The William Harvey Research Institute, Saint Bartholomew's and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, Charterhouse Square, London EC1M 6BQ and
1 51 Woodbourne Avenue, London SW16 1UX, UK
Correspondence to:
J. Botting.
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Introduction |
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The meeting began with a historical overview of rheumatology in the UK from 1948 to the present day given by Dr Allan Dixon, followed by a historical perspective from the USA by Dr Stephen Crane of the Arthritis Unit, Massachusetts General Hospital in Boston.
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Historical perspectives |
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The story in the USA was similar. The American Rheumatism Association was formed in 1937 and many young scientists were encouraged to involve themselves in the study of rheumatic diseases. Cortisone was synthesized and glucocorticoids made available for treating rheumatic conditions. Scientists at the Massachusetts General Hospital elucidated the structure of collagens and their cleavage by collagenase enzymes. Many significant discoveries followed, including the characterization of crystal-induced arthropathies, evaluation of the role of rheumatoid factor and characterization of Lyme disease.
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Current trends in therapeutics |
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At least as early as 3500 yr ago, rheumatism was treated in ancient Egypt and Greece with decoctions or extracts of herbs or plants such as willow bark or leaves, most of which turned out to contain salicylate. In England, in 1763, the Reverend Edward Stone of Chipping Norton performed the first clinical trial of powdered willow bark and found it to be effective in 50 patients with fever. He wrote to the President of the Royal Society that he had no other motive for publishing this valuable treatment than that the world may benefit from his discovery. However, the bitter taste of salicylates made them relatively unpalatable drugs and in 1897 the German chemist, Felix Hoffman, acetylated salicylic acid to form aspirin and thereby improve the palatability. Thus, 50 yr ago, aspirin, together with steroids which were very toxic, was the mainstay of anti-rheumatic drug therapy.
In the 1960s, the importance of a newly characterized group of lipid mediators called the prostaglandins in inflammation, fever and pain became evident. Also at that time, other anti-inflammatory, antipyretic and analgesic drugs, such as phenylbutazone and indomethacin, were developed by drug companies. The discovery, by Vane in 1971, that all the chemically diverse members of this group of NSAIDs acted by inhibiting the key enzyme in prostaglandin biosynthesis (which we now call cyclooxygenase or COX) provided a unifying explanation for their therapeutic actions and shared side-effects (such as gastrointestinal and kidney toxicity).
The clarification of the mode of action of NSAIDs also gave the pharmaceutical industry a new tool for finding novel drugs, using the COX enzyme in vitro as an initial screen. However, the side-effects, especially on the stomach, were still a major problem and some companies began to screen for new compounds which were anti-inflammatory, but were less damaging to the gastrointestinal tract. Meloxicam and nimesulide were discovered in this way.
The next important advance in our understanding of the NSAIDs came in 1991, when several laboratories showed that COX exists in two isomeric forms: COX-1 and COX-2. The constitutive isoform, COX-1, has clear physiological functions. Its activation leads, for instance, to the production of prostacyclin, which when released by the endothelium is anti-thrombogenic and when released by the gastric mucosa is cytoprotective. COX-1 in platelets produces thromboxane, which promotes platelet aggregation and leads to thrombus formation. The second isoform is COX-2, which is inducible in a number of cells by pro-inflammatory stimuli. It is encoded by a different gene from COX-1. Its amino acid sequence shows 60% homology with the sequence of the non-inducible enzyme. Both enzymes have the same molecular weight of 70 kDa, but their mRNAs are different. Small differences in the structures of the active sites of COX-1 and COX-2 have been exploited in order to design inhibitors with selectivity for COX-2. The larger active site of COX-2 will accept compounds too large to enter the active site of COX-1 easily, thus conferring selectivity.
Since COX-2 is induced by inflammatory stimuli and by cytokines in migratory and other cells, it is attractive to suggest that the anti-inflammatory actions of NSAIDs are due to the inhibition of COX-2, whereas the unwanted side-effects, such as irritation of the stomach lining and toxic effects on the kidney, are due to inhibition of the constitutive enzyme, COX-1. Clearly, to treat inflammation, the NSAIDs must be used in doses that suppress COX-2. Interestingly, at such doses, the effects on COX-1 vary depending on the drug involved. Thus, drugs which have the highest potency on COX-2 and a better COX-2/COX-1 activity ratio will have potent anti-inflammatory activity with fewer side-effects on the stomach and kidney. The COX-2/ COX-1 ratios of a range of NSAIDs have been measured in several ways and the human whole-blood assay is now generally used. Results obtained with an improved whole-blood assay show a strong correlation between the COX-2/COX-1 ratios and published figures of epidemiological data for gastric damage in humans, proving the concept that inhibition of COX-2 accounts for the anti-inflammatory actions of the NSAIDs, whereas inhibition of COX-1 causes the damage to the stomach mucosa.
The development of more selective inhibitors of COX-2 will clearly provide important advances in the therapy of inflammation. Conventional NSAIDs lead to gastrointestinal side-effects, which include perforations, ulcerations and bleeds (PUBs) that lead to the hospitalization of >100000 patients every year in the USA alone. About 15% of these patients die in intensive care. The evidence is strong, both from animal tests and from the clinic, that the selective COX-2 inhibitors will have greatly reduced side-effects. Meloxicam is the first selective COX-2 inhibitor to be licensed in the UK, and its efficacy and safety have been tested in large numbers of patients. The extensive clinical trials comparing the gastric toxicity of meloxicam with that of diclofenac and piroxicam have demonstrated the superior safety of meloxicam. However, the next generation of COX-2 inhibitors, such as celecoxib and rofecoxib, clinical trials of which are still in progress, have even greater selectivity for COX-2 with much less effect on COX-1, and may have even less frequent incidence of gastric damage.
New uses will also be found for selective COX-2 inhibitors. For example, aspirin is effective in the prophylaxis of colon cancer and we now know that it is the induction of COX-2 in colon cells which is associated with this condition. Knockout of the COX-2 gene prevents the growth of colonic polyps in mice with a genetic mutation for colon polyps which ultimately develop into colon tumours. Selective COX-2 inhibitors prevent the growth of tumour cells in experimental carcinogenesis and the major drug companies developing celecoxib and rofecoxib have entered their compounds into clinical trials in colon cancer.
There is also some less direct evidence that Alzheimer's disease is associated with an upregulation of COX-2. However, clinical trials with celecoxib and rofecoxib have also been initiated in Alzheimer's patients.
COX-2 is induced during labour and initiates parturition, through producing prostaglandin F2 , which contracts the uterus. The selective COX-2 inhibitor, nimesulide, prevented pre-term labour in women who had already suffered several early abortions, and meloxicam, with a longer half-life, may well provide a better alternative.
Therefore, highly selective inhibitors of COX-2 will be of enormous benefit to rheumatism sufferers, providing an efficacious treatment for the reduction of inflammation and pain without the currently expected accompanying gastrointestinal symptoms. In addition, they may have a future use as prophylactic drugs in the treatment of colon and other cancers, and for retarding the progression of Alzheimer's disease. Their use in delaying premature labour has already been tested and found to be effective. The use of aspirin as an anti-inflammatory drug will decline, but its unique action in preventing heart attacks and strokes by inhibiting platelet aggregation, through inhibition of COX-1, will no doubt continue to expand.
Professor Paul Dieppe outlined the problems facing sufferers from OA and in particular the arthritic pain and incapacity which afflicted Oliver Bird.
Oliver Bird developed OA in 1941, when he was 61 yr of age, and from photographs showing him walking with two sticks, one assumes that he suffered from arthritis of the hips. This accelerated form of the disease is very painful and currently the only effective treatment is hip replacement. Oliver Bird tried many different remedies for his OA, including physical therapies and spa treatments, but obtained only temporary relief. He particularly recommended a `vaccine' treatment for chronic rheumatic disease practised at the Charterhouse Clinic. Not much more is known about Oliver Bird's OA, except that he suffered a `long and trying incapacity', as mentioned in a letter from A. A. Miller, the first administrator of the Oliver Bird Fund.
Fifty years ago, in 1948, at the time the fund was set up, OA was considered to be a `wear and tear' and `degenerative' disease, largely untreatable except for the spa treatments used by Oliver Bird. The present concept of the disease is that it is an `age-related disorder of evolution; a mechanically-driven but chemically mediated active disease process'. It is now potentially preventable as well as treatable and over 80% of lower limb prostheses are for OA, mostly for replacement hip joints, but increasingly for knee joint replacements as well.
