International Journal of Drug Development and Research

Key words

Multiple sclerosis, Experimental autoimmune encephalomyelitis, Gene expression, Cytokines

Introduction

Multiple sclerosis (MS) is a debilitating neurological disease with unknown etiology that has a strong autoimmune component and affecting about two million people worldwide. The inflammatory phase of MS likely involves activation and migration of myelin-reactive T cells across the blood–brain barrier (BBB), resulting in damage to myelin and axons in the central nervous system (CNS) (Wang C et al; 2010)[1] and in MS clinical signs and symptoms are very variable and depend on the parts of the CNS it affects, that is, the brain and spinal cord, and include motor, sensory, autonomic and cognitive disabilities (Vosoughi R et al; 2010). [2] CD4+ T helper cells reactive to myelin, which produce proinflammatory cytokines, such as interferon-E (IFN-E), osteopontin (OPN) and tumour necrosis factor-G (TNF-G), are known to have a leading role. In the complex of Th1/Th2 paradigm, MS and EAE, several experiments support a role for Th 1 responses in promoting of diseases and Th 2 responses in suppressing of disease and the adoptive transfer of myelin-reactive Th 2 cells to immune-deficient mice to induce EAE and eosinophils regarded as Th 2- associated cells also contribute in disease development (Friese MA et al; 2003). [3]

Classification of Multiple Sclerosis

1. Relapsing–remitting (RRMS)

2. Secondary progressive (SPMS)

3. Primary progressive (PPMS)

4. Progressive relapsing (PRMS)

Relapsing–remitting (RRMS)

Almost 80% of MS patients suffered from RRMS disease course, It is the most common phenotype, starts with a single mono- or multi-focal demyelinating episode (clinically isolated syndrome, CIS) with partial or full recovery and about 80% of MS patients suffered from RRMS disease course. In this, similar or varied types of relapses occur in time after initial attack. It is dominated by overt inflammation, demyelination, clinical attacks and the formation of new lesions which appears on MRI; though sub-clinically damage to neurons and axons, gradually diminishing the ability to sustain further events without acquiring disability.

Secondary progressive (SPMS)

It involves slowly progressing phase with or without superimposed relapses (SPMS) and about 50% of patient developed SPMS after 10–15 years disease duration. At this stage of the disease inflammation appears less prominent, and instead the accumulated damage to neurons and axons starts to show itself clinically as neurological progression.

Primary progressive (PPMS)

It is recognized by its progressive course from the outset, with a paucity of inflammation detected in the CNS compared with RRMS and about approximately 15% of MS patients develops PPMS.

Progressive relapsing (PRMS)

It involves relapse-like activity (PRMS) developed over time in about 5% of patients and patients have clinical and radiological evidence of inflammation, but no proven effective treatment (Pedotti R et al; 2003). [4]

Common factor of MS

The cause of this disorder is not known, but there are several factors linked to higher risk of developing the disease:

1) Genetics may play an important role and several alleles have been identified as predisposing factors for the disease, most of them related to the human leukocyte antigen (HLA) system;

2) MS is more prevalent in women than in men, suggesting importance of hormones in its pathophysiology.

3) Psychological stress has been implicated in triggering exacerbations, and some metaanalyses suggest a positive relationship between stressful events and higher risk for relapse and increased risk of new gadoliniumenhancing (Gd+) brain lesions (Perez-Nievas BG et al; 2010). [5]

4) Viral factor causes development or progression of MS, such as human herpes virus-6 (HHV-6) or Epstein Barr Virus (EBV). The bacterium Bordetella pertussis causes whopping cough in humans and produces pertussis toxin (PTx) as its main virulence factor (Weber MS et al; 2010). [6]

Common symptom of MS

A) Visual disturbances, which include eye pain, distortion or loss of vision in one eye, or impairment of color perception.

B) Difficulty in walking or performing tasks that require coordination.

C) Loss of sensation.

