The Cause Of Autoimmune Disease Biology Essay

Multiple sclerosis (MS) is a chronic inflammatory disease with neurodegeneration especially affecting the central nervous system (CNS), including the brain and spinal cord. It is one of the most common human autoimmune diseases. It damages the myelin sheath, the material that surrounds the axons of the CNS and protects the nerve cells, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms. MS is a complex disease with different kinds of clinical and pathological phenotypes. MS may present clinically as a steady, progressive, neurological disability or a relapsing-remitting disease.

There are four main pathological characteristics of MS (Constantinescu et al., 2011): (1) inflammation, which is in general thought to be the major trigger of this disease resulting in tissue damage of CNS in most cases, despite recent finding shows that initial neurological damage can trigger the formation of new symptomatic lesions sometimes (Barnett and Prineas, 2004); (2) demyelination, which means the loss of myelin and can cause the myelin sheath or the cell body of the oligodendrocytes destroyed by the inflammatory process; (3) axonal loss or damage; and (4) gliosis, which is an astrocytic reaction to CNS damage (Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012).

The cause of MS is still not clear, but it has been widely accepted that a combination of genetic susceptibility and environmental factors plays a role. Both factors together result in a self-sustaining autoimmune disorder, leading to recurrent immune attacks in the CNS. Geographic variation in the incidence of MS supports the probability that environmental factors are involved in its aetiology.

Animal models for human diseases utilize diseases in non-human species (often rodents), which closely resemble their human counterparts and are studied in order to better understand and treat human diseases. Experimental Autoimmune Encephalomyelitis (EAE) is an inflammatory autoimmune disease of CNS and has been a widely used animal model for studying the human demyelinating disease, MS. Strictly speaking, EAE is not MS and is a single disease in a single species. A major difference between EAE and MS is that EAE requires an external immunization step to develop diseases, whereas MS does not need the sensitization to autoantigen in humans (Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012). In EAE, sensitization to myelin antigens takes place by using complete Freund’s adjuvant, containing the bacterial components (Mycobacterium tuberculosis) that are able to activate the innate immune system via the pattern recognition receptors such as Toll-like receptors (Libbey and Fujinami, 2010). The antigens for inducing EAE are known, while no unique antigens are identified in MS. Thus, the difference between MS and EAE may be due to the different ways in which antigen-specific T cells are primed and activated (Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012)(Takeshita and Ransohoff, 2012). However, the different forms of EAE resemble the various forms and stages of MS very closely.

There are a number of benefits in studying MS by using the EAE model. It is possible to study demyelination, which is a process underlying the symptoms of MS, in the ways that would be neither ethically acceptable nor possible in studies of human MS. Also, the CNS sample can be collected to study histological symptoms. The potential treatments for MS can be tested for their efficacy and safety in EAE animals before using them on human patients. Different methods of inducing EAE can be utilized in order to find the potential causes of human MS. Additionally, a large number of rats/mouse can be studied at the same time because of their short breeding cycle.

There are mainly two different ways to induce EAE. Firstly, EAE can be induced by active immunization with the whole or parts of various CNS antigens, and this is referred to as active EAE. Several proteins are used as CNS antigens to induce active EAE including: myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). Secondly, it can be induced by passive transfer of myelin autoreactive T cell blasts, and this is called passive EAE. Compared to active EAE, the advantage of the passive EAE model is that the donor encephalitogenic T cells can be tagged by using allelic markers (such as CD45.1 and CD45.2), to track their route in vivo. Also, active EAE consists of two phases, an induction phase and an effector phase; whereas passive EAE consists only of the effector phase. The most commonly used CNS antigen is myelin oligodendrocyte glycoprotein35-55 (MOG35-55), which is expressed in the CNS and can cause lesions that have similar features to MS. EAE can be induced in susceptible mice, such as the C57BL/6 mouse strain, by the administration of MOG35-55. The EAE mice show the chronic and progressive disease phenotypes in clinical symptoms, including limp tail, hind and/or front limb weakeness, complete paralysis or death. The MOG35-55-induced EAE C57BL/6 mouse model is regarded as being a suitable method to experimentally address the different mechanisms leading to the chronic, progressive, and inflammatory autoimmune pathology in the CNS which are observed in MS patients, and also can be used to evaluate the therapeutical efficiency of investigational compounds to cure or prevent MS. In our studies, the MOG35-55-induced EAE C57BL/6 mouse model was used.

There are two barriers that protect the CNS from cellular infiltration: the blood-cerebrospinal fluid (CSF) barrier that surrounds the choroid plexus in which CSF is synthesized (Figure . Anatomical structure of the brain showing the possible routes for activated T cell entry. (A) and meningeal venules (Figure . Anatomical structure of the brain showing the possible routes for activated T cell entry. (B), and the blood-brain barrier (BBB) that surrounds the parenchymal venules (Figure . Anatomical structure of the brain showing the possible routes for activated T cell entry. (C). However, in the case of CNS inflammation, the barrier characteristics of the blood-CNS barriers are altered, leading to the recruitment of inflammatory cells into the CNS (Goverman, 2009).