Thus, OA is an age-related disease involving areas of high stress, such as the hip, knee and thumb base, which are underdesigned for the use to which we put them with our upright, bipedal posture and prehensile grip. The main drive to the arthritic process is therefore mechanical, supporting the focal nature of the disease, followed by chemically mediated destruction of the joints.
In a community study of 2000 OA patients over 5 yr, the different risk factors associated with initiation and progression of the disease were identified. Risk factors for initiation of OA include obesity, being in pain, past injury and past heavy sporting activities, whereas the only significant risk factor for progression of the disease is obesity. Osteoporosis is negatively associated with initiation or incidence of the disease. This indicates that initiation and progression involve different processes.
Many of the clinical symptoms of OA are due to an active attempt by the injured joint to repair itself and this repair process can progress in different ways which form a continuous spectrum with hypertrophic OA at one extreme and atrophic OA at the other. Hypertrophic OA is characterized by thickening of the capsule and new bone formation, leading to `tight joints', lessening of the pain and a good prognosis. Atrophic OA is a complete failure of repair, loss of bone and soft tissues, and loose joints, and the prognosis is bad. This emphasizes that different tissues are involved.
It has recently been suggested that the initiation, progression and clinical expression of OA are largely related to muscle power. Development of somatopenia in older people may result in destabilization of joints and thus provide the continued mechanical drive for degeneration of the joint and progression of the disease.
More research into the causes and mechanisms of OA is crucial. Future advances in the understanding and treatment of OA will include high-technology approaches such as quantitative imaging by MRI of structural changes which take place in the joints and the development of biochemical markers of the disease process. There will be increased awareness of primary prevention, which will include strengthening of muscles by exercise, avoiding obesity and joint injury, and careful selection of sporting activities. New therapeutic approaches will be tried for secondary prevention with structure-modifying agents such as growth hormone, cartilage transplants and the use of protease inhibitors. Tertiary prevention will still continue, including joint replacement and physiotherapy. It has been suggested that joint replacement can be avoided in some cases by the use of external prostheses, perhaps placed in shoes, which alter the position of the joint and take pressure off the injured part. In a recent, remarkable study by New York surgeons, it was found that shoes with a lateral heel wedge were as effective as a knee replacement in 30% of cases. No comparative, randomized, controlled clinical trials of the beneficial effects of joint replacements have yet been performed and many are needed.
Finally, it may be possible to influence the initiation and progress of OA by nutritional means. Already, major trials are in progress in the USA with glucosamine and chondroitin sulphate products for reconstructing the injured joints, and increasing the intake of vitamins C and D by drinking more milk. It appears that atrophic OA may be associated with low vitamin D levels. So future therapy for OA will come from exercise, wearing trainers and drinking more milk, perhaps in the form of Bird's Custard.
When answering questions, Professor Dieppe did not consider that present treatments for increasing synovial viscosity conferred any therapeutic benefit and agreed that although exercise improved the function of OA joints, it had little effect on the accompanying pain, whereas joint replacement primarily removed the pain.
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Basic mechanisms of major diseases |
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The characteristic feature of this disease is the development of a spectrum of autoantibodies, particularly against a series of intracellular particles. These include antibodies against the nucleosome comprising DNA, the histones, basic elemental units of chromatin, spliceosome particles and the Ro/La small cytoplasmic RNP particles. In addition, autoantibodies form against components of the cell membrane, especially anti-phospholipid antibodies and some which bind to plasma proteins such as rheumatoid factors and anti-C1q antibodies.
An important aspect of the immunopathology of the disease is the deposition of antibodies in many tissues, particularly the kidney, in the form of immune complexes with complement. Thirty per cent of SLE patients have high titres of autoantibodies to the first component of complement, C1q, so there is a connection between the development of SLE and the complement system. In a closely related syndrome to SLE called hypocomplementaemic urticarial vasculitis (HUVS), patients develop urticarial rash with underlying vasculitis, they become severely hypocomplementaemic, some develop glomerulonephritis and arthritis, and the prognosis is poor. Patients with HUVS have high titres of precipitating antibodies to C1q.
In SLE patients, there appears to be a close negative correlation between the levels of anti-C1q autoantibodies and levels of complement proteins such as C1q, C4 and C3. Thus, C1q antibodies are associated with evidence of classical pathway complement activation. Clinical observations in a rare group of patients (only 41 of whom have so far been identified) with an inherited type of SLE have identified a hereditary deficiency of C1q protein. Of these patients, 36 had SLE, two suffered from cutaneous discoid LE and three were reported to be healthy. The SLE in these patients is characterized by early disease onset with a median onset of 6 yr, a severe rash which is typically photosensitive, major internal organ disease, e.g. of the central nervous system or kidney, and a typical spectrum of autoantibodies.
Both the severity and susceptibility to development of SLE seem to depend on the position of the deficient protein in the activation pathway of complement. Ninety per cent of patients with a homozygous deficiency of C1q develop a very severe form of SLE. C2 deficiency is relatively common in Caucasoid populations and has a prevalence of ~1:20000 of the population. It is associated with SLE of medium severity and approximately one-third of C2-deficient individuals have the disease. The `autoimmune' phenotype with an inherited deficiency of C3 protein occurs rarely, and is milder than other complement protein deficiencies. This is surprising since C3 is the major protein of the complement system in quantitative terms, normally present in serum in a concentration of 1 g/l, increasing by 0.5 g/l during an acute-phase response. These patients develop a rash after severe pyogenic bacterial infections, but fewer of them have autoimmune phenomena or glomerulonephritis than C1q- or C2-deficient patients. Of 24 subjects with C3 deficiency, 18 were prone to pyogenic bacterial infections, eight developed glomerulonephritis, eight developed non-SLE rashes and three had an SLE-like illness. The conclusion from this is that there is a physiological activity of the early part of the classical complement pathway which protects against SLE.
The complement proteins are important for clearing immune complexes from the body, and a defect in clearance of antigens, which act as autoantigens for the formation of autoantibodies in SLE, may be the underlying cause for the development of this disease. Thus, if the clearance function fails due to an absence of complement, immune complexes deposit in tissues, cause inflammatory injury with release of autoantigens and build up a cycle of tissue injury. Knockout mice with a homozygous deficiency of the C1q gene developed typical autoantibodies and 25% of the first cohort died of glomerulonephritis between 5 and 8 months of age. Electron micrographs of their kidneys showed dense immune deposits in the glomeruli similar to those of SLE.
There is evidence that C1q protein may also be involved in the clearance of apoptotic cells. Glomeruli of patients with SLE and of C1q-deficient mice are filled with apoptotic debris previously known as `haematoxylin bodies'. Large numbers of the typical SLE autoantigens are cleaved during apoptosis by the proteases involved and C1q may become an autoantigen complexed with apoptotic cells in SLE. It has recently been shown that C1q binds to apoptotic keratinocytes and this may be the link between C1q deficiency and SLE. Thus, the autoantigenic drive in SLE may come from apoptotic cells and the inability to process the apoptotic particles may be sufficient to cause the development of the disease.
In answer to a question from the audience, Professor Walport pointed out that mechanisms of repair are also important and knowing how inflammation resolves is essential for a full understanding of chronic inflammatory conditions.
The possibility that study of T-cell biology might offer a therapy for rheumatoid arthritis was addressed by Professor Avrion Mitchison (University College, London, UK). Driving T-cell differentiation to form Th2 rather than Th1 cells should in theory provide a viable therapeutic strategy in view of the more favourable cytokine profile of the former [interleukin (IL)-4, IL-10] compared to the latter [interferon gamma (IFN-), IL-2]. Although some experimental evidence does not support this contention, e.g. models of autoimmune disease generated in IFN-
knockout mice get worse instead of better, and IL-4 knockout mice are not made worse, as would be expected, study of genetic variation that can occur in Th1/Th2 differentiation tends to support the view that this circuitry is important in disease susceptibility. Thus, variation in the expression of the IL-4 receptor, IL-10, the IL-12 receptor or MHC class II results in increased or decreased susceptibility to autoimmune disease, consistent with the view that favouring Th2 cell differentiation would be beneficial for therapy.
