cells to naïve animals, whereas humoral factors and CD8+ cells are relatively ineffective.1, 19, 26, 32 In addition, mice deficient in the Th1-polarizing cytokine, IL-12, are resistant to the development of EAE.33

However, many studies also suggest that this Th1/CD4-centric view might be an oversimplification. For instance, CD8+ cells appear to be the predominant T cell type found in MS lesions, and their cytolytic activities have been implicated in more aggressive forms of the disease.34, 35, 36, 37 In addition, MHC I and not MHC II appears to be expressed on oligodendrocytes and neurons, and MHC I is upregulated on these cell types in more severe cases of MS.38 Some reports also indicate that blocking Th1 cytokines might enhance the severity of the disease and that MS-associated neurodegeneration may even precede inflammatory responses.39, 40, 41 B cells and plasma cells are found in MS lesions, as well as associated co-deposition of immunoglobulin (Ig) and complement molecules.42, 43, 44 Autoantibodies, including those directed against myelin-associated proteins, can be found in MS lesions and cerebrospinal fluid (CSF).42, 45, 46 Regardless of the complexities associated with MS pathogenesis, the infiltration of immune cells and their activities likely contribute to the loss of myelin and axonal degradation that accompanies progression of the disease. Therefore, it is critical to understand the mechanisms involved in the transmigration of leukocytes into regions of the CNS where they are normally excluded.

Blood-Brain Barrier

Figure 1. The selective permeability of the BBB protects the CNS from circulating molecules and cell types that could negatively impact neuronal activity.
View Larger Image 		Figure 1. The selective permeability of the BBB protects the CNS from circulating molecules and cell types that could negatively impact neuronal activity. Vascular endothelial cells are joined by tight junctions. Perivascular cells and basement membrane surround the blood vessel. Astrocytic processes, termed endfeet, help to maintain BBB integrity.

The BBB is a critical line of CNS defense, limiting the access of circulating solutes, macromolecules, and cells that could negatively impact neuronal activity. The BBB is permeable to water, oxygen, and non-ionic molecules including alcohol and certain drugs, while other molecules like glucose and amino acids require transport into the CNS.47 Several anatomical features contribute to the selective permeability of the BBB (Figure 1). CNS blood vessel endothelial cells are joined by intercellular tight junctions that restrict the flow of solutes. They are also ensheathed by a basement membrane consisting primarily of collagens, glycoproteins, proteoglycans, and laminin. Pericytes found within the basement membrane surround the vessels, regulating morphogenesis and possibly BBB permeability.48, 49, 50 Astrocytic structures, termed endfeet, directly interact with the CNS microvasculature, an activity important for maintaining the integrity of the BBB.51, 52, 53, 54 Although leukocytes are not normally found in the CNS, pathological conditions including bacterial and viral infections can result in trafficking into the nervous system. In addition, inflammatory disorders of the nervous system, including MS, may result in significant accumulation of leukocytes. Consequently, many studies have delved into the mechanisms underlying the disruption of the BBB and the infiltration of immune system cells into the CNS.

Adhesion Molecules

One of the first steps in diapedesis involves changes in the expression and activities of adhesion molecules on both the blood vessels and cells of the immune system. Several classes of adhesion molecules play significant roles in mediating leukocyte adhesion and are likely to play important roles in MS leukocyte trafficking. Adhesion molecules that may have prominent roles include those associated with endothelial tight junctions, members of the Selectin family, Cell Adhesion Molecules (CAMs) of the Ig superfamily, and the Integrin family (Figure 2).