Figure . Anatomical structure of the brain showing the possible routes for activated T cell entry. (A) Activated T cells can enter the subarachnoid space (SAS) by migrating from blood vessels into the choroid plexus stoma and then crossing the blood-cerebrospinal fluid barrier surrounding the stroma of the choroid plexus, which consists of the epithelial cells joined by tight junctions. (B) Activated T cells can also enter the SAS by extravasating through the meningeal venules, which comprise endothelial cells connected by tight junctions. (C) Activated T cells can migrate across the blood-brain barrier (BBB) surrounding the post-capillary venules and then infiltrate into the brain parenchyma. After crossing the BBB, T cells accumulated in the perivascular space, which is the region between the endothelial cell basement membrane (BM) which is connected to the blood vessel endothelial cells and the parenchymal BM overlying the astrocyte endfeet (not shown). (Modified from Goverman, 2009.) (Goverman, 2009)

Activated T cells can enter the CNS via different routes. T cells can migrate from the bloodstream to the CSF across the choroid plexus or meningeal vessels to the subarachnoid space (SAS), which is a region between the outer arachnoid and inner pial membranes. T cells can also migrate from the bloodstream to the perivascular space that separates the endothelial cell basement membrane (BM) and the parenchymal BM. To initiate CNS inflammation and damage, myelin-specific T cells must be first activated in the periphery and migrate into the CNS, and then be reactivated by antigen presenting cells (APCs) presenting self-antigen in CNS (Gerard and Rollins, 2001; Glabinski et al., 1995). T cell reactivation can trigger many inflammatory cells to produce the soluble mediators, which can further recruit other inflammatory cells into the CNS. However, only when the endothelium of the blood vessel is activated by inflammation and then expresses adhesion molecules such as P-selectin and E-selectin , can T cells adhere to the vascular component of the BBB (Piccio et al., 2002). In the absence of inflammation, the vascular endothelium in the brain does not express these adhesion molecules, thus, T cells cannot enter the CNS in this case (Piccio et al., 2002; Ransohoff et al., 2003). However, memory T cells which are previously exposed to antigen can carry out the CNS immune surveillance, because they can express adhesion molecules, chemokine receptors, and integrins, which allow them to cross the CNS barriers (Ransohoff et al., 2003). The selectins and adhesion molecules are constitutively expressed in the subarachnoid space (SAS); therefore, memory T cells are reactivated by antigen in the SAS before the onset of EAE (Kivisakk et al., 2009). CD4+ T cells, which had been previously activated in the periphery, are reactivated by major histocompatibility complex (MHC) class II+ APCs first in the SAS; and the recognition of the ligand results in T cell proliferation. The T cells are largely aggregate in the SAS. Therefore, EAE inflammation is firstly detected in the SAS, being the initial site of T cell entry (Kivisakk et al., 2009; Lassmann and Wisniewski, 1978). In the case of EAE, there are two routes by which inflammatory cells infiltrate the CNS. The first route is through the choroid plexus, leading to T-cell re-stimulation by antigen in the SAS, early CNS inflammation, and stimulation of the BBB vasculature (Figure 2) (Brown and Sawchenko, 2007; Reboldi et al., 2009). The second route is initiated by activation of the endothelium of the blood vessel, leading to a large number of inflammatory cells infiltrating the CNS parenchyma by migrating across the BBB (Figure . The second route for T cell entry into the CNS, migrating across the BBB).

For the first route of T cell entry into the CNS during EAE, T cells migrate across the blood-CSF barrier through the choroid plexus. CC-chemokine receptor 6 (CCR6), which is expressed by a subtype of pathogenic T cells termed Th17 cells, is critical for the initial T cell entry into the CNS (Reboldi et al., 2009). CCR6 knockout mice do not develop EAE after active EAE induction. However, CCR6-deficient T cells accumulate in the choroid plexus after immunization with myelin proteins (Reboldi et al., 2009). The choroid plexus is a region of the brain that is located in the ventricular system and is rich in capillary blood vessels. CSF is actively secreted by the choroid plexus epithelial cells and circulates from the ventricles through the SAS. CC-chemokine ligand 20 (CCL20), which is the ligand for CCR6, have constitutive expression on epithelial cells of the choroid plexus. Therefore, CCL20 must signal through CCR6 for Th17 cells to enter the CSF through the choroid plexus (Figure . The first route for initial T cell entry into the CNS during the onset of EAE). The entry of Th17 cells, in turn, begins the process that culminates in brain and spinal-cord inflammation (Ransohoff, 2009 ; Reboldi et al., 2009).

Figure . The first route for initial T cell entry into the CNS during the onset of EAE. CCR6-expressing effector and memory T cells escape from the blood vessels of the choroid plexus into the supporting tissue stroma, then migrate towards the choroid plexus epithelial cells, which express the chemoattractant molecule CCL20. Signalling by CCL20 through CCR6 allows T cells to cross the blood-CSF barrier (the tight junctions between the choroid plexus epithelial cells) and enter the cerebral ventricles, from which they migrate to the subarachnoid space (SAS). Restimulation of T cells by macrophages in the SAS leads to the production of growth and inflammatory factors (cytokines), increased T cell numbers, and stimulation of BBB vasculature, which initiates the second route of inflammatory cell infiltration into the CNS. (Taken from Ransohoff, 2009.) (Ransohoff, 2009 )

For the second route of CNS inflammatory cell entry, T cells migrate across the BBB (Figure . The second route for T cell entry into the CNS, migrating across the BBB). Firstly, activated T cells adhere to the inner walls of the activated cerebral vessels and then crawl in continuous contact with activated endothelial cells, most often in the opposite direction to the blood flow. Crawling allows these cells to identify vascular exit sites (Schenkel et al., 2004). Then, activated T cells migrate across the endothelial cell monolayer and the underlying endothelial basement membrane (BM), which together comprise the endothelial barrier. These activated T cells accumulate in the perivascular space, which is the space between the endothelial barrier and parenchymal barrier. Some T cells are reactivated in the perivascular space by the perivascular antigen presenting cells (APCs), including macrophages and dendritic cells. Next, T cells continue to migrate across the parenchymal BM and the layer of astrocyte endfeet, which collectively constitute the glia limitans, into the CNS parenchyma. Some T cells are also reactivated in the CNS parenchyma by either the resident microglial cells or activated macrophages and dendritic cells. Finally, in the CNS parenchyma, activated T cells, together with activated macrophages and microglial cells, secrete soluble mediators, such as different chemokine and cytokines, which can trigger demyelination and lead to axon destruction and tissue damage (Goverman, 2009). Only upon penetration of the parenchymal border do the clinical symptoms occur and the disease becomes apparent.