There are some potential therapies directed at driving Th2 differentiation. Vitamin D, ß-adrenoceptor agonists and prostaglandin E inhibit synthesis of IL-12 and thus promote Th2 rather than Th1 formation, and antibodies against the CD4 receptor similarly drive Th2 differentiation. Experiments on mouse lymphocytes in culture show that anti-CD4 antibodies, in conjunction with inhibitors of Src-related tyrosine kinase downstream of the receptor signalling mechanism, induce Th2 differentiation denoted by a switch in cytokine production towards IL-4 and away from IFN-. The administration of anti-CD4 antibodies directed against the membrane-proximal domain in vivo in mice results in a high-frequency, but low-level induction of Th2 cells. Although initial trials of anti-CD4 antibodies in man were encouraging, tests in large numbers of patients gave poor results. However, these studies used antibodies to the distal domains 1 and 2 of CD4; antibodies to the 3 and 4 membrane-proximal domains should be more effective. Since CD4 ligation is only one of the co-stimulatory activating mechanisms influencing Th1/Th2 differentiation, there are many more targets in this paradigm to attract therapeutic interest.
Professor Richard Eastell illustrated the value of epidemiological studies to approach the genetic causes of osteoporosis. Osteoporosis is classically defined as a systemic skeletal disease characterized by low bone mass, increase in bone fragility and susceptibility to fractures. Such a definition, however, is of little help in epidemiology, and an understanding of the genetic basis of the disease is better served by examining fracture syndromes and what they tell us of the causes of osteoporosis.
The classical fractures of osteoporosis are those of the vertebrae, proximal femur and the wrist. However, the epidemiology of these is quite different. Hip fractures increase exponentially with age in both men and women, suggesting a multifactorial cause. Forearm fractures rise sharply after the menopause in women, then reach a plateau, which suggests a single cause. Clearly, each fracture should be studied as a separate entity, and not simply be regarded as a general symptom of osteoporosis.
Osteoporosis is an important disease; although wrist fracture carries no risk of mortality, mortality increases after hip fracture by 17% and by 18% after vertebral fracture. All three fractures have morbidity, loss of independence and economic cost. Osteoporosis is common; a 50-yr-old woman has a lifetime hip fracture risk of 18%, and a risk of any fracture of 3040%. This big public health burden will certainly worsen, since with increased longevity the incidence is projected to increase over the next 50 yr in North America, Europe and in particular Asia.
Reduction in bone density provides not only a more reliable means of detecting those at risk, and thus a rationale for treatment, but also a more precise way of understanding the genetics of the condition. Therefore, WHO guidelines suggest that a bone density of 2.5 S.D. below that of the mean of young, healthy women should signify a diagnosis of possible osteoporosis; occurrence of a fracture signifies established disease. Certainly, there is a strong relationship between bone density and fracture risk, individuals in the lowest quartile of bone density are eight times as likely to endure a fracture (a stronger correlation then that between high cholesterol and coronary heart disease); however, even reliance on bone density measurements has some limitations in epidemiological studies.
Bone mass increases steadily in childhood, accelerates during puberty, and peaks between 20 and 40 yr. Rapid bone loss occurs at menopause, bone mass loss continues into old age in both men and women. Fracture results from low bone density and trauma. Low bone density can be a consequence of inadequate peak bone mass or increased bone loss. The latter has three components: menopause, ageing and sporadic causes such as corticosteroid therapy. Both peak bone mass and bone loss are under genetic control, as evidenced by racial differences in bone density, twin studies and the study of animal mutations. The genetic influence is reflected in studies of fracture rates, e.g. Black women have a higher bone density than Caucasians and a lower incidence of hip fractures. Even amongst Europeans, fracture rates vary greatly between women of different nationalities; no doubt, this is partly due to environmental factors, but genetic differences are significant.
Candidate genes for the cause of osteoporosis include that for the vitamin D receptor, collagen type I, transforming growth factor beta (TGF-ß), the oestrogen receptor and the IL-1 receptor antagonist, all of which may be associated with low bone mineral density and fracture. Continuing epidemiological studies, and research into the various gene polymorphisms involved in the development of osteoporosis, will enhance our knowledge of the aetiology of the disease and open avenues for its prevention and treatment.
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Biomolecular basis of rheumatic diseases |
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Vitamin D-resistant rickets is still a serious disease of genetic origin. The hypocalcaemic form is linked to the absence of the 1 hydroxylase enzyme in the kidney which is necessary to form the active derivative of vitamin D. Fortunately, this can be easily treated by replacement of the active metabolite. However, some patients have a mutation in the gene expressing the receptor for which the vitamin D metabolite is a ligand. The various mutations of this receptor have been identified, but as yet there is no effective treatment. Hypophosphataemic rickets is a less severe form of the disease characterized by a defect in the proximal renal tubule, whereby phosphate reabsorption is reduced, and a defect in osteoblasts. Study of both the human disease and of a spontaneous mutation in mouse (the hyp mouse) which manifests a condition homologous to the human established that expression of PHEX, a neutral endopeptidase with an as yet unknown substrate, is virtually absent in the osteoblasts of the mutant mice. Thus, the osteoblast appears to be the primary target of this mutation and examination of the link between PHEX and osteoblasts will eventually provide a clue to the nature of this condition.
Inherited diseases of bone density are also a problem. Osteopetrosis is characterized by excessive mineralization of bone, which in consequence is totally inefficient and becomes brittle. The defect is lack of activation of osteoclasts, which though present in the human disease, are not functional. In the spontaneous mutants or engineered mouse models of osteopetrosis, e.g. the cfos knockout mouse, osteoclasts are absent or deficient in number. These various animal models have established several genes to be important in the development of the mature osteoclast from the stem cell, but none so far has provided an explanation of the cause of the human form of osteopetrosis.
The most common form of osteoporosis in children is osteogenesis imperfecta. This is a common disease, affecting ~25000 children in North America and a similar number in Europe. It is characterized by inadequate mineralization of bone, which varies in degree; in its most severe form, it is a serious crippling condition. Linkage studies have shown a close association with mutations in the genes coding for the major constituent of bone matrix, collagen type I. These mutations affect either the structure of collagen or the amount of collagen produced. Mutations relating to change of structure are the more severe, since the correct orientation of the collagen molecule is crucial for adequate mineralization of bone. Other forms of inherited osteoporotic syndromes involve defects in collagen type II, collagen type X or collagen oligomeric matrix protein. Disorder of collagen formation may not be the only cause of some forms of osteogenesis imperfecta. Recent clinical studies have exposed the existence of a type of the disease in eight patients, in which linkage studies point to a gene on chromosome three, which does not have a collagen gene.
Mouse gene knockout studies continue to provide clues to the control of osteoclast and osteoblast development. Knockout of the transcription factor CBFA1 leads to the total absence of bone in homozygous animals with the retention of a partially calcified skeleton, while heterozygotes manifest a condition analogous to cleiodocranial dysplasia syndrome seen in man, characterized by delayed membranous bone development. Mutations of the CBFA1 gene have subsequently been characterized in the human condition. Besides providing clues to the aetiology of human disease, and perhaps ultimately to a treatment, these studies have established that the development of intramembranous and endochondrial bone are separately controlled.
The studies described by Professor Glorieux are an outstanding example of the value of careful analysis of heritable disease in humans, allied to the examination of spontaneous or engineered murine mutations, to the treatment of distressing and life-threatening conditions. Some treatments have already emerged, such as replacement therapy for rickets and the use of biphosphonate for osteogenesis imperfecta. Ultimately, gene therapy, whereby abnormal genes will be switched off by some form of antisense therapy, or normal genes inserted to correct an abnormality, will become a reality.
In order to manipulate the processes of T-cell development and differentiation with drugs, it is necessary to understand the biochemistry of how the cells make these changes. T cells, when they encounter antigenic stimulation, undergo basal proliferative expansion and, under the control of different cytokines, they differentiate into various types of effector cells. Cytokines produced by these cells then go on to control immune responses.
Dr Doreen Cantrell (Imperial Cancer Research Fund, London, UK) has concentrated on elucidating the functions of one particular signalling process in lymphocytes, that of the GTPase proteins. The GTPases form a group of molecular switches which become activated when T cells are triggered through their antigen receptors. This activation causes GTPases to cycle between the active GTP-bound state and the inactive GDP-bound state. In the active state, they can bind to a variety of effector molecules, thereby initiating distinct effector pathways and regulating different biological processes such as cytokine gene expression, control of adhesion and control of lymphocyte migration.