Tight Junctions

Tight junctions are comprised of transmembrane adhesion molecules including Cadherins, Junctional Adhesion Molecules (JAMs), Occludin, Claudins, and associated intracellular proteins that mediate interactions with the Actin cytoskeleton.55 The association between tight junction adhesion molecules on adjacent cells and factors that regulate their activities play direct roles in the integrity of the BBB. Although leukocyte transmigration may occur without perceivable changes in tight junctions, other studies suggest that alterations in tight junction integrity might facilitate migration of cells through the BBB.56, 57, 58, 59, 60 Tight junction breakdown has been observed in tissues with active MS lesions, and endothelial cell exposure to serum from relapsing MS patients can downregulate the expression of the junctional proteins Occludin and VE-Cadherin.61, 62 The mechanisms are unclear, although these observations could be due to endothelial cell exposure to inflammatory cytokines such as IFN-gamma and TNF-alpha.63, 64 The effects of inflammatory cytokines on tight junction proteins appear to be blocked by IFN-beta, a molecule with therapeutic effects in MS patients.65

Selectins

The Selectins comprise a family of Ca2+-dependent carbohydrate binding proteins including Endothelial (E)-Selectin, Leukocyte (L)-Selectin, and Platelet (P)-Selectin. E- and P-Selectins are expressed by blood vessel endothelial cells in response to inflammatory stimuli. They are well known for mediating the initial weak interactions and rolling of leukocytes along the endothelia in the direction of blood flow prior to diapedesis.66, 67 Selectins are characterized by N-terminal C-type lectin-binding domains that determine binding to specific carbohydrate moieties such as Sialyl-LewisX (sLeX) and Sialyl Lewisa (sLea) found on core proteins including P-Selectin Glycoprotein Ligand 1 (PSGL-1).66, 68 Other structural features of Selectins include an Epidermal Growth Factor (EGF)-like region, a number of Complement Regulatory Protein-like repeats, a membrane spanning domain, and a short C-terminal cytoplasmic sequence.67

 

Figure 2. Transendothelial migration requires upregulation of several adhesion molecules.
View Larger Image 		Figure 2. Transendothelial migration requires upregulation of several adhesion molecules. Adhesion molecules join to form the tight junctions between CNS endothelial cells. Selectins mediate the initial weak interactions between leukocytes and blood vessels (rolling). Chemokines upregulate the adhesiveness of Integrins, enhancing their interaction with CAMs, and providing the attachment necessary for diapedesis.

Relatively few published reports have addressed the role of Selectins in mediating leukocyte adhesion to MS brain microvessels. Most have centered on soluble (s) forms of L-, P- and E-Selectins in both blood and CSF, many of which are upregulated in MS patients.69, 70, 71 However, differences may exist depending on whether the disease takes on a relapsing remitting form, a progressive form, or is in remission.69, 72, 73, 74, 75 Using magnetic resonance imaging, elevated levels of sL-Selectin correlate with the size of gadolinium-enhancing lesions, and treatment of MS with methylprednisolone is associated with sE-Selectin downregulation.70, 76, 77 CD8+ T cells from MS patients showed increased expression of the Selectin ligand PSGL-1 and in a model system of rolling and adhesion, exhibit increased rolling on P-Selectin.78 CD4+ T cells also have elevated PSGL-1, but in contrast, rolling appears to be primarily dependent upon VCAM-1.78

There is evidence to suggest that Selectins are important for leukocyte infiltration into the inflamed CNS. For instance, lymphocyte transmigration across activated brain endothelia in vitro is inhibited by E-Selectin-neutralizing antibodies.79 In addition, E- and P-Selectin-deficient mice are protected from inflammatory cell infiltration and enhanced BBB permeability in models of inflammatory meningitis.80 Whether Selectins play a role in EAE development remains unclear. Injection of fluorescent E- and P-Selectin antibodies reveals the presence of these Selectins on cerebral vessels during EAE progression in vivo, and P-Selectin has been described in brain and spinal cord homogenates of EAE mice.81, 82 In addition, blocking antibodies to E- and P-Selectin suppress lymphocyte rolling on inflamed EAE vessels.81, 82, 83 However, other immunohistochemical and in situ hybridization studies suggest a lack of P- and E-Selectin on EAE brain endothelium.84 In addition, E-Selectin and P-Selectin neutralizing antibodies apparently have little effect on EAE-induced inflammatory cell infiltration or the progression of EAE.84 The role of the Selectin ligand, PSGL-1, also appears to be a matter of debate. While PSGL-1 is expressed on EAE lymphocytes, and neutralizing antibodies inhibit lymphocyte rolling on activated endothelial cells, PSGL-1-deficient mice still exhibit inflammatory cell infiltration and develop EAE.81, 83, 85, 86