D) Fatigue and/or weakness.

E) Loss of bowel or bladder control (Ringold S et al; 2006). [7]

Experimental autoimmune encephalomyelitis (EAE) is currently the most commonly used animal model for the study of MS. This model causes inflammation and demyelination that is similar to the MS and to simulate different disease progressions where clinical symptoms (monophasic fashion), either acute or chronic, or in a relapsing form (Peiris M et al; 2007). [8] It is an inducible model of CD4+ T cell driven demyelination and axon damage. Myelin antigen reactive CD4+ T cells dominate the CNS infiltrate and initiate disease upon adoptive transfer into naive animals (Bettini M et al; 2009). [9]

Cytokines have a key role in the EAE process providing the necessary signals to activate T cell specificity against self-antigens and suggest that the T cells mediating EAE are of the Th1 phenotype, producing IL-2, IFN-E, and TNF-G is related to disease-promoting role, while TGF-M and IL-10 are related to the s recovery of EAE (Castro GM et al; 2004). [10]

In early phase of MS, autoreactive T cell crosses the blood brain barrier (BBB) and are estimated by local antigen presenting cell (APC). This results in release of cytokines such as IFN-E, TNF-G and IL-17 which initiates and perpetuates an inflammatory response by activating microglia and recruiting macrophages and BBB is breached and allow for the influx of more T lymphocytes, additional immune cells and the deposition of humoral components. Subsequently, this process leads to myelin destruction, induction of oligodendrocyte death, axonal degeneration and eventually to the development of severe functional deficits (Wiist S et al; 2008). [11]

Various EAE model for MS

A large number of EAE model exist, each parameter reflecting pathologies observed in CNS of MS patients. With different strains of mice, rats or other mammals and immunizing with different epitopes (peptides, whole proteins or complex mixtures like spinal cord homogenate) to achieve a range of pathological and temporal outcomes (Jones MV et al; 2008). [12]

Different types of EAE

Importantly, by stepwise reduction of the complexity of the antigenic material from crude brain tissue and protein extracts through various central myelin protein such as

i. Myelin basic protein (MBP) such as MBP1-37, MBP1-11, MBP1-9, MBP83-99.

ii. Myelin oligodendrocyte glycoprotein (MOG) such as MOG55-75.

iii. Proteolipid protein (PLP) such as PLP139-151.

iv. Myelin-associated oligodendrocytic (MOG) basic protein and 2´,3´-cyclic nucleotide 3´- phosphodiesterase.

Some of other myelin constituents such as neurofascin NF 155, other are expressed on myelin and axons such as the neuronal membrane protein neurofascin NF 186, the neuronal cytoskeletal protein neurofilament-M and the astrocyte-typical Ca+2binding protein S100M.

Neurofascin NF 186 caused axonal injury without enhancing inflammation and demyelination in MOGEAE. In contrast to MOG-EAE, contactin-2/TAG-1- specific T cells induced inflammatory lesions prefentially in the cerebral cortex and the spinal cord white and gray matter.

Timelines of milestones of the development of increasingly specific EAE models were:

i. Induction of EAE by transfer of total lymph node cells, isolated MBP-specific T cell-line cells or IL-23-dependent PLP-specific CD4+T helper IL-17 cells into naïve rats or mice, respectively, establishing distinct forms of adaptive transfer EAE

ii. Generation of transgenic mice with deletion or over-expression of pathogenetically relevant genes. For examples genes are those encoding T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, cytokines as well as neurotrophic factors and their receptors.

