From the National Magazine

LUPUS a disease of defective waste disposal, but why is it so difficult to diagnose?

Dr Paul Eggleton, Senior Lecturer, Autoimmunity and Inflammation, Musculoskeletal Group, Institute of Biomedical and Clinical Science, Peninsular College of Medicine and Dentistry, Exeter, Devon

The symptoms of systemic lupus erythematosus (SLE) can present in many different ways, from mild migraines to acute kidney failure. But why does the disease produce so many diverse symptoms and why does it take so long to diagnose someone with lupus? The answer to this frequently asked question perhaps lies in the way the disease develops within the body and provokes the immune system to generate large amounts of antibodies against the sufferers own cells and organs. But can we take advantage of the over stimulation of the body's defence mechanisms to help make a more rapid and accurate diagnosis? One of the main complaints of sufferers of SLE is that their condition was not diagnosed for several years. A reason for this is the diverse number of symptoms presented and the lack of accurate diagnostic tests that can say with certainty that an individual is suffering from SLE.

Cell death and the immune system. In this environmentally conscious age of recycling, it may come as a surprise that our own bodies have been recycling their cells and the molecules within, for millions of years. Our bodies consist of billions of cells, the majority of which die each year (with some notable exceptions - for example, brain and nerve cells). An individual may recycle and exchange 70% of their cells for new ones each and every year. But what happens to the old cells and how are they disposed of in a safe manner? All individuals recycle their old cells by a process termed - Apoptosis, a term originating from the Greek to mean 'falling leaves'. The analogy with leaves is quite apt, because when trees lose their leaves each autumn, even the youngest child knows the tree itself is not dead and come the spring, the tree will produce new leaves. Similarly, when our cells die within our bodies they do so in a very systematic way that normally does not lead to inflammation or provocation of the immune system. Typically during the process of apoptosis, the cells shrink and produce small blebs, packed full of DNA, proteins and other molecules that are recognised by phagocytic cells, such as macrophages that can engulf and dissolve the contents of the blebs. This is a natural process and does not lead to inflammation. Our work at Peninsula College of Medicine and Dentistry (PCMD-University of Exeter) and the work of others, have shown that in patients with SLE, the ability to produce apoptotic cells is not impaired, but the ability to remove the dead cells from the blood circulation appears to slow down. This leads to an accumulation of dead and dying cells in lupus patients, which are potentially a target for immune surveillance cells to attack in a more rigorous fashion. This accumulation of waste cells in the blood and other organs is effectively a breakdown of the body's waste disposal system. Normally, the immune system of the body is designed not to attack its own tissues. However, if the damage cells and tissues are disguised or altered in some way, then the immune system assumes such cells are 'foreign' and will trigger a full blown attack on the altered cells, DNA or proteins, etc. So why are the apoptotic cells in SLE patients not recognised for removal? This is a question that we are focusing our research on at PCMD.

We believe that some of the major proteins in the blood that normally recognise apoptotic cells (for example proteins named C1q and calreticulin) can no longer do so. This could be due to a variety of reasons, either the proteins that recognised the dead cells have been altered and no longer function correctly or, the cell surfaces themselves have been altered so that the apoptotic recognition proteins can no longer detect and help the macrophages remove the cells and their debris. There is much support and evidence for both hypotheses from lupus researchers around the world. For example, many of the so-called autoantigens (patient's molecules that induce an autoantibody response) are concentrated in the small cellular blebs that are retained in the blood of SLE patients. Individuals who are genetically deficient for C1q develop SLE. The consequence of leaving dead cells lying around the body's vital organs, is that they are susceptible to chemical modification by the body's oxidative stress mechanisms. This can lead to chemical and structural changes in the cells and their contents, rendering them 'foreign' by the body's immune system. This can have serious consequences for patients with autoimmune disease.

Our own molecules can be seen as foreign by our own immune system. The majority of the time our multi-complex immune system performs the job it was designed to do, that is, recognise and remove foreign material in the form of bacteria, fungi and viruses. The immune system can do this because microorganisms appear 'different' to our own cells and can be recognised as such. Very early on during development, our bodies eliminate immune cells that can react with 'self' molecules and cells to prevent our bodies mounting an immune response against itself. A problem arises when our cell components change due to inflammatory processes, such as oxidative stress. An analogy would be if a white square shaped piece of material was cut into two pieces diagonally and dyed black, to another individual the material would then appear as two black triangular shaped items and they would have no indication that the material was originally a white square. Similarly, once produced, our proteins and DNA can be modified by a process called post-translational modification. If they are chemically and structurally altered, they may be perceived as foreign or non-self by the immune system. There are many examples of proteins being modified. For example, the amino acid building blocks that are used to construct our proteins can be modified allowing sugar molecules to bind to them. On occasion, such modifications can make the protein appear more self -reactive to our T lymphocyte cells. This is the case with type II collagen in collagen-induced arthritis. In lupus, several of the well characterised autoantigens - the sm antigens (Sm-D1 and Sm-D3) have a single amino acid called arginine, that is susceptible to modifications and when it is modified, these proteins act as targets for autoantibodies in SLE. Clearly, it can take only minor modifications in single amino acids of proteins to evoke an immune response.The biological processes that can cause these posttranslational modifications are a focus of research at PCMD. Several of our research groups (led by Paul Eggleton and Paul Winyard) in association with our consultant rheumatologists, Richard Haigh in Exeter and Nick Viner in Torbay are investigating whether such modifications are a result of inflammation, apoptosis or aging.

