Biomedical subjects
Publications and source records attributed to J T Dingle.
The damage to articular cartilage, characteristic of arthritic disease, is usually ascribed to increased degradative activity by enzymes or free radicals from locally activated cells. We propose that inhibition of matrix synthesis, and consequential impairment of the natural repair process, may be at least as important in chronic joint disease.
The response of the rabbit knee joint to a brief episode of cytokine induced damage is described. After three intra-articular injections of catabolin/interleukin-1 all joint cartilages showed an immediate extensive loss of proteoglycan (glycosaminoglycan), which was gradually replaced over three to four weeks. Glycosaminoglycan biosynthesis (measured by 35SO4 uptake) was initially depressed, but at one week had almost doubled its rate as compared with the normal side. This increased synthetic activity was further maintained throughout the duration of the experiment (28 days), though the rate gradually fell. Histological cartilage metachromasia to toluidine blue mirrored the glycosaminoglycan changes. No disturbance of the articular cartilage collagen network was found. It is considered, therefore, that during treatment for arthritis the indigenous chondrocyte must continue to be capable of carrying out regenerative matrix repair and that antiarthritic agents should first be screened for interference with that process.
A polyclonal antiserum which recognizes surface epitopes on IL1-activated pig chondrocytes has been used to immunolocalize chondrocytes responding to IL1 produced during co-culture of pig synovium and articular cartilage. Activation of the chondrocytes by the cytokine was restricted to the articular and subarticular region of the cartilage adjacent to the synovium. Chondrocyte activation was also seen when human rheumatoid synovium was co-cultured with the cartilage. The presence of IL1 in some synovial cells was confirmed by immunolocalization using antisera specific for IL1 alpha and IL1 beta.
Monoclonal antibodies have been used to demonstrate the induction of intercellular adhesion molecule 1 (ICAM-1) on chondrocytes in human articular cartilage. ICAM-1 was found not to be constitutively expressed but could be induced by exogenous interleukin 1 alpha(IL1- alpha) at concentrations ranging from 0.01 to 20 ng/ml during in vitro culture. Maximum expression was observed with 2-5ng/ml. In time-course experiments ICAM-1 was not expressed after 4h in culture with IL1 alpha. Expression was induced by 16h and was sustained for a minimum of 6 days in the continued presence of the cytokine. The endothelial leukocyte adhesion molecule (ELAM-1) was not expressed on chondrocytes and was not induced by IL1-alpha.
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The structural integrity of the matrix of human articular cartilage is maintained by a dynamic equilibrium between synthesis and degradation. In osteoarthritis (OA), synthesis may be inhibited by the presence of subnanogram quantities of the cytokine interleukin 1 (IL-1), leading in the longterm to loss of matrix and susceptibility to mechanical damage. IL-1 may also inhibit the potential for repair processes to take place in this cartilage if continued synthesis and secretion of the cytokine occurs. Evidence is presented that animal and human cartilages are sensitive to the action of certain nonsteroidal antiinflammatory drugs (NSAID) in inhibiting the synthesis of cartilage proteoglycan and also diminishing the repair activity of cartilage recovering after IL-1. In OA cartilage, the sensitivity to action of NSAID may depend on the state of the tissue in terms of glycosaminoglycan (GAG) turnover and GAG synthetic activity of the indigenous chondrocytes. Preliminary investigations of the prostaglandin analog misoprostol on the synthetic repair activities of animal and human cartilage in the presence of NSAID are reported.
Proteoglycan levels were measured in a series of synovial fluid samples from patients with acute and chronic joint diseases using a modified chemical dye binding method. Levels found in 50 miscellaneous inflammatory arthritis fluids (mean = 173.2 +/- 90.9 micrograms/ml) were higher than found in either 50 with rheumatoid arthritis (96.3 +/- 31.3 micrograms/ml) or 50 with osteoarthritis (83.8 +/- 27.3 micrograms/ml). For comparison, proteoglycan levels were measured in 15 cadaver synovial fluids (98.9 +/- 44.2 micrograms/ml) and 12 synovial fluids from patients with sports injury (163.7 +/- 79.4 micrograms/ml). Patients were recruited into a trial where synovial fluid was aspirated as often as possible over a 6-month period during which the patients were followed using a number of well proven clinical parameters. No correlation was found between the degree of joint destruction as measured by X-ray damage and the concentration or total amount of proteoglycan in the synovial fluid. In addition, there was no correlation between the level or total amount of proteoglycan and any clinical parameter of disease activity.
Interleukin-1 activated chondrocytes have been identified in pig articular cartilage by immunolocalisation using a polyclonal antiserum recognising cytokine induced surface epitopes. Although chondrocytes with membrane staining were noted in all zones of the cartilage, only a proportion of the cells were positive within the range of antiserum dilution used (1:100-1:2000). Maximum staining was found after four days' treatment of cultured cartilage with pig IL-1 alpha, though weak staining was just detectable after treatment for one day at high antiserum concentration. Immunoreactivity was also shown in cartilage which had been cultured with human recombinant IL-1 alpha.