In 1990, it was shown that the GTPase encoded by the Ras superfamily of proteins could become activated in response to triggering of the antigen receptor on T lymphocytes. In addition, GTPases of the Rac and Rho families act as molecular switches at several critical points in lymphocyte development and function. Ras and Rac are both involved in the regulation of signalling pathways that cooperate with calcium/calcineurin signals to the nucleus which control transcription of cytokine genes.
The GTPase proteins are critical for T-cell function and the GTPase Rho is able to regulate survival signalling pathways in T cells. One of the functions of Rho is to control survival in lymphocyte progenitors, the thymocytes. This role was discovered by eliminating the function of Rho GTPase in the thymus using Clostridium botulinum toxin to inactivate the protein. Transgenic mice were made expressing the C3 enzyme from C. botulinum in thymus cells. This enzyme specifically ribosylates Rho at the region which interacts with the downstream effector proteins and prevents Rho activity. These C3 transgenic mice had a very small thymus containing 510% of normal thymocyte numbers and almost no peripheral T cells. The thymocytes were also dying before they could differentiate and proliferate, so it became clear that Rho GTPase was necessary for cell survival.
A rescue of the early progenitor cell populations could be effected by crossing the C3 transgenic mice with transgenic mice expressing bcl-2, the survival protooncogene. However, total thymocyte numbers could not be restored and Rho is probably required for cell cycle progression in further populations of cells. The survival of these cells is normally controlled by IL-7 and the data suggest that Rho is a component of the signalling pathways used by IL-7 to control cell survival. It has been proposed that IL-7 regulates survival signalling pathways in thymocytes by controlling levels of Bcl-2.
The clarification of the functions of second messenger systems is a rapidly evolving field. Important functions have been identified and quite different functions for molecules have been found in different cell populations. For example, Rho GTPase controls production of actin stress fibres in fibroblasts, whereas it regulates survival in T cells. This has major implications for potential drug therapy since a drug which modulates a signalling pathway which has different functions in various cells is likely to manifest complex side-effects.
Cartilage destruction is the ultimate, crippling stage of joint disease. Professor Tim Cawston (University of Newcastle upon Tyne, UK) described work on the elucidation of the aberration in control of cartilage turnover in joint disease, research which will eventually produce therapies to limit cartilage breakdown and loss of joint function. Early in vitro studies demonstrated that live synovial tissue is necessary for cartilage degradation, and that factors released from these cells presumably activate degradative enzymes. Research was then directed to identify the relevant cytokines, and the enzymes they switch on, in order to devise ways to inhibit the process.
Cartilage is an avascular, aneural and alymphatic tissue containing chondrocytes that maintain the collagen network and proteoglycan content of the tissue. The synthesis and breakdown of these two substances are finely balanced: oversynthesis causes fibrosis, the reverse leads to loss of extracellular matrix. Proteoglycan loss from cartilage is rapidly replaced, but when collagen is lost it is not regenerated, thus the preservation of collagen is vital for cartilage survival. The enzymes that break down collagen are the zinc-containing matrix metalloproteinases (MMPs); formed as proenzymes, they require activation before they can act on a substrate. The active forms of the enzymes can be inhibited by endogenous inhibitors called tissue inhibitors of metalloproteinases (TIMPs), polypeptides which bind to MMPs with high efficiency in a 1:1 ratio. Cytokines cause upregulation and conversion of the proenzyme to the active form. If excess TIMP is present, the action of MMP is blocked; thus, sufficient enzyme must be activated to swamp the available TIMP before tissue destruction can occur.
To establish the cause of collagen destruction, and to ascertain if it can be prevented, bovine nasal cartilage was incubated in culture for 14 days and the loss of proteoglycan and collagen measured. Addition of IL-1 caused loss of proteoglycan, but significant loss of collagen occurred only after the addition of oncostatin M (a member of the IL-6 family) in addition to IL-1. Loss of collagen was accompanied by upregulation of the proenzyme. Similar results are obtained with human cartilage, where oncostatin M together with IL-1 reproducibly releases proteoglycan, although collagen release is somewhat less. Inhibitors of MMPs abolish release completely, thus all effects are due to MMPs. The relevance of these studies to human disease is suggested by the detection of oncostatin M in rheumatoid synovium, where it is apparently present in macrophages.
There are three collagen-degrading MMPs present in humans: MMP-1, MMP-8 and MMP-13. MMP-1 and MMP-13 can be released by chondrocytes, MMP-8 is stored in neutrophils. It is not yet known which enzyme is responsible for joint damage despite a plethora of data on relative distribution and activation by cytokines. Professor Cawston ventured his own opinion that MMP-8 is involved in septic arthritis, MMP-13 is likely to be involved in normal turnover of cartilage and probably the enzyme mostly responsible for tissue destruction in OA, and that MMP-1 is implicated in rheumatoid disease. However, it is likely that in any one disease tissue breakdown will involve all three enzymes to some degree.
Inhibitors of MMPs, as potential chondroprotective drugs, have been developed based on the knowledge of the structure of the collagen cleavage site, and have been shown to be effective in vitro. The question of whether selective inhibitors of MMPs would be preferable to non-selective agents is not yet resolved. Selective inhibitors would presumably be useful if it emerged that one MMP enzyme was involved in a single disease, since general inhibitors would be expected to manifest side-effects due to inhibition of other metalloproteinases. It is likely that MMP inhibitor therapy would be best combined with other therapies directed at different stages of the disease.
Numerous intercellular messengers, or cytokines, can promote damage in rheumatic disease and thus provide targets for the development of effective therapies. Chemokines, cytokines that potently attract white blood cells into tissues, are currently attracting much interest in view of their ubiquity in chronic inflammatory conditions. Dr Joost Oppenheim (Frederick Cancer Research and Development Center, Maryland, USA) presented an overview of the nature and action of chemokines, and how chemotactic activity of chemokines may be modified by an acute-phase protein, serum amyloid A (SAA).
Chemokines are pro-inflammatory peptides that induce migration and regulate the movement of leucocytes, endothelial cells and fibroblasts. Besides being present in every acute and chronic inflammatory disease, they also participate in immune responses, allergy, cell trafficking, cell homing, haematopoiesis and angiogenesis. The 50 or so chemokines so far identified form four subfamilies according to the presence or absence of a varying number of amino acid residues between the first two cysteine molecules in the peptide chain. The two major families are termed CC or CXC. Every leucocyte can express a number of chemokine receptors designated CXCR15 for CXC chemokines, CCR18 for the CC family or CX3CR1, the receptor for the sole CX3C chemokine, fraktalkine. Leucocytes not only express many chemokine receptors, but also the receptors generally react to many chemokines. Thus, there appears to be much redundancy and overlap in in vitro chemokinereceptor interactions; however, gene knockout studies of individual receptors produce particular defects and mice with unique phenotypes, which indicate that chemokines have specific roles in vivo depending on their distribution and kinetics of production.
Chemokines act upon G protein-coupled 7 transmembrane receptors (STM), which have three extracellular and three intracellular loops. The intracytoplasmic tail is rich in serine and threonine, which become phosphorylated upon activation and with more extensive phosphorylation results in desensitization. Activation of the receptors triggers the formation of GTP, activation of phospholipase C and the generation of two messenger pathways IP3 and DAG, and hence activation of the ras MAP kinase, resulting in chemotaxis, adhesion, degranulation and maybe desensitization. Heterologous desensitization can also occur, whereby an alternative ligand, acting on another STM receptor by activating the PKC pathway, results in the phosphorylation of an unoccupied STM which becomes unresponsive to subsequent exposure to its ligand. For example, immunosuppressive opiates which use µ and STM have been shown to inhibit the chemotaxis induced by chemokines, an effect blocked by the opiate receptor antagonist, naloxone. Since the opiate was therefore not directly competing with the chemokine, the inhibition was presumably through heterologous densensitization of the chemokine receptor. This was subsequently established by the direct demonstration of phosphorylation of the chemokine receptor by opioids that was reduced by the antagonist naloxone.