Figure . The second route for T cell entry into the CNS, migrating across the BBB. There are five steps involved in this process. (1) Activated T cells crawl along the vessel wall, mainly against the direction of blood flow, presumably in search of a suitable exit site, and then adhere to the endothelial cell monolayer of CNS postcapillary venules that express the adhesion molecules. (2) Activated T cells migrate across the endothelial barrier, including the endothelial cell monolayer and the underlying endothelial BM. (3) T cells migrate across the parenchymal barrier, including the parenchymal BM and the layer of astrocyte endfeet, which collectively constitute the glia limitans. T cells accumulate between the endothelial barrier and the parenchymal barrier within the perivascular space. (4) T cells are reactivated in the perivascular space by the perivascular antigen presenting cells (APCs), including macrophages and dendritic cells (4a), or in the CNS parenchyma by the resident microglial cells or infiltrated APCs (4b). (5) Activated T cells, together with activated macrophages and microglial cells, secrete soluble mediators that trigger demyelination in the parenchyma. (Modified from Chuan Wu, 2009 and Goverman, 2009.) (Goverman, 2009)

EAE is a CD4+ T cell-mediated autoimmune disease of the CNS. CD4+ effector T cells (Teffs), including interferon (IFN) -γ-producing Th1 and interleukin (IL) -17-producing Th17 cells, mediate disease pathogenesis in EAE. After activation by professional APCs, naive CD4+ T cells can differentiate into the different phenotypes of CD4+ helper T (Th) cells, depending on the microenvironment (Figure . Th1 and Th17 cell differentiation and trafficking into the CNS.). In the presence of IL-12 and IFN-γ, naive CD4+ T cells differentiate into Th1 cells, which can cause EAE. Th1 cells upregulate IFN-γ via Stat4, leading to IFN-γ-mediated Stat1 activation and induction of the Th1 lineage transcription factor T-bet (Figure . Th1 and Th17 cell differentiation and trafficking into the CNS.A and C) (El-behi et al., 2010). In the presence of IL-6, naive CD4+ T cells differentiate into Th17 cells, which also can cause EAE. IL-6 activates Stat3 and the lineage-determining transcription factors RORγt (Figure . Th1 and Th17 cell differentiation and trafficking into the CNS.A and C) (El-behi et al., 2010) . However, when IL-6 is present together with TGF-β, the differentiated Th17 cells can inhibit the expression of T-bet, finally resulting in non-encephalitogenic Th17 cells that lose the ability to induce EAE (Figure . Th1 and Th17 cell differentiation and trafficking into the CNS.A and C) (Lovett-Racke et al., 2011). The ratio of infiltrated Th1 versus Th17 cells determines the location of the CNS inflammation. When the Th17 cells outnumber Th1 cells, T cell infiltration into the brain parenchyma occurs. By contrast, T cells showing a wide range of Th17 versus Th1 ratios induce spinal cord parenchymal inflammation (Stromnes et al., 2008). The absolute number of Th1 cells infiltrating into the CNS during EAE is significantly higher than the number of Th17 cells (Figure . Th1 and Th17 cell differentiation and trafficking into the CNS.B). Figure . Th1 and Th17 cell differentiation and trafficking into the CNS. (A) Naive CD4+ T cells can differentiate into Th1 or Th17 cells. During Th17 cell differentiation, IL-17 production can be enhanced by TGF-β, but this decreases the capacity of these cells to induce EAE. (B) During EAE, Th1 and Th17 cells have the different distribution in the CNS. Th1 cells preferentially home to the spinal cord, while Th17 cells preferentially appear in the brain. The number of Th1 cells is significantly higher compared to the number of Th17 cells in the CNS during EAE. (C) The transcription levels of Th1 and Th17 differentiation is regulated by the signaling pathways, which may be very important in the development of encephalitogenic T cells. The transcription factor found in the encephalitogenic Th1 and Th17 cells is T-bet. During Th17 cell differentiation, the presence of TGF-β contributes to inhibit the expression of T-bet, resulting in non-encephalitogenic Th17 cells if primed with TGF-β and IL-6 together. (Taken from Lovett-Racke et al., 2011.) (Lovett-Racke et al., 2011)

Naive CD4+ T cells can also differentiate into regulatory T cells (Tregs) in the presence of predominantly TGF-β, and these are characterized by the expression of CD25 and the transcription factor Foxp3. These regulatory T cells contribute to the natural protection against autoimmunity and participate in the spontaneous remission of disease. They are active both in the peripheral lymphoid organs and in the inflamed CNS during EAE (Anderton and Liblau, 2008).

The balance between Teffs and Tregs is critical during EAE development. Teff cell numbers have a moderate increase in the pre-symptomatic and initial phases of the disease and revert to baseline levels during the peak phase of disease, whereas Treg cell numbers are constant during the course of EAE in the peripheral lymphoid organs (Korn et al., 2007). However, in the CNS, Teff cells reach their peak at the peak phase of disease and then go down. By contrast, Treg cells reach their peak at the recovery phase of disease in the CNS (Korn et al., 2007).

Macrophages are the critical antigen presenting cells (APCs) and immune effector cells in both innate and adaptive immunity. They have been classified into two main groups, including M1 and M2 macrophages (Gordon, 2003). M1 macrophages, which are classically termed as being activated macrophages, are immune effector cells that are activated by lipopolysaccharides (LPS) and IFN-γ, and secrete high levels of IL-12 and low levels of IL-10; M2 macrophages, which are also called activated macrophages, are activated by IL-4 and produce high levels of IL-10 and low levels of IL-12, and play a role in wound healing and tissue repair (Mantovani et al., 2007).

Macrophages are also involved in EAE. There are two different kinds of macrophages in the CNS of EAE mice: infiltrating peripheral macrophages, that are called perivascular macrophages (PVMs), and the resident macrophages of the CNS, which are referred to as microglia cells. They can be distinguished from each other by the level of CD45 expression: PVMs have higher levels of CD45 compared with microglia cells (Aloisi, 2001; Fabriek et al., 2005).

