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Neuromyelitis optica is a severe demyelinating autoimmune disease. The recent discovery of NMO-IgG positive serum on most patients with the disease , has given new momentum to the study of how the disease develops and the exact mechanisms of development. A significant feature of NMO is the activation of complement which causes loss of AQP4 and the formation of lesions. This experiment injects NMO-IgG into mouse brain tissue expressing AQP4 or AQP1 along with human complement. The results showed that when NMO-IgG was injected onto AQP4 expressing cells with human complement a greater number of cell death was recorded. Conversely there was no increase in cell death when no human complement was added. As a control the experiment was also carried out with AQP1 cells which showed no signs of increased cell death in either conditions.
Background of Neuromyelitis optica Page 2-3
Background of Aquaporins Page 3-4
Production of antibodies Page 4-5
Activation of complement Page 5
Methods Page 5-7
Hypothesis Page 5
Results Page 7-8
Discussion Page 8-
Keywords: Devics; Neurolmyelitis; Complement; Aquaporin; Immunoglobulin.
NMO- Neuromyelitis optica
MRI- Magnetic resonance imaging
FAB-Fragment antigen binding
Fc Region- Fragment Crystalizable region
Background on NMO
Neuromyelitis optica (NMO) is a progressive autoimmune disorder. It is an inflammatory demylinating disease of the spinal cord and optic nerve called myelitis and optic neuritis respectively.(1) They can occur together or separately. 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. 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)
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
NMO was first discovered in the late 19th century by Eugene Devic. 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 and where 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). 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). It is 73% sensitive and can distinguish NMO from classical MS with an accuracy of 91% (13). This has lead to the belief the onset of disease may be caused or exacerbated by the immune system (1). Furthermore the development of MRI has allowed the lesions to be viewed in vivo on living patients.
Treatment of NMO
Currently 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).
Of late there has been increased interest in the role of aquaporins 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) Aquaporins (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). 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 proving 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). 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).
Production of antibodies.
The human body's defense mechanisms are 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) One mechanism of the innate immune system is the use of antibodies. These belong to the immunoglobulin super family. (14) They consist of 2 upper light chains and 2 heavy chains. 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 the different antibodies depending on the stimulus which caused it to mature.
The main Ig isotype we shall discussed 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 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 way in which IgG works is via opsonization and activation of the complement system.
When an antibody detects a pathogen of correct specificity it bind to it with the FAB regions of the light chains. This leave 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.
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. 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.
What is not clear is whether the presence of NMO-IgG is enough to cause demyelination or whether it requires other factors present in NMO typical patients, but which we have not discovered yet. The aims of this study is therefore to determine whether NMO-IgG can cause lesions and inflammation in mouse tissue with or without human complement.
The hypothesis for this experiment is that there will be a statistically significance in the increase of cell death when NMO-IgG is injected into cells expressing AQP4 along with human complement.
Isolating IgG from patient serum: Serum was obtained from five patients (P1 - P5) with an established diagnosis of NMO and strong AQP4-Ab serum positivity. We also used three different pooled non-NMO control sera (C1 - C3). Clinical details of the NMO patients are shown in Supplementary Table 1. Five ml of each serum (P1 - P5) or pooled serum (C1 - C3) was diluted 1 in 4 in PBS and loaded onto a Protein-A column (Sigma, Poole, UK). After washing with PBS the bound IgG was eluted with glycine pH 2.3 and immediately neutralised in 1 M Tris pH 8.0. The positive fractions were pooled and dialysed against Hartmann's solution. They were concentrated by dialysis against polyethylene glycol, dialysed again against Hartmann's solution and stored at 4oC. IgG concentration in the samples were 6 - 38 mg/mL. We term IgGNMO the total IgG isolated from the serum NMO patients (that contains AQP4-Ab) and IgGCON the total IgG isolated from the serum of non-NMO subjects. The presence or absence of AQP4-Ab in each sample was confirmed by immunocytochemistry on cultured Chinese Hamster Ovary (CHO) cells expressing AQP4 or AQP1 (as control). AQP4-Ab titres were independently measured by fluoroimmunoprecipitation and cell-based assays (Supplementary Table 2). Each mouse was injected with IgG from a single sample (P1 - P5 or C1 - C3). In experiments involving several mice, we used at least three different IgGNMO samples (from P1 - P5) and at least two different IgGCON samples (from C1 - C3).
Human and mouse complement: Non-haemolysed blood was collected from human volunteers or CD1 mice in a plain glass tubes and allowed to clot at room temperature for 30 min. The samples were centrifuged at 1,000 r.p.m. and the serum supernatant was collected, aliquoted and stored at -80 0C. Serum collected in this way preserves complement activity and is termed human (hC) or mouse (mC) complement in the manuscript.
Cell culture: CHO K1 cells stably transfected with plasmids encoding M23 AQP4 (CHO-AQP4) or AQP1 (CHO-AQP1) were grown on coverslips in F12 medium with 10 % FBS (Invitrogen, Paisley, UK). More than 95% of cells expressed the respective proteins in their plasma membranes (Saadoun et al., 2005).
Immunocytochemistry: For AQP1, AQP4 and C5b-9 immunostaining, CHO cells were washed in PBS, fixed in 4% neutral buffered formaldehyde (Sigma, Poole, UK) for 5 min followed by rabbit anti-AQP1 (1:200, Chemicon-Millipore, Livingstone, UK) or rabbit anti-AQP4 (1:200, Chemicon-Millipore, Livingstone, UK) or rabbit anti-C5b-9 (1:100, Abcam, Cambridge, UK) primary antibody for 1 h at 25 0C. Cells were 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 or mC, and where stated 0.25 mg/mL C1inh (Biopur, Bubendorf, Switzerland) final concentration. After 1 h, some coverslips were immunostained for C5b-9. 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 f
uorescent red. Coverslips were examined using a BX-51 Olympus epifluorescence microscope and the number of red and green cells were counted.
Fig 2: AQP4 staining with Dapi and Red
Fig 1: AQP1 staining with Dapi and Red
Fig3: Control IgG + AQP1 Fig 4: Control IgG + AQP4
Fig 5: NMO- IgG + AQP1 Fig 6: NMO-IgG + AQP4