Human collagenase: identification and characterization of an enzyme from rheumatoid synovium in culture.

Synovial tissue from patients with rheumatoid arthritis produces lysis of gels of reconstituted collagen fibrils in culture and releases soluble collagenase when cultured in collagen-free medium. Collagen molecules in solution at neutral pH at 20 degrees and 27 degrees C are cleaved by the synovial enzyme into (3/4) and (1/4) length fragments. In this respect the action of synovial enzyme is similar to that of amphibian collagenase and distinct from that of bacterial collagenase. At 37 degrees C reconstituted collagen fibrils and native fibers are attacked by the enzyme and further degraded to polypeptides of low molecular weight. These polypeptides are produced only after denaturation of the larger fragments, which occurs at temperatures near 37 degrees C.

Studies on collagenase from rheumatoid synovium in tissue culture.

Fragments of synovium from patients with rheumatoid arthritis survive in defined tissue culture medium in the absence of added serum and, after 3-4 days, release into the medium enzyme capable of degrading undenatured collagen. Maximal activity is observed at pH 7-9 but the enzyme is inactive at pH 5. At temperatures of 20 degrees and 27 degrees C, collagen molecules in solution are cleaved into 3/4 and 1/4 length fragments with minimal loss of negative optical rotation, but with loss in specific viscosity of approximately 60%. Above 30 degrees C the fragments begin to denature and denaturation is complete at 37 degrees C. If the enzyme is not inhibited at this stage the large fragments are broken down further to polypeptides of low molecular weight. Reconstituted collagen fibrils and native fibers at 37 degrees C are cleaved to the low molecular weight fragments, although the fibrils are resistant to breakdown at lower temperatures (20 degrees -27 degrees C). It is proposed that the production of such an enzyme by inflamed and proliferating rheumatoid synovium may be responsible for some of the destruction of collagenous structures that accompanies rheumatoid arthritis.

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.

Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue.

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.

Detection and treatment of psychiatric illness in a general medical ward: a modified cost-benefit analysis.

This article describes a prospective, randomized, controlled trial of screening and treatment for psychiatric disorder in medical in-patients. The study has assessed whether increased recognition of psychiatric disorder among medical in-patients improves clinical outcome and reduces the costs of care, and whether routine involvement of a psychiatrist in the assessment and care of medical in-patients with probable psychiatric disorder is superior to the efforts of the physicians alone. A total of 218 medical in-patients who scored over the screening threshold for psychiatric disorder on the General Health Questionnaire were randomly allocated to one of two intervention groups or a control group. Six months later their mental health, subjective health status, quality of life, and costs of care was reassessed. Mental health and quality of life at 6 months were similar in the two intervention groups and the control group. Patients whose physicians were told the results of the screening test had lower costs for subsequent admissions, but this was probably due to differences between the groups in terms of employment status. Treatments recommended by psychiatrists broke down when patients were discharged home, leading to inadequate treatment of psychiatric disorders. We have not been able to show that routine screening for psychiatric disorder produces any benefit, either in better outcome for patients or reduced costs for the NHS. Further research should: consider examining a more homogeneous group in terms of costs of care; screen only for disorders likely to respond to a specific treatment; and ensure that treatment recommendations are carried out.

Collagenase and its natural inhibitors in relation to the rheumatoid joint.

This report attempts to summarize our present knowledge of rheumatoid synovial collagenase and its natural serum inhibitors, beta1-anticollagenase and alpha2-macroglobulin, in relation to cartilage collagen resorption in the rheumatoid joint. Immunolocalization of collagenase across the cartilage/pannus junction is described, and in the light of the finding of the specific, small molecular weight beta1-anticollagenase we propose a model of cartilage erosion based on the interaction between collagenase and its natural inhibitors.

Inflammatory cytokines stimulate glucose uptake and glycolysis but reduce glucose oxidation in human dermal fibroblasts in vitro.

Interleukin-1 alpha (IL-1 alpha) produced alterations in human dermal fibroblast glucose metabolism in vitro of the type seen in severe sepsis in man. Glycolysis and glucose uptake were increased but the oxidation of glucose within the tricarboxylic acid (TCA) cycle was reduced. The combined addition of tumour necrosis factor alpha (TNF alpha) with interferon-gamma (IFN-gamma) similarly increased the dependency for cellular energy provision from an oxidative to the glycolytic state. These cytokine-induced changes in glucose metabolism were unaffected when prostaglandin production was inhibited with a cyclo-oxygenase inhibitor, but were significantly reduced by the steroid dexamethasone. Thus, the inflammatory cytokines IL-1 and TNF alpha reportedly detected in the circulation during severe sepsis may directly affect not only glucose uptake but also its subsequent metabolism within tissue fibroblasts.