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The rate of loss from the synovial cavity of proteoglycan subunit, a major constituent of cartilage, has been measured in rabbits with and without experimental arthritis. The effect of aggregation between proteoglycan and hyaluronic acid upon the rate of elimination has also been investigated. The results indicate that proteoglycan subunit has a clearance half life of around 12 hours in normal joints and that this rate is not significantly altered in the presence of an active synovitis. Neither injection of proteoglycan as an aggregate, nor in a form incapable of aggregation, had any significant effect on clearance rates. These data indicate that loss of proteoglycan from the joint is not retarded by its ability to aggregate with hyaluronic acid in the synovial fluid and, together with measurements of synovial fluid proteoglycan, may enable rates of cartilage breakdown in vivo to be calculated.
Human synovial tissues have been assayed for the production of cartilage degrading activity (CDA). This activity is thought to be homologous with catabolin/interleukin 1 (IL1) produced by porcine synovium and leucocytes and by human leucocytes. The CDA of 26 rheumatoid (RA) and 41 non-rheumatoid synovia was measured on a dry weight basis. The rheumatoid synovia showed a threefold increase in activity over the non-rheumatoid, but there was no significant overall difference on a deoxyribonucleic acid (DNA) basis. The rheumatoid synovia appeared to consist of two populations; in over half the samples CDA was not related to cellularity, but eight patients had a high CDA and a high cellularity.
A partially purified porcine synovial catabolin interleukin 1 (CF) preparation was injected intra-articularly into rabbit stifle (knee) joints. Radiolabelled CF was rapidly cleared from the joint (0.4 h). Repeated injections of CF caused a marked loss of articular cartilage glycosaminoglycan (GAG) and a great increase in synovial fluid GAG. 35SO4 uptake was inhibited. Time course experiments after a single injection produced similar loss of GAG from knee cartilages, which was maximal three days after injection. The above changes were significantly less with heat inactivated preparations. Loss of articular cartilage metachromasia was found histologically, and an acute synovitis occurred together with lymphocytic foci and plasma cell infiltration.
The injection of partially purified porcine synovial catabolin/IL-1 alpha intra-articularly in rabbits resulted in a 50% loss of glycosaminoglycan (GAG) after 3 days. An increase in the synovial fluid content of GAG was found, and 35SO4 incorporation into proteoglycan was inhibited. Measurements were also made of the GAG loss from articular cartilage into the synovial fluid in human rheumatoid (RA) and osteoarthritic (OA) patients. Very high levels of GAG content in the synovial fluid was found, and calculations were made of the half-life of the cartilage proteoglycan during the active phases of the disease. Estimation of the synovial fluid GAG is believed to be a simple and quantitative method for monitoring the effectiveness of cartilage-"sparing" anti-arthritic drugs.
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Cultured porcine heart valves secrete a factor that induces cells to degrade their extracellular matrix. This activity was routinely monitored by measuring the release of glycosaminoglycans from proteoglycan in cultured bovine nasal cartilage. It was observed that factor-mediated matrix degradation was dose dependent and required live target cells, while factor production by valves was inhibited by cycloheximide and declined with reduced cell viability. The effect of various steroidal and nonsteroidal anti-inflammatory drugs on the production and activity of the factor is discussed with reference to similar cytokines. It is concluded that there is a close similarity between the cardiac catabolic factor described here and catabolin, a protein isolated from porcine synovia and leukocytes, as shown by the neutralization of biological activity with antisera raised to porcine catabolin (interleukin-1).
A cardiac catabolic factor (CCF) has been partially purified from serum-free medium conditioned by minced porcine heart valves. CCF was prepared by a series of chromatographic techniques and compared directly with porcine synovial catabolin purified by the same protocol. CCF displayed a somewhat higher molecular weight (Mr 21,000) and isoelectric point (pI 5.2) than did synovial catabolin (Mr 18,000 and pI 4.8), but the two factors clearly resemble one another closely. CCF stimulated the release of glycosaminoglycans from cultured cartilage and mitral valve and provoked porcine valves to degrade their own collagen extracellular matrix. The release of hydroxyproline was inhibited by corticosteroids, whereas proteoglycan breakdown was not. Partially pure preparations of CCF and synovial catabolin stimulated murine thymocyte proliferation; moreover, that activity was almost totally abolished by an antibody raised against pure porcine interleukin-1. These observations suggest that CCF may represent a catabolic factor that belongs to the interleukin-1 family and that it could potentially regulate the composition of valvular connective tissue.
A specific antiserum to rabbit bone collagenase was raised in a sheep and shown to react with collagenase from several mammalian species. This antiserum was used to demonstrate that only active collagenase binds to and can be immunolocalized on collagen fibrils and, by indirect immunofluorescence, the secretion of latent collagenase by stimulated rabbit chondrocytes and cells of the post-partum involuting rabbit uterus. The ionophore monensin was used to demonstrate intracellular accumulation of collagenase in the Golgi apparatus of both stimulated chondrocytes and involuting uterine cells. Collagenase was not detectable in either normal cartilage or non-gravid uterus. These results are discussed in relation to other studies of collagenase immunolocalization reported in the literature.
Explants of pig kidney cortex and medulla release a catabolin-like factor (CLF). The CLF of both kidney cortex and medulla can be precipitated with ammonium sulphate, mainly in the 60-95 per cent fraction. By gel chromatography the kidney CLF showed a major active fraction at a molecular weight of around 22 500. Whole glomeruli and dissociated glomerular cells in culture also released into the medium a CLF that could be bioassayed in live cartilage but displayed no effect on dead cartilage. The possible role of such a local hormone is discussed.