PVMs are located between the endothelial cell BM and parenchymal BM of blood vessels in the CNS, and exert their function in controlling innate and adaptive immune responses in the CNS (Chastain et al., 2011; Fabriek et al., 2005). Depletion of peripheral macrophages by intravenous (i.v.) injection of mannosylated liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP) abrogated EAE (Huitinga et al., 1995; Tran et al., 1998). In those animals, T cells, monocytes/macrophages, and an unusually high proportion of MHC II+B220+ B cells accumulated in subarachnoid spaces of the leptomeninges around the spinal cord but did not infiltrate the CNS parenchyma, suggesting an important role for macrophage infiltration in the development of CNS inflammation and demyelinization (Tran et al., 1998).

Microglia are bone marrow-derived resident macrophages and are a more stable and irradiation-resistant subpopulation of monocytic cells in the CNS, and have similar functions to the periphery-derived macrophages in the CNS during inflammation (Perry and Gordon, 1988). During the progression of EAE, microglia become activated and then accumulate around demyelinated lesions. The inhibition of microglial activation leads to a delayed onset of EAE but increased severity and delayed recovery from neurological dysfunction, suggesting that microglial activation is harmful during the onset of the disease but beneficial in the recovery phase (Lu et al., 2002; Luo and Chen, 2012). The inhibition of microglial activation during onset of EAE significantly decreased EAE progression, while the stimulation of microglial activation prior to the onset of EAE promoted a less severe EAE and an earlier recovery from symptoms (Bhasin et al., 2007). These studies demonstrated the different roles for microglial activation during different phases of EAE and that different timing for microglial activation can obviously affect whether microglia are neuroprotective or injurious (Bhasin et al., 2007; Luo and Chen, 2012).

EAE is a T lymphocyte-mediated autoimmune disease of the CNS. However, besides infiltrating T cells and macrophages; B cells, plasma cells, and the antibodies they produce are also important for CNS inflammation in EAE mice, and might play roles during EAE to facilitate CNS damage and promote disease progression (Weber et al., 2011).

B cells can contribute to the pathogenesis of EAE in several different ways: (1) B cells exert their functions as APCs for autoreactive T cells, (2) Plasma cells can secret autoantibodies that recognize target structures on the myelin sheath, and then contribute to axonal damage and demyelination, (3) B cells can produce pro-inflammatory cytokines and chemokines, which are involved during EAE, (4) A rare IL-10-producing CD1dhighCD5+ regulatory B cell subset (B10 cells), which represents 1%-2% of spleen B cells, has the function in inhibiting EAE initiation (Matsushita et al., 2008; McLaughlin and Wucherpfennig, 2008).

B cells have the reciprocal regulatory roles during EAE, which has been shown by using CD20 antibody treatment to deplete B cells before EAE induction or during EAE progression (Matsushita et al., 2008). The B cell depletion before EAE induction significantly exacerbated EAE symptoms and also dramatically increased encephalitogenic T cell infiltration in the CNS. The depletion of B10 cells has been shown to be responsible for this increased severity, because the EAE phenotype were normalized by the adoptive transfer of splenic B10 cells into B cell-depleted mice before EAE induction. Also, the adoptive transfer of MOG35-55-specific B10 cells into wild type (WT) mice dramatically reduced the initiation of EAE, but it did not suppress ongoing EAE (Matsushita et al., 2010). Taken together, those findings suggest an important function for B10 cells in EAE initiation depending on their production of IL-10, which can inhibit EAE development (Fillatreau et al., 2002). However, B cell depletion during EAE progression suppressed disease severity dramatically (Matsushita et al., 2008) . B cells are important for encephalitogenic T cell activation and also for antigen-specific T cell proliferation by functioning as APCs. B cells can present antigen to MOG-specific T cells, whereas B cell depletion significantly reduced MOG-specific CD4+ T cell proliferation, followed by decreased encephalitogenic T cell entry into the CNS and, finally, leading to decreased disease severity (Matsushita et al., 2008). Therefore, B cells are required for the generation of CNS autoantigen-specific CD4+ T cells and the encephalitogenic T cell infiltration into the CNS during disease progression.

Plasma cells are terminally differentiated B cells that provide protective immunity through the continuous production of antibodies. Plasma cells develop from B cells that are made in the bone marrow. During MOG35-55 induced EAE progression, plasma cells can secrete MOG-specific antibodies. Previous studies have shown that the antibodies specific to MOG35-55 enhance demyelination and disease severity (Lyons et al., 1999; Pollinger et al., 2009). MOG-specific antibodies are critical to EAE. The administration of a monoclonal antibody specific for MOG exacerbated clinical and histological symptoms in both acute and chronic EAE (Schluesener et al., 1987). The injection of this antibody into EAE animals caused fatal relapses in SJL mice with chronic relapsing EAE and a hyper-acute inflammatory response, together with demyelination in Lewis rats with acute EAE, indicating that the relapses and demyelination can be induced by administration of an antigen-specific monoclonal antibody. In transgenic mice endogenously producing high levels of myelin-specific antibodies, EAE severity was substantially increased with greater inflammation and CNS demyelination (Litzenburger et al., 1998).

B cell depletion can efficiently improve disease progression in EAE partly through ablation of IL-6-secreting pathogenic B cells (Barr et al., 2012). The levels of IL-6 were elevated in B cells from EAE mice compared with naive mice. The B cell-specific IL-6 deficiency mice showed reduced disease severity than the mice that had WT B cells (Barr et al., 2012). Also, IL-6 is essential for the development of EAE, since IL-6-deficient mice are completely resistant to EAE induction (Okuda et al., 1998; Samoilova et al., 1998); and the antibody response to MOG is significantly impaired in the mice lacking IL-6 (Okuda et al., 1998). This is further evidence to support the important role of IL-6 in EAE, indicating that MOG-specific antibodies are also crucial for EAE development.