Heterologous densensitization may provide the basis for the immunosuppressive effects of the acute-phase protein SAA. SAA is produced by the liver, small intestine and macrophages, and can be released by various cytokines and endotoxin. It is chemotactic for neutrophils, monocytes and T cells, but at rather high concentrations. SAA is inhibited by pertussis toxin, which suggested that it may act on an STM, and prior exposure to SAA did inhibit chemotaxis in response to the chemokine IL-8, presumably by cross-phosphorylation of the CXCR1 and CXCR2 receptors. Thus, although SAA can induce inflammation when applied locally, the high plasma concentrations manifest during the acute-phase response could downregulate chemokine responses by heterologous densitization.
Some progress has been made towards the identification of the SAA receptor. In cells transfected with FMLP receptors FPR or FPRL1, cross-desensitization studies suggested that the low-affinity FMLP receptor FPRL1 may be the SAA receptor. SAA binds competitively to FPRL1 and can only be partially displaced by FMLP, and cells transfected with FPRL1 manifest a chemotactic response to SAA but not FMLP. SAA also interacts with higher affinity with FPRL1 than FMLP, but does not interact at all with FPR. Professor Oppenheim concluded that although SAA can be locally pro-inflammatory, it may downregulate systemic inflammation by receptor desensitization. SAA gene mouse knockout experiments may shed further light on the significance of this acute-phase protein.
The relationship between genetic variation in the production of the cytokine, IL-1, and the incidence of inflammatory diseases was addressed by Professor Gordon Duff (University of Sheffield, UK). Three protein ligands act upon the IL-1 receptor; IL-1 and IL-1ß stimulate and are thus pro-inflammatory, IL-1 receptor antagonist (IL-1ra) blocks the receptor and is thus anti-inflammatory. A clue that there was genetic variation in the control of IL-1 emerged from early studies of the production of IL-1ß by monocytes obtained from a large cohort of individuals. The release of IL-1ß varied greatly and was later shown to be associated with particular genotypes.
The association of a particular genotype with susceptibility to diseases is fraught with difficulty and necessitates large cohort sizes and replication studies. Such studies have, however, indicated a link between erosive rheumatoid arthritis and overproduction of IL-1ß, with a high frequency of the allele producing high levels in the patient group. Similarly, both genetic and experimental studies indicate that the rarer allele of the IL-1ra gene, which is associated with low production of the anti-inflammatory protein, is overrepresented in inflammatory bowel disease. IL-1ra content in colonic biopsies from patients with ulcerative colitis substantiates the association, since patients who are homozygous for the rarer allele have less of the anti-inflammatory protein, IL-1ra.
Periodontal disease, which is a disease of the gums resulting in early tooth loss, is one of the most common inflammatory conditions, and is also associated with an IL-1 genotype. In mild disease, the genotype occurs in 25% of patients, in severe disease in 66%, representing a relative risk of seven. Such a high relative risk suggests clinical utility as a genetic diagnostic in the management of the disease. The epidemiology of periodontal disease indicates a link with coronary heart disease, which raises the possibility of an association of the IL-1 genotype with this condition as well. Study of a large cohort of patients with single or triple coronary vessel disease revealed that the IL-1 genotype could differentiate between single and multivessel coronary disease, a particular genotype conferring a relative risk of three for single-vessel disease. Single-vessel coronary disease resembles an arteritis and is considered by many cardiologists to be a distinct disease, not merely an earlier stage of multiple coronary occlusion. Substantiation of the relationship between the IL-1 genotype producing low amounts of IL-1ra and single-vessel disease was obtained in mice in which the IL-1ra gene was knocked out. Heterozygotes appear normal, but homozygotes begin to die at 2 months, none surviving beyond a year. Death in these animals is due to aggressive inflammation of major arteries with aneurysms in the aorta or its first-degree branches, including the coronaries. There thus appears to be substantial epidemiological and genetic evidence for overrepresentation of the particular IL-1ra allele associated with decreased formation of IL-1ra in patients with single-vessel coronary artery disease.
An increase of our understanding of the genotypes, or combination of genotypes, that predispose to various diseases will ultimately enable physicians to devise strategies of risk avoidance or risk reduction for individual patients. Customization of treatments based on genetic diagnostics (pharmacogenomics) may also provide a more efficient use of resources and should greatly increase the efficacy of treatment.
The accumulation of white blood cells into tissues is central to the pathology of inflammatory disease. The processes by which these cells migrate from the blood vessels to, for example, the synovium are enormously complex, but are initiated by the adhesion of the cells to the inner cell layer of the blood vessel, the vascular endothelium. The mechanisms by which blood cells adhere to endothelial cells were reviewed by Professor Dorian Haskard (Cardiovascular Medicine Unit, NHLI Hammersmith Campus, Imperial College School of Medicine, London, UK).
Early in vitro studies with cultured endothelial cells demonstrated that adhesion of monocytes, lymphocytes or neutrophils could be upregulated by cytokines, and many putative adhesion molecules were defined by use of monoclonal antibodies. It thus appeared that the apparent redundancy of the system would preclude simple blockade of adhesion as a means of preventing leucocyte migration and hence a treatment for inflammatory disease.
However, the realization that white cells do not bind with the endothelium under static conditions, and examination of the phenomenon of adhesion and migration in vivo, e.g. in the vasculature of the exteriorized rat mesentery, revealed that adhesion was a multiphased process. Initially, the white cells become partially tethered to the endothelium and thus roll along the surface before being swept on by the circulation. In the presence of a further stimulus, e.g. the addition of FMLP, the rolling leucocytes adhere firmly to the surface of the endothelial cell and eventually migrate into the underlying tissues. These observations clearly indicated the limitation of static, in vitro studies. Also, the cDNA cloning of the molecules defined by the antibody studies showed that their structures were quite different and thus that they had distinct functions in the adhesion and migration process. Some were involved in the initial tethering and rolling, others for firm adhesion and migration. E-, L- and P-selectins, expressed by endothelial cells, leucocytes and platelets, respectively, are responsible for the tethering and rolling of white cells. Whereas these molecules cannot immobilize the cell, they do expose it to the surface of the endothelium, where it may be activated, perhaps by locally attached chemoattractants, to adhere to the surface via activation of other adhesion molecules termed integrins. The integrins are not merely sticky molecules, they can activate the cell through various signalling systems to cause migration out of the vascular system. The significance of this process in combating infection is well illustrated by the clinical picture of those patients whose neutrophils lack the ability to express ß2 integrins. In leucocyte adhesion deficiency disease, the neutrophils cannot enter tissues, and accumulate in increased numbers in the circulation. Such patients suffer recurrent infections and die from sepsis. The endothelial cell ligands for the neutrophil integrins are ICAM-1 and -2, other white cells express the integrin VLA-4 which binds to the endothelial cell ligand VCAM-1. Once the white cells are in the tissues, integrins and other adhesion molecules govern whether the cell remains sessile, migrates or even if it survives or undergoes programmed cell death.
Since the tissue infiltration of white cells is responsible for many of the symptoms of inflammatory disease, the prevention of white cell migration could be a therapeutic target. With respect to cell adhesion, it is now clear that there is no great redundancy and that interference with any part of the mechanism may have a therapeutic effect. Antibody studies in animal models have suggested that blocking selectins, ß2 or 4 integrins or ICAM-1, show potential. Some non-peptide antagonists to adhesion molecules are available, but since their use may result in infections, their usefulness in the therapy of chronic rheumatic diseases is open to question. Ongoing selective gene knockout studies will presumably throw further light on the crucial targets of the adhesion/migration mechanism.