Induction of EAE models in different species

The most common mode of EAE induction is based on the injection of an encephalitogenic peptide, mostly MOG35-55 or PLP139-151, which is emulsified in CFA containing mineral oil and Mycobacterium tuberculosis strains H37RA, followed by intraperitoneal injections of pertussis toxin. The resulting phenotype depends mainly on the antigen source and the genetic background of the animal species and the strains used. For example, PLP139-151 induces a relapsing-remitting EAE in SJL mice, whereas MOG35-55 triggers chronic-progressive EAE in C57BL/6 mice. Crossing of C57BL/6 mice, which over-express MOG-TCR and MOG specific B cells, resulted in a severe form of EAE with inflammatory lesions of optic nerves and spinal cord. MOG-TCR transgenic mice backcrossed to SJL/J background develop a relapsing-remitting form of EAE with episodes altering between optic nerve, cerebellum and spinal cord. All EAE models are directly accessible to investigation of the immune and nervous system, which interact during the pathogenesis of the disease and which are both targeted by established and experimental therapies (Mix E et al; 2010). [13]

Various form of EAE has been induced depending on the immunizing neuroantigen and the redent strain used.

Lewis rat EAE showed a monophasic and selfremitting neuroinflammation in the CNS. Swiss- Jackson Laboratories (SJL) mice and Dark Agouti (DA) rats EAE showed a relapsing and remitting course of the disease.

MOG induced EAE in the C57BL/6 mice showed a chronic progressive course and other EAE model induced in NOD and DBA/1 mice showed a chronic progressive and severe course of secondary progressive/primary progressive form of MS (Donia M et al; 2010). [14]

Behavioural analysis of animals after EAE induction

EAE scoring (Clinical scoring)

Immunized animals are observe daily by measuring their body weight, feed weight and assessing clinical signs of EAE, starting at 7 days post immunization.

Grip strength test

Strength of forelimb and hindlimb are tested for grip strength by using Grip Strength Meter. In Grip strength, grip power of hindlimb primarily and majorly affected and forelimb less affected in EAE induced animals.

Rotarod performance

Rotarod test is used to assess motor co-ordination and balance of animals. Each mouse is placed on stationary rod and start the rotation of rod at 4 rpm and accelerated 4 rpm every 30s. The time and speed at which the mouse falls down from rod is recorded in EAE induced animals.

Open field activity test

Open field activity test is determined by using Open Field Photobeam Activity System. It is used to assess total activity and rearing activity of animals. Mice are placed into the clear plastic chamber for the assessment of interruption of infrared beams. In EAE induced mice, the total activity and rearing activity both are decreased consistently (Jones MV et al; 2008). [12]

Gene Expression Profile of EAE model

Microarray analysis technique is usually used for expression of gene in MS and EAE. Oligonucleotide microarray represents 11000 genes (6000 full-length gene and 5000 expressed sequence tags) which are expressed in inflamed spinal cord of EAE mice at the time of onset and the peak of disease determined by the clinical evaluation of the disease. The oligonucleotide microarray data are generally validated by the use of quantitative RT-PCR method to analyse a subsets of gene either down-regulated or up-regulated. The microarray technique enables to identify differentially express gene with a high degree of validity. Increased expression of genes for extracellular matrix, cell adhesion molecules and molecules involved in cell division, death and transcription, differential regulation of molecule involved in signal transduction, protein synthesis and cell metabolism in inflammation.

Genes are group according to their presumed biological functions and to the fold change in gene expression using normal spinal cord as a baseline (Ibrahim SM et al; 2001). [16]

Neural dysfunction in Spinal Cord during EAE

The protein levels of plasma membrane Ca2+ ATPase 2 (PMCA2), an essential ion pump expressed exclusively in grey matter and involved in Ca2+ extrusion, synapsin IIa and syntaxin 1B, important regulators of vesicular exocytosis decreased coincident with the onset of clinical symptoms and changes in the expression of several other ion pumps, vesicular proteins, mitochondrial enzymes and sodium channels also occurred in disease. Glutamate and Kainic acid significantly reduced PMCA2 mRNA levels. In EAE pathogenesis, suppresses neuronal PMCA2 expression leading to Ca2+ dyshomeostasis at initial clinical phases.

