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Horror autotoxicus was the term first given to describe autoimmune diseases by Paul Ehrlich. He however believed it was impossible for the human body's immune system to attack itself due to the horror it would create (14). We now know that this is not the case. An autoimmune disease is normally caused by an inappropriate reaction of the immune system towards its own cells. Certain subsets of T cells, coined T regulatory cells, exert great control over the immune system (14). Unlike immunodeficiency diseases autoimmune diseases are generally caused by polygenic mutations as opposed to single gene mutations, although there are exceptions (14). Currently over 80 autoimmune diseases have been categorised (25). In the past autoimmune diseases were classified as B or T cell mediated. Now however this classification is no longer used as it appears both are required for autoimmunity (14).
Genetic and environmental susceptibility.
There is great knowledge to link both genetic and environmental factors with increased susceptibility to certain autoimmune diseases. Many studies have shown a higher incidence of autoimmune diseases in monozygotic twins than dizygotic twins. However as mentioned above the polygenic nature of autoimmune diseases means even if someone has one of the required mutations it is unlikely to be severe as it normally required several mutations for a autoimmune response (27). It is generally accepted that although a person may have the correct genetic mutations for an autoimmune disease the disease tends to only manifest after a certain environmental trigger (27). This is shown by studies which have shown that the incidence of both Multiple sclerosis (MS) and type 1 diabetes changed as the members of the population moved to different locations (27,28,29).
Pathogenesis of Neuromyelitis optica
Neuromyelitis optica (NMO) is an idiopathic autoimmune disorder(4). It is an inflammatory demylinating disease of the spinal cord and optic nerve called myelitis and optic neuritis respectively.(1) Symptoms can occur together or separately(22). Optic neuritis is known to cause complete visual impairment with a low incidence of recovery. (2) Myelitis is more severe as it affects the spinal cord and depending on the location of the lesions can affect a large part of the lower body causing pain in the limbs and bladder paralysis(1). In severe case if the lesions travel upwards they can damage the brain stem and cause neurogenic respiratory failure. (2)
It has also got a very high rate of relapse, 85%, especially when compared to Multiple Sclerosis. (4) Women compromise more than two thirds of reported cases of NMO with a mean age of onset in the late 30's(20), ten years later than the average onset of multiple sclerosis(MS)(22). Recent studies in population variance have shown that the disease is far more predominant in Asians than in Caucasians with 30%-40% of MS cases in Asia though to be attributed to NMO(36). As with most autoimmune diseases there appears to be an abundance of environmental triggers. Many case studies have been published describing patients who had suffered from either syphilis, HIV type 1, Chlamydia and varicella, and have then gone on to develop symptoms of NMO (39,40,36). These are described as post infectious NMO.
T2-weighted (left) and T1-weighted (right) sagittal MRI of the cervical spinal and upper thoracic cord in a patient with
NMO, showing longitudinal extensive transverse myelitis with swelling, necrosis and linear gadolinium
Discovery of NMO and advancement
NMO was first discovered in the late 19th century by Eugene Devic(21). It was after viewing several patients all whom suffered from loss of vision, paralyses and loss of bladder control. The most striking characteristics observed by Eugene Devic was that the same patients developed non organ specific autoantibody conditions. (3) The conditions were found to be a result of inflammation in the spinal cord and optic nerve. This was discovered during postmortems which showed immunoglobulin deposition and complement activation. (3) For many years after Eugene Devic recorded his findings, NMO was still mistakenly classed as a subtype of multiple sclerosis (1). It was not until Clifford Allbutt in the late 19th century, studied Eugene's work and associated the symptoms with a separate disorder (12).
The biggest achievement on NMO so far has been the discovery of the NMO-IgG autoantibody. It was discovered in 2004 by Lennon VA et al (24). They reported a detection rate of almost 75% in patients already diagnosed with NMO and almost 50% in patients who were deemed to be high risk. In other scientific research it has been shown to be 73% sensitive and can distinguish NMO from classical MS with an accuracy of 91% (13). As NMO-IgG is quantifiable it allows for the measurement of the effectiveness of treatment as well as disease progression (24). NMO-IgG is an autoantibody specific to the Aquaporins 4 protein found on cell surfaces in the brain.
Differences between NMO and MS
As mentioned above for many years NMO was mistaken as MS(1), and even today many in Asia still refer to it as optic-spinal MS (36). The main distinction NMO has over MS is the detection of an autoantibody to the water channel protein aquaporin 4 (24). This was only discovered in 2004, but there has also been clinical and neurological evidence supporting this theory before the discovery of the autoantibody. When viewed with MRI typical MS often appears as widespread regions with large amounts of inflammation in the white matter, they also appear more commonly in cerebellar and pervientricular regions (36,38) . This is contrary to NMO where the lesions are typically seen in the diencephalon and very rare to find lesions in the white matter for patients suffering from NMO (40).