Although we have a fair knowledge of what adhesion molecules are involved in various conditions, Professor Haskard emphasized that we need to ascertain precisely how the endothelial cell is regulated to express these molecules. Stimulation of endothelial cells with IL-1 and tumour necrosis factor alpha (TNF-) results in the expression of E-selectin, chemokines, ICAM-1 and VCAM-1 through gene activation and de novo protein synthesis. However, there is differential expression with distinct kinetic differences. E-selectin expression occurs with a rapid rise peaking at ~5 h, then declines despite the continued presence of the inducing factor. VCAM-1 shows a more delayed rise that persists, ICAM-1 has some constitutive expression, increases and also stays high. Professor Haskard described recent experiments designed to investigate the control of leucocyteendothelial cell interaction in an in vivo model of chronic inflammatory disease. The MRL lupus-prone mouse (MRL/lpr) develops widespread infiltration of monocytes in many organs, particularly lungs and kidney, and dies at ~2022 weeks of age from renal failure. The significance of adhesion molecules for the pathology is exemplified by the fact that if the mice are crossed with ICAM-1 knockout animals, then survival is prolonged. Unlike normal mice, the MRL/lpr mice show a gradual rise in serum levels of ICAM-1 and VCAM-1. Injection of radiolabelled antibodies to these molecules and subsequent `organ count' demonstrated an increased expression in brain, kidney, lung and heart, showing that the endothelium was gradually and chronically activated. There was some apparent constitutive ICAM-1 in the lung and VCAM-1 in the kidney. Cytokine assays showed that serum TNF-
, but not IL-1, was raised by 14 weeks, and that IL-1
and ß were also raised at the time of death, suggesting that TNF-
is the primary trigger of the regulation of the endothelium. Administration of antibodies to the cytokines confirms these results since anti-TNF at 14 weeks inhibits the expression of ICAM-1 and VCAM-1, whereas in the advanced disease the anti-IL-1 antibody is also required to reduce adhesion molecule expression.
The transient increase, then decline, of selectin expression and the maintained level of ICAM-1 and VCAM-1 suggest that selectins may not be involved in the chronic disease, although they can be induced by injecting endotoxin. The induction of selectins by infection or other triggers may explain the flares that occur in lupus and other diseases. The priming of the endothelial cells by the accumulation of monocytic cells in the chronic condition may produce an enhanced accumulation of neutrophils and inflammatory response to the superinfection.
Although study of adhesion molecules has not yet been translated into benefit to patients, our knowledge of this aspect of cell biology has burgeoned over the last 10 yr. Recent experiments have demonstrated that gene knockout of a single integrin can abolish leucocyte migration to the gut and an integrin antagonist is being formulated as an inhaled preparation for trial in asthma, which suggests that some selectivity of effect may be achieved for the treatment of conditions such as inflammatory bowel disease and asthma.
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Professor Mark Ferguson (School of Biological Sciences, University of Manchester, UK) presented an account of his studies of the active endogenous substances mobilized during wound healing, studies which have resulted in the development and preliminary clinical use of agents to prevent scarring.
A significant initial observation was that fetuses operated upon in the first third of pregnancy showed no scarring when delivered at term, but undetectable repair of the damaged area. To determine exactly when scar-free healing of the fetus changed to scar healing, and the cellular and molecular correlates of this change, incisions were made in mouse fetuses at various times during the 20 day pregnancy. At term, it was noted that surgery prior to day 16 resulted in scar-free neonates. After this day, a gradual increase in scarring occurred. Since the immune system matures late in pregnancy, marker antibodies for various white blood cells were used to determine the white cell accumulation in sections of the damaged area before and after day 16. After this time, there was a gradual increase in those cells capable of producing oxygen-derived free radicals, etc. There were also differences in cytokine profiles; TGF-ß1 and TGF-ß2 were found in the wounds of mature animals, but were absent in early fetal wounds. It thus appeared that TGF-ß might have a central role in wound healing, and hence scarring, in mature mice.
TGF-ß is initially released from a stored form from degranulating platelets. It is chemotactic for endothelial cells (which can result in angiogenesis) and for monocytes and macrophages which enter the wound and release cytokines, including further amounts of TGF-ß. TGF-ß can also upregulate its own synthesis, further amplifying the concentration of this cytokine in the wound. Clearly, TGF-ß is a key agent in rapid wound healing and hence manipulation of levels of TGF-ß in adult wounds was investigated.
Intradermal injections of neutralizing antibodies to TGF-ß1 and TGF-ß2 were made into incisions prepared in the backs of adult rats. The antibody treatment, provided it was administered before 48 h, resulted in a striking decrease in scarring, a reduction in collagen in the incised region (but similar strength in the healed area), reduced fibronectin, lower levels of monocytes and macrophages, and reduced angiogenesis. Analysis of the involvement of the members of the TGF superfamily, using selective antibodies, revealed that neutralizing TGF-ß1 had some effect, neutralizing TGF-ß2 had no effect, but neutralizing both produced a marked reduction in scarring. Human recombinant phage antibodies to TGF-ß1 and -ß2 are now being used in the clinic to reduce scarring incurred during surgery on the cornea and in surgery for glaucoma (where scarring can prevent filtration). They are also being used in neurosurgery to encourage neuronal reconnection and will soon be tried in skin wounds.
Neutralizing antibodies to the third member of the TGF family, TGF-ß3, make scarring worse, whereas administration of this cytokine itself reduces scarring. Similarly, murine fetuses in which the gene for producing TGF-ß3 is knocked out scar even if damaged early in pregnancy. Together, these data suggest a possible role for TGF-ß3 in the prevention of scarring.
A further strategy for the reduction of scarring emerged from a study of the regulation of growth factors. This control is post-translation by the cleavage of the growth factor from its association with a latency-associated peptide. The peptide has three carbohydrate side chains, in two of which the terminal four sugars are mannose-6-phosphate. To release the active growth factor, the mannose-6-phosphate binds to a receptor, a conformational change ensues which exposes the protease-sensitive site, with the result that the active molecule is cleaved free. Elucidation of this mechanism raised the intriguing possibility that mannose-6-phosphate, a simple molecule, might prevent the release of TGF-ß and hence limit scarring. Indeed, the application of mannose-6-phosphate to experimental incisions in rat skin did prevent scarring, whereas mannose-1-phosphate, which does not bind to the relevant receptor, is inactive. The method of application of mannose-6-phosphate is crucial due to its short half-life in tissues, but the optimal formulation has a good, dose-dependent anti-scarring effect accompanied by reduced fibronectin in the wound and a dramatic reduction in the levels of monocytes and macrophages.
These experimental studies on rats and mice have now been repeated in pig skin and in human studies, where similar results have been obtained. Antibodies to TGF-ß1 and 2, TGF-ß3, mannose-6-phosphate and some novel molecules are now entering phase I or phase II clinical study. Developed assay techniques to measure the concentration of TGF at various stages of wound healing, and of computer three-dimensional constructs to quantify the degree of scarring, will ensure adequate assessment of the benefit of drugs to reduce scarring.
Scarring is thus the unfortunate result of an evolutionary need to obtain rapid healing in potentially infectious situations. These experiments into the basic mechanisms of healing will ultimately lead to repair of surgical or other traumatic damage without the unfortunate aesthetic or functional problems that can ensue. Ultimately, perhaps treatments for systemic fibrosis might also emerge.
Dr Sam Okret (Senior Scientific Officer at the Karolinska Institute, Stockholm, Sweden) spoke on his major research interest, the molecular mechanisms involved in the anti-inflammatory actions of glucocorticoids (GC).
Despite the wide use of GC since the 1940s, very little is known about the molecular mechanisms of their anti-inflammatory action. This has probably been due to the greater research interest in how GC activate target genes than how they repress gene expression. However, recent results have suggested that repression of target genes is a more important effect of GC in normal physiology than their activation. Transgenic mice with the wild-type GC receptor (GCR) replaced by a receptor which can repress, but not activate, target genes, develop normally, whereas mice with a total knockout of GCR die post-natally, mainly due to impaired lung maturation.
In inflammation, GC repress the transcription of a number of cytokine, adhesion molecule and other genes involved in the inflammatory response. Most of these genes are activated by an inducible transcription factor complex called nuclear factor B (NF-
B), which belongs to a family of several member proteins all characterized by homology of domain in the N-terminal region. This region is responsible for binding the proteins to DNA and for dimerization and nuclear translocation of proteins. Some members of the NF-
B family contain a transactivation domain in their C terminal region and these activate target genes. Other members of this family, called inhibitory
binding (I
B) proteins, act as inhibitors of the DNA-binding proteins. They form complexes in the cytoplasm, which prevents the DNA-binding forms from translocating into the nucleus.
A number of genes involved in inflammation contain promoters with binding sites for NF-B. Stimulation of the NF-
B pathway with TNF, bacterial lipopolysaccharide, viruses or other cytokines results in activation of these genes. Many genes which can be stimulated by NF-
B can also be repressed by GC hormones. GC regulate gene activity by binding to an intracellular GCR. In the unactivated state, GCRs are attached to heat shock proteins, e.g. heat shock protein 90, which keeps them unactivated and permits them to bind ligands. After binding, the heat shock protein dissociates from the receptor, the receptor dimerizes and undergoes a conformational change which allows it to react with DNA and especially with GC responsive elements. Binding of ligand to the same GCR can activate or repress transcription.