Expression of Glutamate-mediated changes in neuronal dysfunction in EAE symptoms

Intracellular Ca2+ level are often increases often and causes cell dysfunction, injury and death in CNS, including Multiple sclerosis, Ischemia, Alzheimer and Parkinson disease. In EAE, loss of voltagedependent channel-mediated Ca+2 balances depicts defect in mechanism that regulate the Ca+2 entry, extrusion or intracellular sequestration. PMCAs are major pumps modulating Ca2+ extrusion and sarcoplasmic and endoplasmic reticulum Ca2+- activated (SERCA) ATPase isoforms causes sequestration of Ca2+ into the endoplasmic reticulum. PMCA2 expression decreases in early phase, which is followed by a reduction in SERCA2 transcript levels clinical phases. The PMCA gene family encodes four isoforms, causes maintenance of basal Ca2+ levels. PMCA2 is expressed primarily in brain and heart, in a region- and cell-type specific manner and in pathological condition of EAE, glutamate receptors are over stimulated and overload of intracellular Ca2+ level.

Role of Aberrant neurotransmitter exocytosis in EAE

The level of Synapsin and Syntaxins are decreases in EAE. Synapsin and Syntaxins are vesicle protein and exhibit important role for exocytosis in presynaptic terminals in clinical symptom of EAE. Synapsins are required for the tethering of vesicles to each other and to cytoskeletal proteins (actin). It is also essential for the formation, maintenance and regulation of the reserve synaptic pool in the vicinity of the active zone and the decreased level of synapsin causes reduced number of synaptic vesicles distal to the active zone, exhibiting synaptic fatigue. Syntaxins belongs to SNAREs class (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and essential for the docking and fusion of synaptic vesicles. Vesicleassociated SNAREs (synaptobrevin) and target membrane-associated SNAREs (syntaxins and SNAP-25) induces membrane fusion. The level of syntaxin 1B and SNAP-25 are reduced but reduction in syntaxin 1B occurred at onset of symptoms whereas SNAP-25 at a later clinical stage. The expression of synapsin and SNARE proteins interfere with normal neuronal communication during EAE.

Role of Mitochondria in cellular injury at EAE

Mitochondrial function plays important role in cellular injury due to the increased level of mitochondrial cytochrome c oxidase subunits IV and Vb. Cytochrome C oxidase used as a marker for neuronal metabolic activity, especially in oxidative stress. Elevated level of cytochrome c oxidase express in Alzheimer's disease, following traumatic brain injury and preceding apoptotic death of spinal cord motor neurons after sciatic nerve avulsion and also reflect aberrant energy metabolism and oxidative damage in the spinal cord during EAE.

Role of sodium channel in impulse conduction during EAE

Na+ channel also played an important role in EAE and MS. The expression of Na+ channels decreases that cause impairment of nerve conduction at advanced stages of EAE. Expression of sensory neuron-specific sodium channels in Purkinje cells changes during EAE symptoms (Nicot A et al; 2003). [17]

Role of Vitamin D

Vitamin D play important role in the immune system particular on T cell-medicated immunity. Receptor of Vitamin D is found on the surface of T lymphocyte and macrophage cells and highest concentration is found in the immature immune cells of the thymus and the mature CD+8 T lymphocytes (Deluca HF et al; 2001). [18]

Animal model of Autoimmunity

In autoimmune disease, T cells attacks on self tissue. T cells are thymus-derived cells and subsets of T cells called the T-helper (Th) cells that express CD+4 markers on their surface and also played an important role in EAE and diabetic model to mice. In these models, level of CD+4 T cells are decreased. EAE is mediated by CD+4 T specially Th1 type (IL-2, TNF- G, IFN-E) cells that recognize protein in CNS but suppress the Th2 type (IL-4, IL-10) cells.