Ig Superfamily Cell Adhesion Molecules

CAMs of the Ig superfamily play key roles in leukocyte transmigration in several contexts, including inflammatory responses associated with MS. They are transmembrane proteins characterized by repeating Ig-like domains, varied numbers of Fibronectin type III repeats, a transmembrane domain, and an intracellular domain that interacts with the cytoskeleton. These molecules have the potential to mediate homophilic or heterophilic cell/cell interactions or associate with the constituents of the extracellular matrix (ECM). They are expressed by a wide variety of cell types and are involved in diverse biological activities ranging from regulating tissue morphogenesis and axon guidance to tumor progression and metastasis. They also play critical roles in the immune response and are upregulated on leukocytes and endothelial cells in response to inflammatory stimuli and contribute to the adhesiveness of transmigrating cells.87 Among the CAMs thought to play important roles in the trafficking of leukocytes through the BBB are Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1).

ICAM-1

ICAM-1, also known as CD54, is best known for mediating leukocyte adhesion via the interaction of its ligand alphaLbeta2/LFA-1, a leukocyte-expressed Integrin. ICAM-1 is upregulated on brain endothelial cells in response to inflammatory cytokines including TNF-alpha, IL-1, and IFN-gamma, as well as the bacterial inflammatory mediator lipopolysaccharide (LPS).88, 89, 90, 91 Several in vitro studies have suggested that ICAM-1 activity is important for the interaction of leukocytes with brain endothelia, and may be enhanced by the accompanied activation of the Gi subtype of G protein-coupled receptors.81, 92, 93, 94 In addition, ICAM-1 expression is increased on the microvascular endothelia in MS and EAE and decreases in response to IFN-beta treatment.95, 96, 97 Although some of the evidence is contradictory, blocking ICAM-1 activity may have protective effects on the development of EAE.98, 99, 100

VCAM-1

Studies have shown that VCAM-1 (CD106) expression increases in response to several cytokines including TNF-alpha, IL-1, IL-4, and IL-13, and it is thought to play an important role in mediating leukocyte interactions with blood vessels.101, 102 It is upregulated on EAE blood vessels, and blocking VCAM-1 activity with neutralizing antibodies suppresses lymphocyte adhesion to the blood vessel endothelia.86, 103 VCAM-1 also appears to be important for MS CD4+ T cell adhesion in a model of leukocyte rolling.78 VCAM-1 is suppressed by methylprednisolone therapy, and inhibition of its Integrin ligand alpha4beta1/VLA-4 may show promise in MS therapy, as discussed in more detail below.104, 105 In contrast, some expression studies question the importance of VCAM-1 in MS-associated leukocyte infiltration. While VCAM-1 expression in MS has been documented in microglia/macrophages, there are conflicting reports for its expression on MS blood vessel endothelia.106, 107

PECAM-1

PECAM-1 (CD31) may play roles in MS-associated leukocyte infiltration into the CNS via direct or indirect mechanisms. It is expressed primarily by platelets, monocytes, T cell subsets, and the blood vessel endothelia.108 Homophilic interactions between PECAM-1-expressing leukocytes and endothelial cells can induce Integrin activation and enhance Integrin-mediated binding of leukocytes to the endothelia. PECAM-1 is also concentrated at the junctions of brain endothelial cells and may play a role in tight junction integrity and/or directly mediate the adhesion of infiltrating leukocytes.108 There are few studies assessing these possibilities. However, PECAM-1 is upregulated on MS monocytes and lymphocytes in patients with active MS lesions.109 In addition, PECAM-1-deficient mice exhibit an early onset of EAE and prolonged endothelial cell permeability.110 PECAM-1-/- endothelial cells support enhanced T cell transmigration in vitro.110 This is independent of PECAM-1 expression on T cells, indicating an importance for homophilic PECAM-1 interactions on the integrity of endothelial cell junctions.110