Chemokines are a group of small molecular weight (8-14kDa) chemotactic molecules released by a variety of cell types. They can be divided into four subfamilies (CXC, CC, C, and CX3C) by their physical structure according to the number and spacing of conserved cysteine residues near the N-terminus (Murphy et al., 2000; Zlotnik and Yoshie, 2000). Chemokine receptors are a large family of seven transmembrane domain G protein-coupled receptors that are expressed in different cell types.

Chemokines exert their biological functions by binding to chemokine receptors on target cells (Takeshita and Ransohoff, 2012). Several chemokines are constitutively expressed or have upregulated expression in the brain during EAE, and the infiltrated immune cells express a variety of chemokine receptors associated with chemotaxis and/or effector function (Wilson et al., 2010). The endothelial cells in the CNS constitutively express CCL19 and CCL21, which is paralleled by the presence of their common receptor CCR7 in the infiltrated CD4+ cells (Alt et al., 2002). Astrocytes and cortical neurons constitutively express CXCL12, which can bind to their receptor CXCR4 and acts to modulate cell migration, differentiation, and proliferation (Bajetto et al., 1999). The epithelial cells in the choroid plexus have constitutive CCL20 expression, which is required for CCR6+ Th17 cell entry into the CNS under non-inflamed conditions through the choroid plexus for the initiation of EAE (Reboldi et al., 2009). Chemokine signalling results in molecular and functional changes in leukocytes. Chemokines and their receptors are involved in multiple steps during leukocyte trans-endothelial migration (Takeshita and Ransohoff, 2012). They are also involved in the trafficking of the immune cells into the CNS during EAE,

Chemokine or chemokine receptor knockout mice, and treatment with antagonists of these interactions in mice have been used to better understand which interactions are likely to be important in the CNS, and the roles of those molecules during EAE development.

Studies of the role of CCL2 during EAE have shown that CCL2 knockout mice on a C57BL6 background were resistant to EAE, together with dramatically impaired recruitment of macrophages into the CNS, indicating that CCL2 plays a critical role in modulating monocyte infiltration into the CNS during inflammation (Huang et al., 2001). Also, the mice lacking the major CCL2 receptor, CC chemokine receptor CCR2, did not develop disease due to defective monocyte recruitment into the CNS. However, adoptively transferred MOG35-55-specific CCR2 knockout T cells were able to induce EAE in WT recipient mice, whereas MOG35-55-specific WT T cells failed to produce EAE in CCR2 knockout recipient mice, suggesting the CCR2 expression on host-derived mononuclear cells is critical for disease induction (Fife et al., 2000; Huang et al., 2001). Interestingly, CCL2 transgenic mice that have constitutively low levels of CCL2 in the CNS showed equal CD4+ T cell and enhanced monocyte infiltration into the CNS during EAE; however, they have significantly milder EAE than the control mice due to the effects of CCL2 on the lymphocytes in the periphery, indicating that the CNS-specific expression of CCL2 in vivo has a protective role for EAE (Elhofy et al., 2005).

CCL3 knockout mice were fully susceptible to MOG-induced EAE and have similar clinical symptoms to WT mice; also, the mice lacking the CCL3 receptor CCR5 developed similar EAE to the control mice (Tran et al., 2000). Leukocyte recruitment during the onset of EAE is CCR1 dependent, as CCR1 knockout mice have a reduced disease severity compared to control mice (Rottman et al., 2000). CXCL10-deficient mice were found to be susceptible to EAE, and the mice lacking the CXCL10 receptor CXCR3 developed an exaggerated disease severity of EAE. Surprisingly, there were no differences in the number of infiltrated leukocytes in the CNS of CXCR3 knockout and control mice, but T cells from CXCR3 knockout mice were found to modulate T cell IFN-γ production and downstream events that contribute to this increased disease severity (Liu et al., 2006).

The utility of these studies into chemokines and their receptors during EAE is the development of small molecular weight antagonists to prevent clinical EAE. For example, studies have shown that a novel non-peptide CCR1 antagonist is efficacious in the inhibition of clinical EAE and may be useful in the treatment of chronic inflammatory diseases (Liang et al., 2000).

Cytokines are small proteins released by numerous cells that have specific influences on the interactions between cells, the communication between cells, or the behaviour of cells. They play an important role in the pathogenesis of inflammatory diseases such as MS and EAE. CD4+ T cells play a critical role in EAE. Naive T cells can differentiate into Th1, Th2, Th17, and Tregs in the presence of different cytokines. Th1 cells produce pro-inflammatory cytokines, mainly IL-2, TNF-α, and IFN-γ, and mediate pro-inflammatory responses during EAE. In contrast, Th2 cells secrete anti-inflammatory cytokines, mainly IL-4, IL-5, IL-10, and IL-13, and participate in the prevention or remission of EAE. Th17 cells produce IL-17 and play a pathogenic role in inducing EAE. When there is a lot of TGF-β in the micro-environment, naive T cells differentiate into Tregs which have protective roles during EAE (Lohr et al., 2006; Villoslada et al., 2012). In addition, the microglia also can produce anti-inflammatory cytokines, such as TGF-β and IL-10, which are associated with inhibition or prevention of EAE (Bhasin et al., 2007; Rott et al., 1994).

By using a murine model, it is possible to enhance or suppress an individual cytokine’s activity in order to study its role during the disease. Also, the single cytokine knockout or transgenic mouse is a useful tool to study the role of single cytokines. However, these studies occasionally yield conflicting results, highlighting the fact that cytokines play complex roles in the disease process. For example, over-expression of IFN-γ in the CNS of mice causes a progressive demyelinating disease (Renno et al., 1998). In contrast, the mice lacking IFN-γ have been shown to be susceptible to EAE (Ferber et al., 1996).

The syndecans are a family of type I transmembrane core proteins that carry heparan sulfate (HS) chains and/or chondroitin sulfate (CS) chains near the tips of their extracellular domains (also called ectodomains) at the cell surface ( A). There are four distinct syndecan genes in mammals: syndecan-1, -2, -3, and -4 (Bernfield et al., 1992).