Vitamin D and Immune system

Vitamin D regulated Ca+2 homeostasis and causes bone formation and resorption. The active form of vitamin D (1,25-(OH)2D3) affected on the immune system. In EAE, level of vitamin D is decreases and upregulated the Th1 cells by increasing proliferation and cytokine (IL-2, TNF-G, IFN-E) secretion. Vitamin D receptors are found in the nuclease of cells and bound to the (1,25-(OH)2D3) and regulated the transcription of targeted genes. Vitamin D act as transcriptional regulator of Th cell cytokine synthesis as a regulator of STAT 1, 4 or 6, as a regulator of GATA-3 or regulate Th cells differentiation (Cantorna MT; 2000). [19]

Cytokines signaling pathways in EAE and Multiple sclerosis

CD4+ T helper (Th) cells play a critical role in MS and EAE (Experimental Autoimmune encephalomyelitis is an animal model for MS). The major types of Th cell are type 1 helper T (Th 1) cells that produce interleukin (IL-2), tumor necrosis factor-G (TNF-G), IFN-E and type 2 helper T (Th 2) cells that produce IL-4, IL-5, IL-10 and IL-13 and type 3 helper T (Th 3) cells that primarily secrete transforming growth factor-M (TGF-M).

Intracellular signaling mechanisms provide the link between the bindings of the cytokine with its receptor and the effect of the cytokine on cellular function. The Janus kinase and signal transducer and activator of transcription (Jas/STAT) family of transducer/transcription-activating factors play a critical role in the signaling of many cytokine receptors. Cytokine binding to the specific receptors activates the Jas molecule associated with the receptor causing phosphorylation of tyrosine residues. This facilitates binding of STAT proteins to the phosphorylated receptor, which dissociates from the receptor and activates transcription of genes containing specific cis-regulatory STAT-binding sequences.

Role of individual cytokines during demyelinating process in MS and EAE

Cytokines play an important role in the pathogenesis of MS because of altered level of cytokines in the CNS and peripheral mononuclear cells of MS patients.

Interferon-: (IFN-:)

It is produced by T cells and Natural killer cells and causes activation of mononuclear cells, differentiation of T cells to Th1 type, induction of MHC I and MHC II expression and apoyptosis of T and other cells types. IFN-E is expressed in the CNS at the onset of EAE and its expression increases during the peak of disease and decreases during disease remission. Over-expression of IFN-E in the CNS causes progressive demyelinating disease. INF-E has an activating role in inflammation in EAE and MS.

Tumor Necrosis Factor-< (TNF-<)

It is produced by activated mononuclear phagocytic cells, natural killer cells, B cell, activated T cells, astrocytes and microglia in the CNS. TNF-G express Th1 type responses and induced activation of variety of cell types and expression of adhesion molecules, chemokines and cytokine. Role of TNF-G is characterized by significantly increases inflammation and demyelination in EAE and MS. TNF-G has a direct effect on the induction of oligodendrocyte apoptosis and demyelination and also promoted the proliferation and induced cell death of oligodendrocyte progenitor cells.

Interleukin-12 (IL-2)

IL-2 is produced by monocytes and the dendritic cells and express Th1 type responses. Over-expression of IL-12 in the CNS resulted in increased inflammation and cellular infiltrates.

Osteopontin (OPN)

Osteopontin is a pleotrophic cytokine that activates macrophage chemotaxis, promotes Th1 responses and activate B-cells. OPN increases plaque formation in the brain of MS patients.

Interleukin-18 (IL-18)

IL-18 is synthesized as an inactive precursor protein and its level increases in the serum of MS patients.

Interleukin-1 (IL-1)

It is produced by monocytes, macrophages, endothelial cells, B cells and activated T cells and upregulated in the CNS and causes increased neuronal cell death and edema. Neuronal apoptosis and neurotoxic effect appear due to expression of iNOS.

Interleukin-3 (IL-3)

IL-3 is a cytokine growth factor produced by CD+4 T cells and microglia. IL-3 mRNA is up-regulated in the CNS of MS patients. Systemic over-expression of IL-3 in the CNS causes degeneration and vacuolation of neurons.

