The majority of this work has been based on close collaboration between the clinicians, laboratory staff and research nurses, which has provided a framework for performing long-term outcome studies. Apart from clinical based studies the laboratory has also been involved in studies on the biology of rheumatic diseases. These include studies on cell growth and behaviour, immunological and biochemical markers, role of micro-organisms and genetic and environmental factors.
In the last few years there has been a considerable shift in emphasis to reflect the rapidly increasing knowledge of the role of genetic variation in the development and progression of disease.
Recent studies by ourselves and others have provided strong evidence that smoking is a risk factor in susceptibility to rheumatoid arthritis (RA), and in the development of more severe disease. Smoking has been associated with features of more severe RA including rheumatoid factor (RF) production, nodule formation and radiographically determined joint damage. Smoking has also been associated with increased functional impairment (HAQ score), lower grip strength and more pulmonary disease. RF concentration has been found to be positively correlated with the number of years smoked, or with the number of pack years (one pack year = 20 cigarettes/day for one year).
The mechanism by which smoking influences RA susceptibility/severity is unclear at present, although it may have direct effects on the disease process by inducing and/or increasing production of RF, and producing alterations in the immune system. We have postulated that genetic factors will have an influence on the association between smoking and RA, and were the first group to demonstrate that glutathione S-transferase (GST) genes associated with detoxification of chemicals in tobacco smoke may influence the severity of RA in people who smoke.
In additional studies we have shown that the production of RF is independently associated with smoking and carriage of the HLA-DRB1*0401 allele. This allele has the strongest association with development of RA and disease severity in the Caucasian populations. We have also shown that smoking and genetic factors have an additive effect on the likelihood RF production. Like RF, the development of subcutaneous nodules is also associated with smoking and genetic factors, and we have shown that nodular disease is associated with an interaction between HLA-DRB1 and TNF genes as well as with smoking. The importance of HLA-DRB1 polymorphisms in the association of smoking with RA has been further indicated by a recent study reporting evidence of interaction between the HLA-DRB1 shared epitope (SE) and long-term smoking in susceptibility to seropositive RA.
We have recently published evidence that smoking is associated with a worse response to treatment with anti-tumour necrosis factor (anti-TNF). It is likely that a number of genes will influence the effects of smoking on RA as well as other inflammatory arthritides such as ankylosing spondylitis (AS). The aim of the current research is to explore further the relationship between smoking and genetic factors on the development and progression of inflammatory arthritis and to define gene-smoking interactions which are relevant to the onset and progression of disease. We also wish to determine whether such interactions may influence response to treatment. Biomarker profiling, gene expression analysis and functional cell studies will be used to further explore the relationship between smoking, inflammation and development of inflammatory arthritis.
We already have a detailed smoking history on over 1000 patients with established RA. There is also an extensive database of long-term outcome on these patients. DNA extracted from these patients has been stored, and most have been genotyped for GSTs, NAT1, NAT2, MMP1, MMP3 and VEGF polymorphisms.
Genetic factors important in RA may have an influence on co-morbidity and mortality. In a study in Spanish RA patients we demonstrated that HLA-DRB1*04 shared epitope alleles are associated with endothelial dysfunction, and may thus predict increased cardiovascular risk. We have recently provided further evidence for this by demonstrating that RA patients carrying two HLA-DRB1 shared epitope alleles are at increased risk of mortality from cardiovascular disease . In particular the DRB1*0401/*0401 genotype is associated with ischaemic heart disease (IHD) and genotypes containing DRB1*0401 are associated with early mortality due to IHD. There was an increased risk of malignancy in patients carrying the DRB1*0101/*0401 genotype, and mortality due to malignancy was associated with genotypes carrying HLA-DRB1*0101. We have also recently shown that a polymorphism in codon 10 of transforming growth factor β1 (TGFβ1) is associated with overall poorer survival in RA patients, and that there is an association with mortality due to malignancy.
Loss of immune regulation in RA is associated with excess production of inflammatory mediators such as cytokines and matrix metalloproteinases (MMPs). A major aim of this research is to determine which molecular pathways are important in the early stages of RA, and whether identification of the molecules involved in such pathways can provide prognostic information or potential therapeutic targets.
We have recently obtained a Bio-Plex Suspension Array System (Luminex xMAP technology) which allows multiple analytes to be measured simultaneously by fluorescent microbead technology. We have carried out initial studies to measure a large panel (32 analytes) of pro- and anti-inflammatory cytokines, soluble cytokine receptors and MMPs from the sera of patients with early RA (less than 6 months). This allows us to investigate whether alterations in the immune system early in disease are associated with particular biomarker profiles that provide clues to the development and progression of RA. This work is only possible because we have previously collected a unique cohort of patients with early RA which have been followed up for many years. We have thus been able to create an extensive database which charts the clinical progress of these patients through a large number of clinical parameters and are able to relate this to various biomarkers in samples collected within months of disease onset and at later stages of disease.