Clinical diagnosis criteria
In the past the diagnosis was based on clinical characteristics such as; optic neuritis, bilateral motor deficits, pain transverse myelitis and many other symptoms (12). Recently however an auto-antibody for AQP4 has been discovered and called NMO-IgG (6). It is significantly accurate and is now considered an accurate marker for NMO (12).. This has lead to the belief the onset of disease may be caused or exacerbated by the immune system (1). Current criteria, requires the presence of both optic neuritis and myelitis (4) (Table 1) as well as other factors. Some believe the factors are too strict, T saida 2009 (30) believes this criteria puts patients at harm as it disadvantages early diagnosis and reduces the possible benefit of treatment.
Table : Criteria for diagnosis of NMO. Adapted from Wingerchuck (2006) (4)
Treatment of NMO and prognosis
Currently treatment for NMO is still limited and similar to that used in MS(15). There are two long term treatments widely used. These are treatment with interferon beta and immunosuppressive therapy(15). Current research suggests that interferon treatment is significantly less effective than immunosuppressive treatment (16). Both treatments however still fail to halt disease development(15). A new drug being trialed, Rituximab, is a monoclonal antibody which works against CD20+ cells and has shown promising results (17). Prognosis for NMO is very poor. 50% of patients become wheelchair bound and 62% become functionally blind at 5 years(15). The sooner the diagnosis the better the outlook for the patient.(23). This is why characterizing the role of NMO-IgG is so vital.
Aquaporins general properties
Due to the discovery of NMO-IgG there has been increased interest in the role of Aquaporins (AQPs) within the brain and the difference between the different isotopes. It has become increasingly important to fully understand the functions and roles water channels have within the brain as strong evidence is emerging suggesting the AQP4 molecule is targeted in NMO. (6) AQPs are a family of transmembrane molecules with 6 membrane domains forming a ring with a pore in the middle with which water can be transported in both directions (5)(figure 2). The majority of work carried out on AQPs has been done on AQP null mice. Experiments by Song, Y. et al (2002)(31) showed that deletion of the AQP5, an AQP known for its role in sweat gland function, led to decreased volume and hypertonic fluid. In general AQPs increase water transport down osmotic gradients, with AQP null mice unable to concentrate urine (5,32), and therefore help to regulate water movement.
Figure 2: a) Shows the monumeric structure of AQPs with the six different domains labeled H1-H6. There is a small gap between them to allow water molecules in and out. b) Shows the tetrameric arrangment of AQPs, made up of 4 monomers is can allow water molecules in and out(5).
Discovery of Aquaporins
AQPs were initially discovered by Petre Agre in 1988 (7) who showed that frog oocytes which expressed AQPs, where far more susceptible to water lysis than thoses not expressing AQPs, hence showing improved water transport (8). Furthermore it has been shown that where brain oedema has occurred the astrocytes associated with that area will show increased expression of AQPs on the cell surface (11). Thus far over 10 different subtypes of AQP have been isolated in mammals (5).The main AQP in terms of abundance in the human brain and importance in NMO is AQP4(11). Brain AQP4 is mainly found in astrocyte foot processes, brain parenchyma and major fluid compartments (9).
M1 vs M23
AQP4 is further subdivided into an isoform of M1 and M23 (10). The two isoforms, M1 and M23, are due to translation initiation differences in the N termini of the first and second methionine amino acid respectively (18). M23 is also 3 times more abundant in the human brain than M1 (19). Functionally no differences have been noted between the 2 isoforms, however they do appear to alter the organization of AQP4 on the intramembrane(10) . This is thought to be due to the M23 isoform forming large square arrays with abundant cross bridges whereas the M1 isoform appears to restrict square array assembly (10). Recent experiments have shown that addition of NMO-IgG to AQP4 expressing cells have resulted in inflammation (6) and this is now the current point of interest.
The immune system
The innate and adaptive immune system
The immune system is described as a collection of mechanisms inside an organism which protect against disease by identifying and eliminating a diverse number of pathogens(34) The human body's immune system is grouped into two main categories, the innate and the adaptive immune system. Whilst they are grouped they work together and rely on one another to work in unison and provide maximum protection for the human body (14). The innate immune system is antigen non-specific and has a rapid response (14). It is the bodies first line of defence. Included in the innate immune system are all the body's natural barriers, phaygocytes and complement among others (14). The adaptive immune system on the other hand is far more antigen specific but has a much slower response of days. The adaptive immune system mainly utilises B and T cells as its main immune components along with antibodies.