Serum IgE anti-cartilage collagen antibodies in rheumatoid patients.

The presence of circulating IgG, IgA and IgM antibodies to native cartilage collagens in some patients with rheumatoid arthritis (RA) suggests that an autoimmune response to cartilage collagens may be involved in the pathogenesis of RA. However, the relevance of such antibodies to the pathological process remains unclear, and it is likely that many humoral and cellular derived factors combined to trigger events leading to the chronicity of the rheumatoid lesion. Since histological and biochemical studies have suggested the involvement of mast cells in the rheumatoid joint, we have studied the frequency of IgE antibodies directed against the cartilage collagens type II, IX and XI in patients with active rheumatoid disease. Of the 91 patients' sera tested, 32 had significant levels of IgE anti-cartilage collagen antibodies when compared with non-arthritic controls. Total serum IgE levels did not correlate with the presence of IgE anti-collagen antibodies, nor were any patients positive for IgE antibodies to fibronectin, a widely distributed extracellular matrix component. These results are consistent with an allergic type I hypersensitivity reaction to cartilage antigens in RA involving mast cell and basophil degranulation.

Contrasting effects of the protein kinase C inhibitor, staurosporine, on cytokine and phorbol ester stimulation of fructose 2,6-bisphosphate and prostaglandin E production by fibroblasts in vitro. Comparative studies using interleukin-1 alpha, tumour necrosis factor alpha, transforming growth factor beta, interferon-gamma and 12-O-tetradecanoylphorbol 13-acetate.

It is known that both interleukin-1 alpha (IL-1 alpha) and 12-O-tetradecanoylphorbol 13-acetate (TPA) promote increases in intracellular levels of the glycolytic regulatory metabolite fructose 2,6-bisphosphate [Fru(2,6)P2] and in the production of prostaglandin E (PGE) by subcultured rheumatoid synovial cells (RSC) and human dermal fibroblasts in vitro. We report here that the protein kinase C inhibitor staurosporine enhanced the IL-1 alpha-induced increase in [Fru(2,6)P2] and PGE production by RSC, whereas in similar concentrations (3-30 nM) this inhibitor decreased the TPA-induced stimulation of these parameters. Staurosporine produced a similar enhancement of the response to IL-1 alpha by normal human dermal fibroblasts. The increased PGE production provoked by tumour necrosis factor alpha (TNF alpha) in RSC was also augmented by staurosporine, but, in contrast, the increases in cellular [Fru(2,6)P2] induced by transforming growth factor beta (TGF beta) and interferon-gamma (IFN-gamma) were diminished. Thus the protein kinase C inhibitor staurosporine discriminates not only between the effects produced by IL-1 alpha and TPA, but also between those of IL-1 alpha and two other cytokines (but not between IL-1 alpha and TNF alpha). These findings suggest that IL-1 alpha and probably TNF alpha act via an intracellular mechanism different from that mediating the action of TPA, TGF-beta and IFN-gamma, and provide evidence that staurosporine is capable of amplifying the IL-1 signal.

Collagenase at sites of cartilage erosion in the rheumatoid joint.

Immunolocalization studies of rheumatoid tissues employing specific synovial collagenase antibody have demonstrated immunoreactive enzyme at the cartilage/pannus junction. Collagenase was not detected in chondrocytes or the cartilage matrix remote from the resorbing front, and relatively little enzyme was observed in the hypertrophied synovial membrane itself. These observations directly support the idea that synovial collagenase participates in cartilage erosion in rheumatoid arthritis.

Paget's disease of bone: diagnosis and management.