Interleukin-6 (IL-6)

IL-6 is produced by mononuclear phagocytes, vascular endothelial cells and fibroblast and promoted Th1 cytokines. It is important for the growth and differentiation of B-cell and also upregulated in the CNS during the induction phases of disease.

Interleukin-4 (IL-4)

IL-4 is produced by CD+4 Th2 cells and involved in differentiation and growth of B-cells. IL-4 inhibits the activation of Th1 cells and decreases the production of IL-1 and TNF-G. IL-4 amplifies the Th2 response through activation of the intracellular signaling factor STAT6, which induces transcription of Th2 related genes.

Interleukin-10 (IL-10)

IL-10 is produced by monocytes, macrophages, B cells and Th2 cells. It inhibits the production of IL-1 and TNF-G cytokines by macrophages and also reduces MHC II and co-stimulatory molecules expression.

Transforming growth factor (TGF)

The TGF family of molecules has 2 principal members that is TGF-G and TGF-M. TGF-G is a polypeptide growth factor for epithelial and mesenchymal cells and TGF-M is a growth factor for cell survival. TGF-M is produced by T cells by T cell, activated monocytes, astrocytes and microglia. It inhibits proliferation of T cells, maturation of cytotoxic lymphocytes, natural killer cell and also inhibits the activation of macrophages and activates the pro-inflammatory cytokines (Imitola J et al; 2005). [20]

Therapies for EAE and MS

The goal of MS is to produce a period of stabilization of symptoms and interrupt the progress of disease. There is no current treatment that can alter neurological damage (Hadaway LC; 1985) [21] but only very few therapeutics that are successful in preclinical EAE trials have shown similar efficacy in MS patients. The majority of new treatments are either less effective in MS patients and worsened disease or caused severe adverse events (Friese MA et al; 2006). [3]

Conclusion

At present, the exact cause of MS is still unknown in pre-clinical EAE studies, which involve in multiple immune cell types, target cells and pathways but conventional EAE play an important role as first line animal model system for the development of novel treatment for MS. Gene expression and cytokines levels play an important role in MS. Oligodendrocyte microassay is a technique which are used for detection of gene expression contributing to earlier stage of diseases. It represents 11000 genes (6000 full-length gene and 5000 expressed sequence tags) in inflamed spinal cord of EAE induced animals, 213 genes are regulated differentially and 100 genes are showed consistent differential regulation throughout the disease. Gene expressions are upregulated in inflammation because increased expression of immune related molecules, extracellular matrix, cell adhesion molecules and those molecules which are involved in cell division and transcription. Alteration in cytokine balance also involved in autoimmune disease pathogenesis. Th2 cytokines (IL-4, IL-5, IL- 10 and IL-13) appears to play a protective role in the survival and differentiation of neurons and oligodendrocyte in the CNS during demyelination injury and Th1 cytokines (IL-2, TNF-G AND IFN-E) play a degenerative role in MS patient by the activation of mononuclear cells, differentiation of T cells, B cell class switching and apoptosis of T and other cell types. The future of multiple sclerosis therapy looks bright, but increasingly complex. There are number of agents who are used for the treatment of MS through orally and parenterally and some are in different stages of investigation or awaiting approval by federal agencies. All of these medications have demonstrated partial efficacy along with different side effect profiles.

Neurodegeneration occurs through noninflammatory mechanisms but depend upon prior inflammation. In these case, anti-inflammatory therapies are best prevent the progression of disability in early course of the disease, before the cascade of events that leads to axon degeneration has been irretrievably established. This raises the difficult prospect of exposing well non-disabled young adults to potentially toxic treatments; a decision made all the more complex by the unpredictable nature of multiple sclerosis. Finally, these drugs continue to inform basic science, revealing aspects of the pathogenesis of multiple sclerosis and reminding us of the complexity of the human immune system.

Conflict of Interest

NIL

Source of Support

NONE

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5 Table 6

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