The core proteins of all syndecans can be broadly divided into three domains: an extracellular domain (ectodomain) with an N-terminal signal peptide, a single hydrophobic transmembrane domain, and a short C-terminal cytoplasmic domain ( B). The ectodomain is an extended extracellular domain of varying size containing covalently attached HS or CS chains. The glycosaminoglycan (GAG) attachment sites in syndecan-1 and -3 occur in two distinct clusters, one is near the N- terminus and the other is near the membrane-attachment site ( A). Because of the extended structure, most structural variations are mainly found in the ectodomain with the exception of some conserved regions for oligomerization, GAG attachment, proteolytic cleavage, and cell interactions (Alexopoulou et al., 2007) The transmembrane domain is highly conserved compared to the ectodomain since only a few amino acids are different among the vertebrate sequences. This domain is important for interacting with other membrane proteins and also for localizing the syndecans to distinct membrane compartments (Rapraeger, 2001). The cytoplasmic domain has a number of important regions, despite it is relatively short. It has two constant regions, a membrane proximal common (C1) region and a C-terminal common (C2) region, which are highly conserved. Between them, there is a variable (V) region with variable length and composition ( B), which is unique for each syndecan, though it is conserved across species (Couchman et al., 2001). The C1 region, which is adjacent to the plasma cell membrane, provides a link to the actin cytoskeleton. It plays roles in syndecan dimerization and binding of several intracellular proteins (Granes et al., 2003; Kinnunen et al., 1998). The C2 region contains two tyrosine residues and a postsynaptic density-95/disc large protein/zonula occludens-1 (PDZ2)-binding site which localizes at the C-terminal end. This binding site can bind to several PDZ-domain containing proteins and is suggested to have a function in trafficking of all syndecans (Ethell et al., 2000; Hsueh et al., 1998; Zimmermann et al., 2001). The V region is unique to each syndecan, endowing each with potential for specific interactions (Zimmermann et al., 2005). According to the sequence comparisons within the transmembrane and cytoplasmic domains, the four mammalian syndecans are divided into two subfamilies: syndecan-1 and -3, and syndecan-2 and -4.

Figure . The structure of the four syndecans in mammalian tissues. (A) Schematically depicted are the four mammalian syndecans, and their glycosaminoglycan (GAG) chain attachments. Listed for each are their reported GAG attachments including heparan sulfate (HS) chains and chondroitin sulfate (CS) chains. (B) Diagram illustrating the structures of the four vertebrate syndecans. The black boxes represent the transmembrane domains. The GAG attachment sites are indicated as black lines. The proteins are aligned at the cytoplasmic domains and the transmembrane domains, and there are gaps in the ectodomains. Syndecans contain a conserved transmembrane domain and a small cytoplasmic domain which consists of one variable region (V) and two conserved regions (C1 and C2). (Modified from Bellin et al., 2002; Lopes et al., 2006.) (Bellin et al., 2002; Lopes et al., 2006)

Syndecan expression is different from tissue to tissue, and also different from cell to cell. The study of syndecan expression in a number of tissues and cell lines has shown that all cells express at least one form of syndecan, with most cells expressing multiple forms (Kim et al., 1994).

There is a distinct spatial and temporal expression pattern for each syndecan, which is highly regulated during development (Bernfield et al., 1992). For example, in the embryonic day 10 (E10) mouse brain, syndecan-1 is the most abundantly expressed syndecan and localized exclusively to neuroepithelial cells in the ventricular zone; Syndecan-4 is expressed at a much lower levels, but also localizes exclusively to cells in the ventricular zone. (Ford-Perriss et al., 2003). However, the adult mouse brain contains a large amount of syndecan-3, a lesser amount of syndecan-2 and syndecan-4, and a small amount of syndecan-1 (Kim et al., 1994). The changes of different syndecan expression that occur during development are closely associated with cell differentiation, morphological transitions, or the changes of tissue organization (Carey, 1997; Lopes et al., 2006).

In the adult tissues, syndecan-1 is expressed predominantly by epithelial cells and plasma cells, and expressed to a lesser degree by other cells types (e.g., endothelial cells, monocytes, fibroblasts). Syndecan-2 is abundantly expressed on endothelial cells and mesenchymal cells. The expression of syndecan-3 is found almost in neuronal and musculoskeletal tissue only, whereas syndecan-4 expression is found in every cell type, although it is expressed at lower levels than other co-expressed syndecans (Couchman, 2003). To better understand the function of different syndecans, single syndecan knockout mice or transgenic mice have been generated. By using single syndecan deficiency or overexpression mice, the published studies have shown several roles that are played by different syndecans during developmental process (Error: Reference source not found) and inflammation (Wang et al., 2012; Xiao et al., 2012).

Mice lacking syndecan-1 (Sdc-1-/-) are ostensibly viable, fertile, and have morphologically normal skin, hair, and ocular surface epithelia. However, they have abnormally slow re-epithelialization during wound healing, indicating essential roles for syndecan-1 in mediating cell proliferation in normal and wounded epithelial tissue (Stepp et al., 2002). Mice overexpressing syndecan-1 showed delayed wound closure, re-epithelialization, granulation tissue formation, and remodelling, suggesting the shed syndecan-1 ectodomain may enhance proteolytic activity and inhibit cell proliferation during wound repair (Elenius et al., 2004). Syndecan-1 can modulate Wnt signalling and is critical for Wnt-1-induced tumourigenesis of the mouse mammary gland because mammary gland-specific expression of Wnt-1 leads to the development of tumours in wild type mice but not in Sdc-1-/- mice. In addition, Sdc-1-/- mice have reduced Wnt-1 mediated hyperplasia (Alexander et al., 2000). Syndecan-1 acts as a negative regulator in leukocyte-mediated inflammatory responses. For example, Sdc-1-/- mice showed increased leukocyte adhesion to endothelial cells after tumor necrosis factor (TNF)-α treatment (Gotte et al., 2002). Thus, syndecan-1 could have use as a therapeutic target for the prevention of pathologic leukocyte-endothelial interactions in angiogenesis and inflammation.