Integrins

The integrins make up a well-conserved family of cell adhesion proteins. In cooperation with ligands that include members of the CAM family, a multitude of studies indicate Integrin involvement in MS-associated leukocyte infiltration. The Integrin receptor complex is made up of two non-covalently associated subunits termed alpha and beta.111 Each subunit contains a large extracellular domain, a single pass transmembrane sequence, and a short non-catalytic cytoplasmic domain that interacts with the cytoskeleton and other associated intracellular proteins. Despite the lack of catalytic activity, integrins can initiate signal transduction cascades that result from their association with multimolecular focal adhesion complexes and the possible activation of several kinases including Focal Adhesion Kinase (FAK), c-Src, PI 3-Kinase, Mitogen-activated Protein (MAP) Kinase, and others.112, 113 Therefore, in addition to their roles in mediating cell adhesion and migration, integrins may also regulate such activities as cell growth and differentiation. At least 18 alpha and 8 beta subunits exist.113 These combine to form approximately 24 different heterodimer combinations, meaning that the association between subunits is restricted, and not every alpha and beta combination is observed. In most cases, one beta subunit combines with several different alpha subunits to form a subfamily of receptors. Some combinations bind several ligands, while others show greater ligand specificity.114

alphaLbeta2/LFA-1

More study is needed to determine the relative importance of the ICAM-1 ligand alphaLbeta2 (LFA-1) in MS pathology. It is expressed on infiltrating lymphocytes and monocytes in and around MS lesions.95 In addition, peripheral blood mononuclear cells from patients with acute relapsing MS exhibit alphaLbeta2-dependent adhesion to cerebral endothelial cells in vitro.115 Like MS, infiltrating perivascular lymphocytes in EAE express alphaLbeta2.116 However, neutralizing alphaLbeta2 antibodies may either suppress, have no effect, or exacerbate EAE.99, 117, 118

alpha4beta1/VLA-4

Much focus has been devoted to alpha4 integrins and their association with MS and EAE. For instance, MBP-autoreactive T cells capable of inducing EAE also express a disproportionately high level of surface alpha4, and blocking alpha4 integrin inhibits MBP T cell clones from inducing EAE.119 In addition, inhibiting alpha4beta1 with neutralizing antibodies, small molecule inhibitors, and antisense therapy, all delay the onset and/or decrease the severity of EAE.120, 121, 122, 123 This affect may result from a disruption in the interaction between alpha4beta1 and its ligand VCAM-1.120 In addition, long-term blockade of alpha4 in animals with chronic EAE results in significant myelin repair and an improvement in motor function.124

In humans, significant reductions in alpha4beta1 expression are associated with the subset of MS patients responding to IFN-beta therapy.125, 126 Elevated expression of alpha4beta1 on both CD4+ and CD8+ T cells from MS patients is associated with an increased MRI T2 lesion load.127 The alpha4beta1 integrin has also been implicated as a target for MS therapy. This results from an observed 66% decrease in relapses in patients treated with monoclonal antibodies (natalizumab) designed to directly target alpha4 integrins.105 Unfortunately, soon after FDA approval the drug was removed from the market after 3 of 3000 patients developed multifocal leukoencephalopathy (MLE).128 MLE is a CNS infection caused by the reactivation of a latent JC polyomavirus infection, a common virus that typically remains dormant in immunocompetent individuals. Despite the large setback, the clinical trial results underscore the possible association between alpha4 integrins and MS pathology.