Antidbody Structure and function
One mechanism of the adaptive immune system is the use of antibodies. These belong to the immunoglobulin (Ig) super family. (14) They consist of 2 upper light chains and 2 heavy chains (figure 4). Depending on their isoform they can be found on the surface of cells or circulating freely within the blood vessels. Immunoglobulins are produced from mature B cells and come in the following isoforms, IgA, IgD, IgE,IgG and IgM and each isotope is further divided into subclasses. They are distinguished by differences in their constant heavy chain regions. This is thought to give each isoform its different biological function, with variation in the lighter upper chains resulting in different epitope specificity. When a B cell matures it will begin to produce different antibodies depending on the stimulus which caused it to mature (14).
The main Ig isotype we shall discuss is IgG. It is secreted out of the cell and equally present in intra and extra vascular surfaces. (14) It has the highest half life of all the Ig molecules with an average life span of 23 days. Furthermore IgG is the only antibody which can pass through the placenta to the fetus giving immunity for first weeks of life. (14) The main mechanisms by which IgG works is via opsonization and activation of the complement system. When an antibody detects a pathogen of correct specificity it binds to it with the FAB regions of the light chains. This leaves the lower heavy Fc region sticking out. Macrophages have receptors which can detect the Fc region and will engulf anything attached to that antibody. This is called opsonization and comes from the Greek opsonin which means to prepare for eating.(14) Furthermore once an antibody is bound both to an antigen and a phagocyte it activates the phagocytes and causes release of Interleukins and cytokines, which mediate an inflammatory response. This is thought to be the cause of inflammation seen around the site of lesions in NMO patients.
Figure 4: Schematic showing the structure of different immunoglobulin molecules (34).
Another way in which antibodies protect the human body from disease is by activating complement. The complement system is a set of 20 or so proteins circulating in the blood. It can be activated via three pathways, the alternate pathway, the lectin pathway and the classical pathway. Eventually all the pathways converge but the initiation is different. Immunoglobulins are excellent triggers of the classical pathway. It was named so as it was the first of the 3 pathways to be discovered. (14) The main proteins are called C1 through to C9, the numbers do not denote order of activation rather the order in which they were discovered. When two IgG molecules are bound to an antigen they cause C1 to cleave C3 and thus begin the cascade. The next complex will result in the cleavage of the next complex with most binding to the pathogens surface. Once C9 is reached a membrane attack complex (MAC) is formed (figure 4). This literally punctures the surface of the pathogen and causes lysis. (14) Again it is this lytic function which is thought to be the cause of lesions and demyelination.
Figure 4: Diagram shows the outlines of the 3 different pathways taken in activating complement starting from recognition and ending in lysis (35).
Isolating the IgG from the serum of patients.
The Serum used in this experiment was obtained from five different patients labelled P1 through to P5. Each patient had an established diagnosis of NMO and was also diagnosed with a strong AQP4-Ab serum positivity. We used three different pooled non-NMO control sera labelled C1 to C3 accordingly. We termed IgGNMO as the total IgG isolated from the serum of NMO patients (that contains AQP4-Ab) and IgGCON as the total IgG isolated from the serum of non-NMO participants.
Non-haemolysed blood was collected from human in a plain glass tubes. This was then allowed to clot at room temperature, approximately 25oc, for 30 min. The samples were then centrifuged at 1,000 r.p.m. The serum supernatant was then collected, aliquoted and stored at -80 0C, until required. When serum is collected in this manner it preserves complement activity, and is termed human (hC).
The CHO cells we used where of the K1 variey. They where transfected with plasmids encoding M23 AQP4. These cells were termed (CHO-AQP1). They were then grown on coverslips in F12 medium (Ham mixture) with 10 % FBS (Invitrogen, Paisley, UK). More than 95% of cells expressed the respective proteins in their plasma membranes.
The CHO cells expressing AQP1 and AQP4 were then washed in PBS and then fixed in 4% neutral buffered formaldehyde (Sigma, Poole, UK) for 5 minutes. We then added rabbit anti-AQP1 (1:200, Chemicon-Millipore, Livingstone, UK) or rabbit anti-AQP4 (1:200, Chemicon-Millipore, Livingstone, UK) primary antibody respectively and incubated them for 1 h at 25 0C. The Cells were then washed with PBS and incubated with AlexaFluor-linked goat anti-rabbit secondary antibody (1:200, Invitrogen, Paisley, UK). For AQP4 immunostaining with IgGNMO, live cells were washed with PBS, exposed to IgGNMO (1:200,15 min, 4 0C in PBS + 5 mM dextrose), washed with PBS, post-fixed in 4% neutral buffered formaldehyde (Sigma, Poole, UK), washed with PBS and incubated with Texas-red-linked anti-human IgG secondary antibody (Vector Laboratories, Peterborough, UK). After secondary antibody incubation, the coverslips were washed with PBS and mounted in Aquamount medium with DAPI (Vector Laboratories, Peterborough, UK). Coverslips were examined using a BX-51 Olympus epifluorescence microscope.