There are no syndecan-2 knockout mice reported in the literature. Syndecan-2 is widely present in the vasculature, particularly during embryogenesis. Zebrafish have been used for most studies of syndecan-2 function, and they have the advantages of rapid development and the ability to observe angiogenesis during development. By using anti-syndecan-2 morpholino oligonucleotides, studies in zebrafish showed defective angiogenic sprouting and demonstrated that syndecan-2 plays its role in vascular development which involves the modulation of vascular endothelial growth factor (VEGF) signalling (Chen et al., 2004). Syndecan-3 expression is prominent in the hippocampus. Syndecan-3 has been suggested to play a role in the development and plasticity of neuronal connections by linking extracellular signals to the regulation of the cytoskeleton. Syndecan-3 knockout mice are healthy and fertile, and have no apparent defects in the structure of the brain. However, they showed impaired performance in hippocampus-dependent memory functioning, suggesting that syndecan-3 acts as an important modulator of synaptic plasticity that influences hippocampus-dependent memory (Kaksonen et al., 2002). Also, syndecan-3 deficiency impairs neural migration in the brain (Hienola et al., 2006). Syndecan-3 knockout mice display a novel form of muscular dystrophy, which is characterized by fibrosis, impaired locomotion, and hyperplasia of satellite cells (Cornelison et al., 2004). Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from WT littermates. Compared with WT littermates, synecan-4 knockout or heterozygous mice have significant delayed skin wound healing and impaired angiogenesis in granulation tissue, indicating that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis, and that syndecan-4 is haplo-insufficient in these processes (Echtermeyer et al., 2001). These is a unique requirement for syndecan-4 in satellite cell function, because the satellite cells from syndecan-4 knockout mice fail to reconstitute the damaged muscle (Cornelison et al., 2004). Syndecan-4 activation plays a direct role in regulating the function of focal adhesions (Wilcox-Adelman et al., 2002). By using transgenic mice expressing syndecan-4 cDNA under the control of the human endothelial nitric oxide (NO) synthase (eNOS) promoter, the study showed that enhanced syndecan-4 expression in mouse cardiac endothelial cells results in preferential augmentation of fibroblast growth factor 2 (FGF2) but not VEGF-A(165)-induced NO release, leading to microvessel relaxation (Zhang et al., 2003). As discussed above, the studies using knockout mice indicat that syndecan-1, syndecan-3, and syndecan-4 are not required for development because all three single syndecan deficiency mice are viable, fertile, and do not show apparent pathologies. However, when the single syndecan deficiency mice were challenged with inflammatory stimuli, they showed dramatically altered phenotypes compared to WT mice (Hayashida et al., 2009; Ishiguro et al., 2001). These findings indicate that other syndecans can functionally compensate each other for the loss of one syndecan during development but not during a certain post-developmental process such as in inflammation, suggesting that each syndecan is important for survival and that they perform specific functions in vivo. Syndecan-1, also called CD138, is a cell surface heparan sulfate proteoglycan (HSPG) which can bind to a wide variety of molecules, including cytokines, chemokines, growth factors, and extracellular matrix components, through their heparan sulfate chains. By this way, leukocyte recruitment, angiogenesis, matrix remodelling, cancer cell proliferation and invasion, microbial attachment and entry, host defense can be modulated by syndecan-1 (Teng et al., 2012).

Inflammation is a host response to tissue injury, which is often caused by invading endogenous or exogenous pathogens. By interacting with many factors, syndecan-1 can modulate the tissue injury and inflammation and affect the outcome of the inflammatory diseases (Bartlett et al., 2007). The significant importance of syndecan-1 during inflammatory responses is highlighted by the striking pathological phenotypes which have been observed in Sdc-1-/- mice when they are challenged with inflammatory stimuli (Error: Reference source not found).

Syndecan-1 regulates leukocyte recruitment in several steps in a wide variety of inflammatory disease models. There are several possible mechanisms that are involved in the inflammatory disease (Error: Reference source not found)

Mechanisms of syndecan-1 action in inflammatory diseases. A) Syndecan-1 can inhibit leukocyte adhesion to activated endothelial cells by inhibiting the interation between integrinβ2 and ICAM-1. B) Cell surface syndecan-1 is shown in both the basal and apical surfaces. Chemokines can bind to the endothelial cell surface syndecan-1, and then a chemokine gradient is generated, which can facilitate the leukocytes trans-endothelial migration across the endothelial cells. C) The shedding of syndecan-1 in alveolar epithelial cells which is mediated by matrix metalloproteinase (MMP)-7 can generate a chemokine gradient to facilitate the trans-epithelial migration of neutrophils. D) The chemokine gradient can be resolved by syndecan-1 shedding either by removing chemokines which are tethered to syndecan-1 on cell surface or by displacing chemokines tethered to other heparan sulfate proteoglycans (HSPGs) through the release of unbound syndecan-1 ectodomain. (Taken from Teng et al., 2012.) (Teng et al., 2012)

Syndecan-1 on endothelial cells or epithelial cells functions as an inhibitor of leukocyte adhesion

Syndecan-1 regulates inflammation by modulating the activity of different cytokines, chemokines, integrins, and other adhesion molecules (Error: Reference source not foundA).