Chemokines

The chemokines make up a large family of small molecules best known for their actions as chemotactic cytokines that regulate leukocyte trafficking. In this capacity, they could have significant roles in MS-associated infiltration of leukocytes into the CNS. In addition to their roles in chemoattraction, chemokines have other putative functions including mediating cell activation, growth, and survival. To date, more than 40 chemokines have been identified and are classified into 4 subgroups based on the number and spacing of cysteines found in the N terminus: CC, CXC, CX3C, and C.129, 130 Chemokine activities are mediated via a subset of 7 transmembrane domain G protein-coupled receptors. Each receptor may have several chemokine ligands but almost always within the same subgroup.129

Chemokines play significant roles in inducing the change from low affinity, Selectin-mediated interactions to high affinity associations between integrin-expressing leukocytes and the blood vessel endothelia. They may also regulate the trafficking of leukocytes in the brain parenchyma through their activities as chemoattractants. The majority of the evidence to suggest that chemokines might regulate leukocyte migration in MS lies in the demonstration that they are present in the correct place and time to regulate these activities. Several cells of the immune system including lymphocytes and macrophages, as well as resident CNS cells such as astrocytes, microglia, and blood vessel endothelial cells are known to express multiple chemokines and/or their receptors (Table 1).131, 132, 133, 134, 135, 136, 137 Furthermore, the importance of chemokines is enhanced by studies examining the effects that their manipulation has on EAE progression.

Table 1. Chemokine and chemokine receptor expression in MS.

Source Chemokine References Chemokine Receptor References
Astrocytes CCL2/ MCP-1 132, 134, 138 CCR3 139
CCL3/MIP-1 alpha 134 CCR5 139
CCL5/RANTES 131, 134 CXCR3 140
CCL7/MCP-3 132    
CCL8/MCP-2 132    
CXCL1/GRO alpha 141    
CXCL8/IL-8 141    
CXCL9/MIG 137    
CXCL10/IP-10 137, 141    
B Cells     CCR5 142
    CXCR3 142
Blood CCL2/MCP-1 143, 144    
CCL5/RANTES 145    
CXCL8/ IL-8 143    
CXCL10/IP-10 144, 146, 147    
CX3CL1/Fractalkine 148    
CSF CCL2/MCP-1 143, 144, 145, 147, 149, 150    
CCL3/MIP-1 alpha 151, 152, 153    
CCL4/MIP-1 beta 149    
CCL5/RANTES 145, 153, 154, 155    
CCL17/TARC 150    
CCL19/MIP-3 beta 156    
CCL21/6Ckine 156    
CXCL8/IL-8 146, 149    
CXCL9/MIG 154    
CXCL10/IP-10 144, 147, 150, 153, 154, 155, 157    
CXCL12/SDF-1 158    
CX3CL1/Fractalkine 148    
Dendritic Cells     CCR5 159
    CCR7 160
Endothelial Cells CCL5/RANTES 134 CCR1 161
    CCR2 139
Macrophages/Monocytes CCL2/MCP-1 134 CCR3 139
CCL3/MIP-1 alpha 131, 134, 136 CCR5 139, 154, 161, 162
CCL4/MIP-1 beta 131, 134 CCR8 163
CXCL9/MIG 137 CXCR3 155
CXCL10/IP-10 136, 137    
Microglia CCL3/MIP-1alpha 136 CCR1 161
CCL4/MIP-1 beta 134 CCR2 139
CXCL1/GRO alpha 164 CCR3 139
CXCL10/IP-10 136 CCR5 139, 154, 161
    CCR8 163
    CXCR2 164
Oligodendrocytes CXCL1/GRO alpha 164 CXCR1 141
    CXCR2 141, 164
    CXCR3 141
T Cells CCL5/RANTES 165 CCR1 166
    CCR2 166, 167
    CCR5 136, 139, 154, 155, 166, 168, 169
    CXCR3 136, 137, 154, 162, 166, 168, 169, 170, 171