Complement activation and cell viability assays:
CHO cells on coverslips were exposed at 37 0C to F12 medium without serum containing (by vol.) 5 % IgGNMO or IgGCON and 5 % hC. After 2 h the cells were stained with a LIVE/DEAD® cell viability kit (Molecular Probes - Invitrogen, Paisley, UK) according to the manufacturer's instructions. Live cells stain fluorescent green and dead cells with damaged plasma membranes stain fuorescent red. Coverslips were examined using a BX-51 Olympus epifluorescence microscope and the number of red and green cells were counted.
The cells where first stained using Texas-red and DAPI to show the effects of the different conditions on the different cells and how the controls would look.
AQP4 red+blue1aqp1 red and blue 1
Figure : AQP1 (left) and AQP4 (right) visualised using Texas-red-linked anti human IgG and DAPI which stained cell nuclei.
Figure : Live cells expressing AQP1, (left) and AQP4, (right) incubated with control IgG and stained with Texas-red-linked anti human IgG and DAPI which stained cell nuclei.
Figure : Live cells expressing AQP1 (left) and AQP4 (right) incubated with NMO-IgG and stained with Texas-red-linked anti human IgG and DAPI which stained cell nuclei.
As mentioned above the cells where stained using LIVE/DEAD® with healthy living cells fluorescing green and cells which were dead or had damaged cell membranes fluorescing red. They were viewed under and microscope and the still pictures were taken. The cells where then counted up based on colour of fluorescence. This data was put into a table and statistically analysed.
NMO is a severe autoimmune disorder (4). The pathogenesis is believed to be the formation of an immunoglobulin which attacks the AQP4 proteins on the surface of astrocytes(6). Like most autoimmune disorders the exact mechanism is only speculation. The discovery of NMO-IgG by Lennon VA et al. 2004,(24) has allowed more accurate detection of NMO, but does not help to explain how the production of the immunoglobulin leads to deymylination. Recent theories put forward have implicated the activation of complement in the disease (6). Therefore this study focuses on establishing a true connection between NMO-IgG and the death of cells expressing AQP4, the most abundant AQP in the brain (11). This study focused on finding signs of cell death or damage in the cells which had been incubated with NMO-IgG from sera positive patients.
4.1 Interpretation and analysis of results
4.1.1 AQP4 expressing CHO cells in the presence of Con-IgG and human complement.
This was the only test conducted with Con-IgG. The reason for this, which is explained in more detail below, is that AQP1 cells showed no reaction in NMO-IgG conditions. This condition mimics a healthy person, thus using the LIVE/DEAD kit we can see very small numbers of cell death. The highest recorded cell death in any sample with these conditions was 16.30%. This can be attributed to natural cell death.
4.1.2 AQP1 expressing CHO cells in the presence of NMO-IgG and human complement.
This experiment was conducted to see if NMO-IgG had any affects on AQP1 proteins. Current research all supports an AQP4 antibody and no experiments have implicated AQP1.Therefore as was expected there was no increased cell death over the control using AQP4. This was also the reason why no Con-IgG was used with AQP1 as it produced no results with NMO-IgG and therefore would have been a waste of time and resources to also do a control, which would inevitably come up blank. As research had shown only AQP4 was affected using AQP1 in the study was in essence its own control as it showed cell death could only be attributed to AQP4.
4.1.3 AQP4 expressing CHO cells in the presence of NMO-IgG and human complement.
Research indicated that NMO-IgG targets AQP4 proteins and in turn causes deymylination. This was the purpose of this study to further support this theory. In this sample the average cell death was 41.85%. This is significantly higher than both the previous experiments. This shows that there is a marked increase in cell death in the presence of NMO-IgG in cells expressing AQP4.
Due to time and resource constraints there where many controls which could have been used in this study but where not. This does not discredit the results found but only means that to progress the results further these controls would need to be taken into account. By identifying controls it not only allows for better understanding on how to develop the results but allows potential influences to be considered and evaluated.
4.2.1 M23 and M1 expressing cells.
The first such was the use of only M23 AQP4 expressing cells. The reason for this is that currently there is no available fluorescent antibody marker for the M1 isoform. In the human brain the two isoforms found are M1 and M23, with M23 being significantly more abundant(10) however the results are not expected to differ greatly as they appear to be functionally identical (10) although they where only conducted in-vitro with no functionality tests done in-vivo.
4.3 In vivo study
Whilst the results gathered are encouraging in supporting the hypothesis that NMO-IgG can activate complement in vitro, it does not help us to understand the effects in vivo. Astrocytes are know to be very resistant to complement lysis as they contain many complement regulatory proteins such as CD46, CD55 and CD59
4.4 General NMO
Therefore, AQP4 antibody is indispensable in the diagnosis and treatment of NMO