Transactivation of genes is a homogeneous process by which the receptor interacts with the DNA as a homodimer, recruits co-activators and stimulates transcription. Transrepression is more complicated and heterogeneous. Either GCR can interact with DNA and inactivate positively acting transcription factors, e.g. by displacement from binding, so the transcription of the target gene is reduced; alternately, GCR does not directly bind to DNA, but through a proteinprotein interaction interferes with transcriptional activation of a positively acting transcription factor. The same NF-B binding site is important both for transactivation and repression by GC.
The DNA binding domain is the most important part of the GCR for NF-B transrepressive activity, so that if it is replaced with the DNA binding domain from retinoic acid, repression is lost. However, DNA binding is not required for repression of NF-
B. There is evidence that GCR interferes directly with NF-
B activity by a proteinprotein interaction. This direct binding can be demonstrated with immunoprecipitation experiments. GCR can also inactivate NF-
B by stimulating the expression of the I
B gene, so that I
B sequesters NF-
B in the cytoplasm, but most data suggest that this is not the main mechanism by which GC repress NF-
B activity in inflammation. GCR and NF-
B have a mutually antagonistic effect so that NF-
B can inhibit GCR activity and GCR can repress NF-
B-dependent transactivation.
GCR also represses other transcription factors, particularly of the AP-1 family. Mutational studies of the GCR and the use of some GCR antagonists such as RU486 or ZK98299 suggest that the GCR-mediated mechanisms for repression of NF-B and AP-1 are different. If the interaction with NF-
B is responsible for the anti-inflammatory effects of GC, and the AP-1 system mediates the side-effects in GC therapy such as osteoporosis or growth arrest, it may be possible to develop new selective GC agonists which will reduce inflammation without the unwanted side-effects of the present hormones. Furthermore, there is still much work to be done investigating other proteins involved in the repressive pathways of GC activity.
Professor Jon Levine (University of California, San Francisco, USA) described the mechanisms of pain and possible new treatments for inflammatory pain.
Since there are several types of pain, there is a need for different approaches to its treatment. The pain associated with inflammation depends on the presence in all tissues of specialized neurones, termed primary afferent nociceptors, which sense tissue injury and create the signal which is ultimately perceived as pain. Any damage to peripheral tissues causes sensitization of the primary afferent nociceptor and hence increases electrical activity of this neurone which sends one branch to the peripheral tissue and another to the spinal cord. The result of sensitization is that the intensity of stimulation necessary to activate the cell decreases, the response of the cell to a stimulus is enhanced and, in a relatively severe inflammatory state, spontaneous electrical activity is generated.
This sensitization and increase in electrical activity after stimulation can be reproduced by injecting PGE2 into the area around the nociceptor or perfusing PGE2 over a single-cell model of the nociceptor in vitro. Transducer elements of the primary afferent nociceptor are sensitive to chemical, mechanical and thermal stimuli, and their activation is linked to changes in the function of ion channels, which may become novel targets in the development of new therapies for pain. NSAIDs are the most commonly used analgesics for inflammatory pain since they block the production of PGs which sensitize the nociceptors. The new selective inhibitors of COX-2 will inhibit PG synthesis at sites of inflammation, but not that of cytoprotective PGs at other sites.
Other pathways exist for sensitization of the nociceptor and mediators of hyperalgesia, in addition to PGE2 , including prostacyclin, leukotriene B4 , 8R-15SdiHETE, adenosine, serotonin, bradykinin, noradrenaline, IL-1, IL-8, nerve growth factor, etc. Many of these mediators act through G protein-coupled stimulatory receptors on the primary afferent neurones. These signal through activation of the cAMP/PKA second messenger pathway. The nitric oxide signalling pathway may influence the PKA function of the primary nociceptor as well as the protein kinase C pathway which affects the activity of the ion channels. There is growing interest in receptors negatively coupled to the adenylate cyclase pathway through inhibitory G proteins, since they could become targets for selective, peripherally acting opioid analgesics which could produce analgesia without the central side-effects of the opiates.
Cellular mechanisms of primary afferent nociceptors can be studied in cultured, small-diameter, dorsal root ganglion neurones by patch clamp electrophysiological techniques. These neurones stain for neurotransmitters such as substance P and calcitonin gene-related peptide, and action potentials can be measured after sensitization with stimulants such as capsaicin. Activation of the tetrodotoxin-resistant sodium channel underlies the majority of sensitization phenomena when exposed to inflammatory mediators such as PGE2 and enhances conductance of current in primary afferent nociceptors. It may be possible to design new analgesics which could selectively block this channel.
Mechanisms of thermal transduction have been studied in isolated, cultured neurones. In vivo, some sensory neurones respond to a heat stimulus of 30°C, whereas nociceptors perceive a thermal stimulus greater than 45°C as an increase in painful sensation. The majority of primary afferent nociceptors respond to all noxious stimuli and are known as polymodal fibres. When the single cultured neurones are heated, they become depolarized and a voltage clamp current passes through the membrane ion channels. Other types of neurones, e.g. sympathetic nerve fibres, do not respond to a thermal stimulus. The second messenger system is via mobilization of intracellular calcium, since decreasing the intracellular calcium concentration attenuated the current. The heat transducer is an intracellular organelle which releases calcium which then sends signals to the cell surface.
A second heat transducer has been cloned and expressed, consisting of a ligand-gated ion channel linked to the vanilloid receptor (VR1) activated by capsaicin. It is similar to the store operated calcium channel in the photoreceptor in Drosophila retina and is also found on small-diameter neurones in trigeminal ganglia. It can be activated by a temperature of 45°C and its activity modulated by a low, acid pH, which is the reason why inflammatory pain or application of capsaicin is experienced as a burning sensation. One treatment for severe clinical pain is to apply a peripheral nerve block with a local anaesthetic and then desensitize the VR1 with large doses of capsaicin.
The small-diameter sensory neurones also have receptors for oestrogens and androgens, and these may be responsible for gender differences in pain perception. Female rats experience greater sensitization and hyperalgesia in response to PGE2 than males, but if they are injected with testosterone, the effect is lost. Similarly, kappa opioid analgesics produce potent effects in female patients, but no analgesia in male subjects.
Primary afferent nociceptors can play an important role in the regulation of the inflammatory process by activating multiple pathways which feed back to inhibit the inflammatory response. This includes activation of sympathetic neurones, the hypothalamicpituitaryadrenal axis and the sympathoadrenal axis. The feedback mechanism is modulated by activity of vagal afferent nerves, since vagotomy increases nociceptor function and the response to inflammatory mediators in a rat hind paw model of inflammation.
Interestingly, it is known that bisphosphonates very effectively relieve metastatic bone pain, but the mechanism for this beneficial effect has not been studied.
Human beings have to build, repair and maintain their structure throughout their lives. Neoplasia is a distortion of this process which is life threatening since it disrupts the architectural integrity of the organism. The genes regulating activity of normal cells, which become corrupted in neoplasia, lead to a whole variety of different pathologies, of which arthritis may be an example. Neoplasia is a clonal disease arising from a single progenitor cell because of the acquisition of mutations in various regulatory genes. Dr Gerard Evan (Principal Scientist, Imperial Cancer Research Fund, London, UK) gave a comprehensive and informed talk about factors regulating the growth of normal and neoplastic cells.
The c-myc gene is involved in human malignancy, but also in normal cell proliferation. It is expressed in proliferating cells and turned off when cells undergo terminal differentiation. It became associated with malignant disease and defined as an oncogene when it was found to cause tumours in chickens. Its usual pattern of expression can be disrupted, turning it from a normal gene into an oncogene, which happens in B lymphomas in chickens, non-AIDS Burkitt's lymphoma, small cell lung carcinoma and severe cases of childhood neuroblastoma. Thus, c-myc is involved in normal cell proliferation unless its function is disrupted when the cell proliferates uncontrollably. For example, serum mitogens turn on c-myc in normal cultured fibroblasts, which then progress through the usual cell cycle. Removing the serum turns off c-myc. Fibroblasts with an experimentally deregulated c-myc cycle obligatorily even in the absence of serum growth factors.