In particular, we are studying in collaboration with Dr Ahuva Nissim and Professor David Perrett at the William Harvey Research Institute, the effect of modifications to C1q and calreticulin, in order to determine if changes in these proteins make them: a) more antigenic, leading to antibodies attacking them, and b) determining if inflammatory molecules damage these proteins, so they can no longer help clear apoptotic cells. With the help of our LUPUS UK funded nurse - Miriam Hass, we have already shown that both these proteins are a target for autoantibodies in SLE patients, and we are attempting to determine precisely what amino acids or fragments of the proteins are involved. This is important for two reasons: firstly, we may be able to develop drugs to mask the autoreactive sites on these proteins to prevent them being targeted by autoantibodies, secondly, if specific sites are always targeted on these and other proteins in patients, then we might be able to exploit this property and improve the diagnosis of lupus.

What autoantibodies should we monitor to help diagnose lupus? Most SLE sufferers are well aware their disease is extremely difficult to diagnose. Patients visiting their GP may not get their disease correctly diagnosed for up to 5 years. Further referrals to a rheumatologist are often required for SLE to be diagnosed with any certainty. This is no reflection on the capability of the physicians involved, but more to do with the fact that SLE lacks a single, unifying diagnostic marker. Indeed, rarely can the diagnosis of SLE be made with absolute certainty due to the diverse number of symptoms presented. External symptoms are easier to detect, e.g. malar rash on the face, oral ulcers, photosensitive skin rash, neurological disorders etc. But internal symptoms such as haematological and immunological dysfunction are more difficult to detect. Of great concern is the early detection of renal complications, brought about by accumulation of immune complexes in this important filtration organ and direct attack by components of the immune system against the kidney itself. Most clinicians request blood samples to be taken from patients suffering from pro-inflammatory disease. This is done so the blood can be tested for abnormal white cell and platelet counts, but also importantly for lupus sufferers for the detection of autoantibodies. Unfortunately there have been some recent studies, which have ascertained that there are over 100 different autoantibodies produced in SLE. What autoantibodies should the physician look for and monitor during disease progression? The most common autoantibody observed in SLE patients is the anti nuclear antibody (ANA), followed closely by antibodies to double stranded DNA. If patients are positive for both these antibodies then a diagnosis of lupus is clearly one to consider. If renal complications are also present then the probability of lupus involvement increases. However, although the presence of high levels of these antibodies are thought to be specific for lupus, antibodies to double stranded DNA and ANA are not always sensitive. It is quite possible to suffer from SLE and have negative tests for ANA and anti-DNA.

So how does research into autoantibody binding to modified antigen possibly help in the diagnosis of the patient? We believe a problem with many diagnostic assays is that they are not sensitive enough. The clinically important autoantibodies generated in the patient against host DNA and proteins may actually be against modified and not native forms of the molecules. So if we can determine precisely the sections of the self-molecules that have provoked the immune response, we can improve the diagnostic capability of current diagnostic tests. As our own research at PCMD suggests the clearance of apoptotic cells is defective in lupus patients, the antigens expressed in and on the surface blebs of these cells form the focus of our research. We are currently establishing if modified forms of the concentrated antigens in these cells act as a source for the main autoantibody provocation. Furthermore, we are attempting to correlate the presence of active kidney disease with a specific selection of antibodies that target components of these apoptotic blebs. Ultimately, we wish to help provide a more specific test for the early diagnosis of lupus, especially SLE with renal complications. This will allow for quicker diagnosis and treatment before long term renal damage occurs. The outcome of this can only be good news for sufferers of this debilitating condition.

Acknowledgements: The work reported here was presented at the LUPUS UK 16th Annual Convention is the culmination of work conducted by Brent Ryan and Dagmara Szestakowska, (PhD students) Professor Paul Winyard, Drs Richard Haigh and Nick Viner and LUPUS UK funded nurse Miriam Haas. I would also like to thank the Devon branch of LUPUS UK for their active support and interest in our lupus research and to Joceline Triner for proofreading the manuscript.

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