In the murine contact allergy model of delayed-type hypersensitivity (DTH), Sdc-1-/- mice showed an increased leukocyte recruitment, following by an increased and prolonged oedema formation (Kharabi Masouleh et al., 2009). The expression of pro-inflammatory cytokines (IL-6, TNF-α,), chemokines (CCL3 and CCL5), and the adhesion molecule intracellular adhesion molecular (ICAM-1) were significantly increased in Sdc-1-/- mice compared to WT mice, and the increased leukocytes adhesion to ICAM-1 in Sdc-1-/- mice was effectively inhibited by using CD18 blocking antibody in vitro (Kharabi Masouleh et al., 2009). The mRNA and protein expression of syndecan-1 were decreased during the DTH process. Taken together, the DTH results show an important function of syndecan-1 as a negative modulator in the contact DTH reaction. Sdc-1-/- mice also showed increased inflammation when they subjected to myocardial infarction, and caused by increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2), increased leukocyte adhesion and transendothelial migration (Vanhoutte et al., 2007). Conversely, mice overexpressing syndecan-1 have reduced inflammation and are protected against cardiac dilatation and failure mainly by reducing transendothelial adhesion and migration of leukocytes (Vanhoutte et al., 2007). In murine dextran sodium sulfate (DSS)-induced colitis, Sdc-1-/- mice exhibited a significantly increased lethality compared to WT controls. Impaired mucosal healing and prolonged recruitment of leukocytes were observed in Sdc-1-/- mice which were accompanied by increased expression of tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP) -1α (CCL3), and vascular cell adhesion molecular (VCAM-1), consistent with the reduced syndecan-1 expression and impaired gut mucosal repair observed in lesions of patients with ulcerative colitis (Floer et al., 2010). This indicates that syndecan-1 has a protective effect during experimental colitis.

These data suggest syndecan-1 plays a negative role in regulating leukocyte adhesion and migration. The possible mechanism behind is that syndecan-1 can inhibit the interactions between integrins on leukocytes and adhesion molecules ICAM-1 and VCAM-1 on endothelial cells. Thus, syndecan-1 expressed on endothelial cells or epithelial cells plays a role in inhibiting leukocyte adhesion and migration.

.2.4.2 Syndecan-1 modulates the generation and activity of chemokine gradients during the process of inflammatory diseases

The major functional domain of the syndecan-1 ectodomains is their heparan sulfate (HS) chains, which can specifically bind to most chemokines, facilitating the formation of chemokine gradients on the endothelial surface (Error: Reference source not foundB). These gradients serve as directional cues to guide the trans-endothelial migration of leukocytes into the sites of inflammation (Bao et al., 2010; Handel et al., 2005).

2.4.3 Syndecan-1 shedding by MMPs facilitates the generation of a CXC chemokine gradient and further facilitates the trans-epithelial migration of neutrophils

Shedding of syndecan-1 can be mediated by MMP-7 (matrilisin) and membrane type matrix metalloproteinase (MT-MMP)-1 (Error: Reference source not foundC) (Endo et al., 2003; Li et al., 2002). Syndecan-1 is the major HSPG of lung epithelial cells. In a mouse model of acute lung injury which is induced by bleomycin, CXCL1 binds to syndecna-1 on the surface of epithelial cells and then MMP shed the syndecan-1/CXCL1 complex into the alveolar space. By this way, a chemokine gradient was generated across the alveolar epithelium in a basal to apical direction. However, neutrophils remained confined in the interstitium of injured lungs and did not advance into the alveolar space in both syndecan-1 knockout and MMP-7 knockout mice. This impaired transepithelial migration was accompanied by a lack of both shed syndecan-1 and CXCL1 in the alveolar fluid (Li et al., 2002). Hence, MMP-7-mediated shedding of syndecan-1/CXCL1 complexes from the mucosal surface directs and confines the neutrophil inflammation to the specific sites of tissue injury where the normal repair is needed, and further limits excessive inflammatory tissue damage. In an allergic lung inflammation model, Sdc-1-/- mice instilled with allergens exhibited exaggerated airway hyper-responsiveness, glycoprotein hyper-secretion, eosinophilia, and lung IL-4 responses, indicating that syndecan-1 can attenuate allergic lung inflammation to some extent (Xu et al., 2005). Syndecan-1 ectodomains bind to the CC chemokines (CCL7, CCL11, and CCL17), which are implicated in allergic diseases, inhibit CC chemokine-mediated T cell migration, and suppress CC chemokine-mediated Th2 cell recruitment to the lung. During this allergic lung inflammation, the anti-inflammatory activity is mediated by the syndecan-1 HS chains, as the administration of purified syndecan-1 ectodomain including HS, but not ectodomain core proteins lacking HS, significantly inhibits these inflammatory responses (Xu et al., 2005).

Syndecan-1 plays an important role in modulating inflammatory responses in Gram-positive toxic shock, a systemic disease that is a significant cause of morbidity and mortality. Sdc-1-/- mice injected intraperitoneally (i.p.) with staphylococcal superantigen showed significantly increased liver injury, vascular permeability, and death compared with WT mice. Increased TNF-α and IL-6 levels, and enhanced T cell accumulation in tissues were also observed in Sdc-1-/- mice undergoing Gram-positive toxic shock. The administration of HS, but not syndecan-1 core protein, can rescue Sdc-1-/- mice from lethal toxic shock by suppressing the production of TNF-α and IL-6, and attenuating inflammatory tissue injury (Hayashida et al., 2008). In an animal model of anti-glomerular basement membrane (GBM) nephritis, a more severe glomerular injury was observed in Sdc-1-/- mice (Rops et al., 2007). During the heterologous phase, glomerular leukocyte/macrophage influx was significantly higher in Sdc-1-/- mice than WT mice, and this was associated with higher glomerular endothelial-specific HS expression. In the autologous phase, the glomerular CD4+/CD8+ T cell influx was higher in Sdc-1-/- compared to WT mice (Rops et al., 2007). There was decreased Th1, and increased Th2 related cytokine and chemokine expression in Sdc-1-/- mice, indicating that syndecan-1 deficiency exacerbates anti-GBM nephritis by shifting the Th1/Th2 balance towards a Th2 response, suggesting that syndecan-1 can exert its function by regulating the Th1/Th2 balance in the inflammatory diseases.