However, cells with a deregulated c-myc do not necessarily transform into neoplastic cells, as c-myc is also an inducer of apoptosis. Thus, if serum growth factors are removed from cells expressing c-myc, they do not stop proliferating, but they also start to die. Apoptosis is an energy-dependent suicide programme which removes unwanted cells and could become the target for anti-cancer therapies in the future. So, a mutation in a growth-regulatory gene like c-myc will only give rise to cancer if the cell is rescued by a simultaneous anti-apoptotic mutation. The first documented example of such a second mutation was the deregulation of the bcl-2 gene, identified in human follicular B lymphomas. Co-expression of the bcl-2 gene prevents apoptosis by c-myc and the cells were able to propagate.
This type of cooperation between oncogenes also occurs between the c-myc and Ras genes. Fibroblasts with c-myc and Ras oncogenes activated singly do not change their phenotype, whereas both genes activated together produce the classic eruption of transformed foci of cells. However, c-myc and Ras cooperate by a different mechanism from c-myc and bcl-2, since oncogenic Ras does not block, but rather exacerbates, c-myc-induced apoptosis. Therefore, to design new cancer therapies, a better understanding of the interaction between these three genes and how they cooperate to support tumour growth is needed. An understanding of the interdependency of tumour genes will no doubt lead to new cancer therapies in the future.
Cell death is the normal outcome of deregulation and overexpression of the c-myc gene, unless survival factors are present to prevent the apoptosis. Survival factors are external cytokines provided by neighbouring cells that activate signal transduction pathways which suppress apoptosis. The supply of survival factors in a tissue will determine whether apoptosis of the cells can be triggered by deregulated c-myc. In order to assess to what extent cells of different tissues are protected by endogenous survival factors, c-myc was activated in various tissues of a living mouse. This was achieved by constructing a switchable c-myc protein, fused to the hormone-binding domain of a modified oestrogen receptor that could only be activated with the synthetic steroid, 4-hydroxy-tamoxifen. Transgenic mice with the switchable c-myc gene targeted to the ß-islet cells of the pancreas became diabetic after 3 weeks of dosing with 4-hydroxy-tamoxifen. The ß-islet cells did not contain enough survival factors to prevent apoptosis by the activated c-myc and were destroyed. In a similar way, the switchable c-myc gene was targeted to the suprabasal layer of skin in a transgenic mouse. This tissue had an abundance of survival factors and painting the depilated skin with 4-hydroxy-tamoxifen caused massive hyperplasia of the epidermal layer when applied every 3 days, and papilloma formation if applied continuously.
c-myc induces apoptosis by sensitizing cells to a wide range of apoptotic triggers such as hypoxia or DNA damage and, in the case of fibroblasts, to stimulation of the CD95/Fas receptor on the cell surface. The continual interaction of Fas ligand with the Fas receptor, both present on the cell surface, constitutes a signal for cell death which only takes effect when c-myc is expressed inside the cell. Another apoptotic trigger, cytochrome C, is released from mitochondria into the cytosol and acts as the focal system to bring together an apoptosome complex involving activated caspases which form the basal apoptotic machinery. The survival factor, IGF-1, prevents the release of cytochrome C from the mitochondria. However, by itself, the release of cytosolic cytochrome C is not enough to trigger cell death unless other signals, none of which will cause death by themselves, intersect this pathway downstream.
The reason why not all cells which express c-myc die is that the viability of cells is also regulated by survival factor pathways. One of these is the IGF-1 signalling pathway which, when activated through the transmembrane tyrosine kinase receptor, recruits survival factors such as Ras and PI 3 kinase. In reality, Ras has multiple intracellular effectors, some of which might suppress apoptosis and others induce it. For example, activation of Ras in fibroblasts activates a Raf kinase-driven pro-apoptotic pathway at the same time as the PI3 kinase, PKB/Akt kinase-driven survival pathway. Thus, the fate of a cell with activated Ras appears to depend upon the balance between these two opposing signals: which one predominates depending upon cross-talk with other signalling pathways obtaining in the cell. Similar opposing signals for growth promotion and growth inhibition are generated by the oncogenic genes, c-myc and bcl-2.
The cooperation between oncogenes and the multiple effects of gene activation make it difficult for cells to become tumour cells through sequential mutations. For example, transgenic mice lacking the tumour suppressor gene p53 do not immediately develop tumours, although clonal neoplasms will form after about 3 months. It is possible that this gene has growth-promoting properties not evident in tissue culture which could be identified in the living organism. Thus, it emerges that key components of signalling pathways with potentially oncogenic outcomes, principally those promoting proliferation or cell survival, are obligatorily interdependent. Exploiting such interdependence may provide a more rational basis for future therapy.
In humans, there are probably ~100000 genes for protein products and most will be sequenced in the next few years. However, it is now apparent that only 50% of these sequences can currently be allocated a function by comparing their sequences with those of homologous proteins.
Professor Tom Blundell (Biochemistry Department, Cambridge University, UK) has an interest in comparative genomics as well as in rational approaches to drug design and protein engineering. He has utilized knowledge of the three-dimensional structures of receptors and other targets as a basis for drug design, identifying various proteins which can be grouped into superfamilies on the basis of their structural rather than sequence homologies. One of the earliest examples was the realization that all insulin molecules from different organisms adopted the same three-dimensional structure and experimental data supported the concept that the tertiary structure of insulin was important for its activity. The characteristic fold of the insulin molecule was found in other insulin-like structures, such as insulin-like growth factor, human relaxin, silkworm bombyxin and a mollusc neuropeptide. All these proteins were able to bind to cell surface receptors and all belonged to the same family. This is now known as the insulin superfamily.
Observations made on nerve growth factor showed that, like insulin, it also contained six cysteine residues, but these were clustered in a different way from the insulin molecule. However, this clustering was repeated in the structures of other growth factors, such as transforming growth factor and platelet-derived growth factor. Over 80% of this protein family are dimeric growth factors which bind to cell surface receptors.
This method of identifying protein superfamilies across species has played a role in the discovery of several drugs now in clinical use. One important example was the development of HIV and AIDS antivirals based on the similarity between HIV proteinase and the aspartic proteinases. Members of this viral proteinase family contain the signature sequence of aspartinethreonineglycine which resembles a similar sequence in the human renin enzyme. Antihypertensive inhibitors of renin also inhibited HIV proteinase and led to the development of successful anti-AIDS drugs, although it is now evident that mutation in HIV makes it necessary to treat AIDS with a cocktail of antiviral compounds.
Another example is the family of collagenases which includes stromelysin, collagenase and gelatinase, targets for anti-arthritic and anti-cancer drugs. An active site pocket in stromelysin forms a useful opportunity for stromelysin-specific drugs. Yet another example is serum amyloid P component (SAP), which is proving useful in the development of anti-amyloid and anti-Alzheimer's drugs. SAP belongs to a superfamily of sugar-binding proteins which have no sequence similarity, but which all bind sugars in the same region.
With the aid of computer programs, structural comparisons can be made between different members of superfamilies and common features recognized. In this way, all known proteins can be clustered into groups according to their similarity of structure. A previously determined metric will indicate whether they are likely to be a superfamily with some similarity of function. Proteins grouped in this way include the retroviral proteinase pepsin family, the lectins and the immunoglobulins.
The use of several databases, such as the domain database, homologous alignment database and superfamily alignment database, can help in relating orphan gene products to known structures with defined functions.
Recent studies of comparative genomics in Cambridge revealed the interesting observation that an intracellular pathway involved in the development of Drosophila has similarities with the IL-1 pathway in humans. Application of the knowledge from the well-documented Drosophila pathway could, by homology, lead to identification of drug targets for the manipulation of the interleukin pathway. Furthermore, the ligand for the Toll receptor in Drosophila resembles human nerve growth factor and study of other ligands may uncover useful proteins for the treatment of human disease.
Thus, an understanding of protein superfamilies can help to identify drug targets not found by sequence searches. Focusing on these targets will enable scientists to make hypotheses about specific ligands for these receptors. This type of approach will be useful in developing treatments for all kinds of diseases and relevant to the search for new treatments for OA.
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Submitted 9 April 1999;
revised version accepted 19 April 1999.