Chemokines

Several cell types may act as sources for MS-associated chemokines. For instance, endothelial cells of CNS vessels express CCL5, and therefore, may contribute to the transmigration of the several cell types expressing the receptor CCR5.134 Cell types with putative roles in MS pathology that exhibit upregulated chemokine expression include macrophages/monocytes (CCL2, CCL3, CXCL10, CCL4), lymphocytes (CCL5), microglia (CCL3, CCL4, CXCL1), and astrocytes (CCL2, CCL5, CXCL10).134, 136, 137, 138, 164, 165 Many studies have also assessed chemokine levels in the CSF and blood of MS patients. Some prominent examples include the upregulation of CCL5, CCL17, CXCL8, and CXCL10 and increased expression of the receptors CCR5 and CXCR3 on CSF T cells.143, 144, 145, 146, 147, 150, 153, 169, 172, 173 In contrast, a number of studies show that CCL2 is downregulated in the CSF and blood of patients with active MS.143, 144, 150, 172 Some suggest that differences in the expression of chemokines (CXCL10) or receptors (CCR5, CXCR3) may be associated with whether MS takes on a relapsing remitting form or a more progressive form, although others show little difference between chemokine/receptor expression in different MS subtypes.144, 155, 167, 172, 174

EAE studies also support a role for chemokines in disease progression. Neutralizing CCL2, CCL3, CCL20, or CXCL10 by administering function-blocking antibodies may suppress EAE onset and/or relapses.175, 176, 177 In addition, antibodies to CCL2 or CCL5 inhibit leukocyte adhesion to the CNS endothelia in vivo.178

Chemokine Receptors

Certain chemokine receptors are commonly associated with MS pathogenesis. In particular, CXCR3 and CCR5 may play an important role. Since T cell infiltration is thought to be an integral part of MS-associated inflammation, of particular note are the many studies examining the expression of CCR5 and CXCR3 on MS T lymphocytes. CCR5 and CXCR3 expression is enhanced on perivascular infiltrating T cells and in T cells from the blood of MS patients with active demyelinating brain lesions.136, 154, 162 Peripheral Th1-type T cells with upregulated CCR5 exhibit increased migratory capacity in vitro in response to CCL3 and CCL5, suggesting a possible mechanism for lymphocyte infiltration into the CNS.179 Similarly, CXCR3-expressing T cells from the CSF of MS patients have enhanced migration toward CXCL10.158 CCR5 and/or CXCR3 are not T cell-specific and may also be expressed by several other cell types thought to be involved with MS pathology including macrophages, microglia, and astrocytes.139, 140, 154, 155, 162 Other prominent receptors associated with MS lesions include CCR2 and CCR3, which are found on monocytes/macrophages and microglia.139

Some MS patients undergoing treatment with IFN-beta, methylprednisolone, or cladribine exhibit changes in chemokine or chemokine receptor expression that could be associated with decreased inflammation.169, 180, 181, 182 The affect of manipulating expression has been more directly assessed using knockouts or neutralizing antibodies in EAE animals. For example, knockout of CCR1, CCR2, or CCR8 results in varying degrees of resistance toward EAE development, while CCR5 knockout appears to have little effect on EAE progression.183, 184, 185, 186

Matrix Metalloproteinases

The Matrix Metalloproteinases (MMPs) constitute a large family of at least 24 enzymes well known for their ability to cleave proteins of the ECM, although other substrates include cytokines, cytokine receptors, and inactive pro-MMPs.187, 188 They are thought to have roles in many biological processes including embryonic development, wound healing, angiogenesis, and tumor cell invasion and metastasis. They are zinc-dependent endopeptidases that exhibit some variability in domain structure between individuals of the family. However, most have a conserved catalytic domain, an inhibitory pro-peptide region, and a C-terminal hemopexin domain.188 The majority are secreted, although a subset are associated with the cell membrane via a transmembrane sequence (MMP-14, -15, -16, -23, and -24) or glycosylphosphatidylinositol (GPI) linkage (MMP-17 and -25).188 MMPs are regulated at the transcriptional and translational levels, as well as post-translationally by proteins that include alpha2-Macroglobulin, RECK, Tissue Factor Pathway Inhibitor-2, and a family of inhibitors known as Tissue Inhibitors of Metalloproteinases (TIMPs).189 Made up of at least 4 proteins (TIMP-1 through -4), TIMPs form non-covalent inhibitory complexes with MMPs. Disruptions in the balance of TIMPs and MMPs favoring MMP activation are implicated in several disease states, including inflammatory disorders.190, 191