Our initial multiplex cytokine and MMP analyses have provided an important insight into the network of cytokines and MMPs present in the circulation of patients with early RA. Our analyses indicate that early RA is extremely heterogeneous in terms of cytokine and MMP patterns in sera, and that different features and severity of the disease along with lifestyle factors such as smoking are associated with different cytokine/MMP profiles. Our results indicate that early RA biomarker profiles may provide prognostic information about the likelihood of developing mild or severe disease. These data have also confirmed the importance of molecules involved in angiogenesis in the early stages of the disease, and provided new information about potential molecular targets in RA.
Our group has provided a major focus for collaborative studies with other groups investigating the role of genetic factors in susceptibility and outcome of diseases such as RA, polymyalgia rheumatica and giant cell arteritis. We have also been involved in collaborative research on markers of oxidative damage in rheumatic diseases, markers of cardiovascular disease in RA, role of parvovirus B19 infection in arthritis, and investigations on the role of smoking in the development and severity of RA.
Future directions in rheumatology laboratory research
We intend to build on the achievements of recent years and concentrate on areas where have developed a strong record of expertise. These include the following:
The role of environmental factors in the development and severity of RA is unclear but various factors (infection, diet, stress, smoking, UV exposure, social deprivation) have been suggested as having an effect. However, knowledge about the influence of environmental factors on RA development and progression is rudimentary, and the mechanisms by which such factors may work are still largely unknown. The aim of future research will be to explore the relationship between certain environmental and genetic factors on the development and progression of RA and to define gene-environment interactions which are relevant to the onset and progression of this disease. We will initially concentrate on looking for further gene interactions with smoking in RA.
Our data suggest that certain genetic polymorphisms are associated not only with severity, but also with co-morbidity and poor survival in RA. We are currently carrying out studies on the role of genetic factors in co-morbidity and mortality on two groups of RA patients. One group involves over 750 patients with long-standing disease, on whom we started collecting data in 1994. A significant number of those patients have since died and we have identified a number of interesting associations between disease survival, genetic factors and circulating biomarkers. We have a large database lodged with the NHS registry at Southport which will flag up future deaths as well as causes of death in this group. We also have data on social deprivation indices in these patients. This is an ongoing study and will provide important data over the next few years. More recently we have set-up another more detailed study to examine the prevalence of all comorbid conditions in RA patients, and to examine the use of a co-morbidity score. These patients are being followed up over a 5 year period, and sera and DNA are being collected to investigate biomarker profiles and genetic associations with comorbid conditions and disease severity.
Research on genetic factors which influence disease susceptibility, severity and drug responses will continue to expand, and increased effort will be focused on identifying how interactions between genes influence disease expression. Research will focus not only on the identification of genetic factors associated with disease, but how these may influence drug responses and the inflammatory response at the cellular level. The role of epigenetic regulation will also be explored.
Recently, there has been special interest in the role of epigenetic regulation in the pathogenesis of RA. Indeed, aberrations in epigenetic mechanisms such as DNA methylation have a well-defined role in other autoimmune diseases, such as systemic lupus erythematosus. Since DNA methylation is primarily concerned with gene silencing, a breakdown in this process (hypomethylation) may contribute to the persistent over-expression of mediators which co-ordinately instruct the chronic destructive inflammatory state.
To date, epigenetic studies in RA, primarily focusing on leukocytes and synovial fibroblasts, have been restricted to the analysis of individual or pre-selected panels of candidate genes. Whilst this is informative, it fails to appreciate both the vast and highly complex network of interactions that occur in RA, and the broad-scale influence of epigenetic regulation across the genome. Thus, with the advent of new technologies capable of simultaneously interrogating the entire genome, we have the opportunity to elucidate for the first time the epigenome in RA. By combining this approach with other more traditional techniques, we hope to extend our understanding of epigenetic processes and their importance in the pathogenesis of RA.
Future work will involve the use of small interfering RNA (siRNA) technology for silencing gene expression in cells. We are involved in collaboration with Professor Bill Farrell and other members of the Human Disease and Genomics group in exploring the use of this technology to investigate the function of particular molecules in the immune response.
Future development of research within rheumatology depends very much on close integration between the clinical and laboratory based research. This will continue to ensure that the Haywood hospital research department remains an important UK centre for studying chronic inflammatory joint diseases.