The main features of Paget's disease are described, together with the indications for medical treatment. A brief summary is given of the drugs available for treatment of Paget's disease with particular emphasis on sodium etidronate (EHDP, ethylidene-1-hydroxy-1, 1-diphosphonate). Sodium etidronate, given at doses between 5 and 20 mg per kilogram per day for 3-6 months, causes a progressive reduction in the biochemical abnormalities (raised plasma alkaline phosphatase and urinary hydroxyproline) and in the histological abnormalities of bone. Clinical symptoms also improve. The usual dose is 5 mg per kilogram body weight per day to be given for not longer than 6 months. Higher doses (10 and 20 mg per kilogram per day) may cause impairment of normal bone mineralisation and should be given for short periods only (1-3 months). Sodium etidronate also has a limited place in the treatment of certain disorders of ectopic calcification, notably heterotopic ossification after spinal cord injury or hip surgery. At the present time there is insufficient evidence to justify its use in the treatment of renal stones or in osteoporosis other than that due to immobilisation after spinal cord injury.

Comparative effects of interleukin 1 and a phorbol ester on rheumatoid synovial cell fructose 2,6-bisphosphate content and prostaglandin E production.

The addition of either recombinant human interleukin 1 (IL1 alpha) or 12-O-tetradecanoyl phorbol-13-acetate (TPA) to cultured rheumatoid synovial cells (RSC) caused dose-related increases in PGE production and cellular fructose 2,6-bisphosphate (Fru-2,6-P2). IL1 consistently produced the greater increases in both parameters. A close association between increases in PGE production and Fru-2,6-P2 was demonstrated for both IL1- and TPA-stimulated cells. The combined addition of IL1 with TPA resulted in an additive increase in both parameters. When IL1 was added together with human recombinant interferon-gamma (IFN-gamma), the resulting Fru-2,6-P2 level was synergistically increased, whilst the combination of IFN-gamma and TPA produced only an additive increase. Thus despite their very similar effects on RSC in culture, the data suggests that IL1 and TPA do not act via an identical intracellular mechanism.

Bidirectional erosion of cartilage in the rheumatoid knee joint.

Specimens of cartilage with contiguous bone and overlying synovial pannus were obtained from 22 rheumatoid knee joints and examined histologically using specific histochemical staining techniques. All showed significant erosions of cartilage by synovial cells, but seven specimens also showed substantial cartilage erosion by cells from the subchondral bone region. This bidirectional attack on rheumatoid knee cartilage did not represent an 'underpinning' of cartilage by synovial pannus, as judged by serial sectioning and the identification of specific cells. Whereas cartilage-pannus junctions had mainly macrophagic or fibroblastic cells, cartilage-bone lesions were usually characterised by chondroclasts and blood vessels. Lymphocytes were generally absent from all sites of cartilage erosion. The bidirectional attack on articular knee cartilage suggests that changes have occurred within the cartilage that make it vulnerable to cellular invasion and erosion. Such changes might reflect a deficiency in 'anti-invasion factors', or the exposure of hidden epitopes and subsequent immunogenicity, or a combination of both.

A neutral collagenase from human gastric mucosa.

Biopsy specimens of human gastric mucosa, maintained in culture for 7 days in the absence of serum, released a collagen-degrading enzyme into the medium. The yield of active enzyme reached a maximum after 2-3 days, and viable tissue, capable of protein synthesis, was essential for its production. 2. At 25 degrees C the enzyme attacked undenatured collagen in solution, resulting in a 55% loss of specific viscosity and producing the two products TCA and TCB characteristic of neutral-collagenase action. 3. Electron microscopy of segment-long-spacing crystallites of these reaction products showed the exact cleavage locus of the collagen molecules to be between bands 43 and 44 (I-43). The larger TCA and smaller TCB products were fragments representing 77 and 23% respectively of the length of the collagen molecule. 4. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a mol.wt. of approx. 38000 was derived from gel-filtration studies. 5. The enzyme was shown to be inhibited by the human serum proteins alpha2-macroglobulin and a smaller component of mol.wt. approx. 40000; alpha1-anti-trypsin was not inhibitory. 6. EDTA, 1, 10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. 7. The gastric enzyme has properties similar to other well characterized collagenases, but differences exist with respect to its molecular size and the site of attack on the collagen molecule.

Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen.

1. The neutral collagenase released into the culture medium by explants of ehrumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine. 2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 mug collagen degraded min-1 mg enzyme protein-1, representing more than a 1000-fold increase over that of the active culture medium. 3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. 4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5. Data obtained from sutdies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent. 7. The collagenase attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). 8. At 37 degrees C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils. 10. The data suggests that pure rheumatoid synovial collagenase at 37 degrees C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases. 11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.