MMPs have the potential to impact many facets of MS pathology including contributing to BBB disruption, activating pro forms of cytotoxic molecules, directly disrupting myelin, or creating cleavage products capable of eliciting an autoimmune response.192, 193 A critical part of leukocyte infiltration into the CNS requires penetration of the vessel-associated basement membrane. The CNS basement membrane contains such proteins as Laminin, Fibronectin, and Type IV Collagen, all of which are susceptible to MMP proteolysis, an activity that could contribute to increased permeability of the BBB.194

MMP-9

An array of MMPs are found upregulated in MS CSF, blood, or tissues/cells (Table 2). MMP-9 (Gelatinase B), in particular, has been widely studied as an important player in MS pathology. Although normally absent from the CSF, it is upregulated in MS patients.195, 196 MMP-9 levels, and/or the ratio of MMP-9 to its inhibitor TIMP-1, are also elevated in the blood of patients with RRMS and SPMS and are associated with gadolinium-enhancing lesions on MRI.197, 198, 199, 200 Several cell types might act as sources for MMP-9. For instance, macrophages, microglia, astrocytes, endothelial cells, and infiltrating lymphocytes associated with demyelinating lesions all express MMP-9.192, 201, 202, 203 Several studies have correlated MS therapies with altered levels of MMP-9 or its inhibitors. For instance, IFN-beta results in significant decreases in the levels of MMP-9 and/or the MMP-9/TIMP-1 ratio, and inhibits the migratory capacity of lymphocytes in vitro.204, 205, 206, 207, 208 Treatment with high-dose methylprednisolone is also shown to reduce CSF MMP-9 and MRI gadolinium enhancing lesions in MS patients.209 In addition, decreased serum MMP-9/TIMP-1 ratio is coincident with autologous hematopoietic stem cell transplantation and patient stabilization.200

Many EAE studies corroborate reports indicating a prominent role for MMP-9 in MS pathogenesis. MMP-9 expression is upregulated in EAE CSF and CNS infiltrates.210, 211 BBB disruption and symptoms are suppressed in response to treatment with exogenous MMP inhibitors.212, 213, 214 In addition, young (<4 weeks) MMP-9-deficient mice are resistant to EAE development, although their adult counterparts are not.215

Table 2. MMP Expression in MS.

MMP Alternative Name Source
MMP-1 Collagenase Macrophages216
PBMC217
MMP-2 Gelatinase A Astrocytes216
Dendritic Cells218
Macrophages216
Monocytes219
PBL220
T cells (Th1)221
MMP-3 Stromelysin-1 Astrocytes216
Dendritic Cells218
Endothelia216
Macrophages216
PBMC217
MMP-7 Matrilysin Macrophages203
PBMC217
MMP-9 Gelatinase B Astrocytes201, 216
Blood198, 199, 200, 222, 223, 224
CSF196, 223, 225, 226
Endothelia203, 216
Macrophages201, 216
PBMC217, 224
T Cells (Th1)221
MMP-12 Macrophage Metalloelastase Macrophages227
MMP-14 MT1-MMP Monocytes219
PBL220
PBL = Peripheral Blood Leukocytes
PBMC = Peripheral Blood Mononuclear Cells

Conclusion

Despite the more than 9000 MS/EAE studies published in the last five years, the mechanisms underlying MS etiology and pathology continue to remain unclear. It is complicated by the tremendous heterogeneity that often occurs between individuals and likely contributes to the fact that there is no universally effective treatment for MS. Ideally, greater understanding of the genetics, epidemiology, and cell biology will allow for the future development of treatments designed specifically for each individual. The molecules that regulate CNS infiltration of leukocytes may provide possible therapeutic targets. However, a greater understanding of the processes underlying transmigration is required. Much of the evidence supporting the roles of specific adhesion molecules, chemokines, and proteases in MS is circumstantial, relying on in vitro evidence or the correlation between expression patterns and MS pathology. EAE studies also help to provide clues, but there are always questions regarding how applicable EAE findings are to understanding the biology of MS. However, collectively these studies emphasize the importance of several molecules with putative involvement in MS-associated leukocyte transmigration and underscore the need for future studies.

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