Rabbit collagenase. Immunological identity of the enzymes released from cells and tissues in normal and pathological conditions.

1. The immunological cross-reactivity between rabbit collagenases from a variety of normal and pathological sources was examined. The specific antibody raised against collagenase secreted from normal rabbit synovial fibroblasts gave reactions of complete identity with collagenases secreted from fibroblasts derived from rabbit skin, and from synovium from experimentally arthritic rabbits. 2. The rabbit fibroblast collagenase was immunologically identical with collagenases obtained from the organ culture medium of normal rabbit skin, synovium, ear fibrocartilage and subchondral bone. 3. Collagenases from the culture media of normal rabbit synovium and from hyperplastic synovium of rabbits made experimentally arthritic were identical. 4. The collagenase secreted from rabbit fibroblasts gave a reaction completely identical with that of a collagenase extracted directly from a rabbit carcinoma. 5. IgG (immunoglobulin G) from a specific antiserum to rabbit fibroblast collagenase was a potent inhibitor of the collagenases obtained from the culture media of the various rabbit cells and tissues. 6. Collagenases from human synovium and from mouse macrophages and bone were neither precipitated nor inhibited by antibodies to rabbit collagenase. 7. No immunoreactive material was found in lysates of rabbit polymorphonuclear leucocyte granules with the specific antisera to rabbit fibroblast collagenase. No evidence for inactive forms of rabbit collagenase in lysates of the rabbit synovial fibroblasts could be found, either by double immunodiffusion against the specific collagenase, or by displacement of active enzyme from inhibition by the IgG.     

Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase.

1. Antisera were raised against the collagenase from rabbit synovial fibroblasts and characterized by immunoprecipitation and immunoinhibition reactions. 2. Immunoglobulins from the antisera were potent inhibitors of the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. 3. The antibody-binding fragment, Fab', produced by digesting the IgG (immunoglobulin G) with pepsin, inhibited collagenase activity just as well as whole IgG. 4. A specific antiserum to the rabbit collagenase was raised by a multi-step procedure. An initial antiserum was made by injecting partially purified collagenase as a complex with sheep alpha2-macroglobulin into a sheep. The non-specific antiserum so obtained was used to produce precipitin lines with the purified enzyme, and these lines were used as antigen for the production of the specific antiserum. 5. An IgG preparation from the specific antiserum was a specific and potent inhibitor of the rabbit synovial fibroblast collagenase. Neutral metallo-proteinase activity secreted by the rabbit fibroblasts was not inhibited by the antibody to the rabbit collagenase. 6. Criteria for determination of the specificity of antisera are discussed.     

Synthesis and release of procollagenase by cultured fibroblasts.

An inactive collagenase was harvested from both serum-free and serum-supplemented fibroblast monolayer cultures in periods of active collagen synthesis. The latent collagenase did not hydrolyze collagen and did not bind the potent collagenase inhibitor alpha2-macroglobulin. Activation with trypsin imparted to the enzyme the ability to hydrolyze collagen at neutral pH in a typical manner and to form an inhibited complex with alpha2-macroglobulin. The molecular weights, determined by calibrated gel filtration, were 78,000 and 60,000 for the latent and active enzymes, respectively. The data indicate that collagenase is released from the cells in inactive form, as a zymogen.     

Purification and properties of a specific collagenase from rabbit synovial fibroblasts.

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.     

Collagenases from human and rat skin fibroblasts purified on a zinc chelating column reveal marked differences in latency as a result of serum culture conditions.

1. Fibroblasts from both human and rat skin were grown in the presence or absence of serum and the collagenase activity in the medium was partially purified on zinc-Sepharose. 2. During chromatography, using a discontinuous elution gradient, the rat collagenase elutes at different pH and ionic strength than the human collagenase. Both latent and active collagenases of both species are retarded by the affinity matrix. 3. Latency of collagenase in media obtained from fibroblast cultures appears to be influenced by the presence of a serum component in the culture medium. 4. The results demonstrate that collagenases secreted by fibroblast cultures established from the same tissue but obtained from different species are biochemically diverse and that, within one species, the amount of active enzyme depends on the presence of a serum factor.     

Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid

In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.     

Biosynthesis of plasma proteins in serum-free medium by primary monolayer culture of rat hepatocytes

Morphologically intact rat hepatocytes separated by collagenase perfusion were cultured in L-15/fetal calf serum medium to form a monolayer. Thereafter the hepatocytes were grown in serum-free L-15 medium in which they produced and continuously released plasma proteins. The secreted plasma proteins were collected, separated and characterized by crossed immunoelectrophoresis. Most of the newly biosynthesized plasma proteins secreted into the medium during incubation for thirty hours had the same electrophoretic mobility, antigenicity and staining characteristics as their counterparts in rat serum. The addition of tritium labelled amino acid mixture to the culture medium revealed that the release of radioactively labelled plasma proteins into the culture medium was essentially linear during the thirty hour incubation period. However, saturation of the intracellular pool took place after ca. ten hours of incubation. Addition of leukocytic endogenous mediator, LEM, to cultures of rat hepatocytes caused a profound increase in the relative concentration of acute-phase proteins secreted into the culture medium.     

Activation of latent rheumatoid synovial collagenase by human mast cell tryptase

The functional role of mast cells in rheumatoid synovium was investigated by assessing the ability of mast cell tryptase to activate latent collagenase derived from rheumatoid synoviocytes. Tryptase, a mast cell neutral protease, was demonstrated in situ to reside in rheumatoid synovial mast cells, by an immunoperoxidase technique using a mouse mAb against tryptase, and in vitro to be released by dispersed synovial mast cells after both immunologic and nonimmunologic challenge. Each rheumatoid synovial mast cell contains an average of 6.2 pg of immunoreactive tryptase and the percent release values of this protease correlated with those of histamine (r = 0.58, p less than 0.01). The ability of purified tryptase to promote collagenolysis was demonstrated in a dose-dependent fashion using latent collagenase derived from rheumatoid synovium, synovial fluid, IL-1-stimulated cultured synoviocytes, and partially purified latent collagenase derived from conditioned media, with between 10 and 92% of the collagen substrate degraded. [3H] Collagen, treated with tryptase-activated latent collagenase, was subjected to electrophoresis on SDS polyacrylamide gels and autoradiography showed the collagen degradation pattern (A, B) characteristically produced by collagenase. Mast cell lysates also activated synovial latent collagenase yielding 24% digestion of collagen substrate. This activator in mast cell lysates could be inhibited by diisopropylflurophosphate or by immunoadsorption of tryptase. Thus, mast cells may activate metalloproteinases and play a role in the catabolism of collagen that occurs in rheumatoid synovium.     

Collagenase synthesis by cloned rabbit pulp cells--molecular and immunological identity of pulp cell and fibroblast enzyme.

Cultures of cloned rabbit pulp (RP) cells without stimulation produced collagenase of a concentration as high as reference rabbit skin fibroblast cultures which were stimulated with phorbol myristate acetate (PMA, 100 ng/ml). The RP cell collagenase was compared with reference fibroblast collagenase in Western blot analysis using monoclonal antibodies prepared against RP cell collagenase and a polyclonal antibody prepared against rabbit fibroblast collagenase. Both enzyme preparations revealed, with either antibody, identical bands of approximate molecular masses 57,000, 52,500, and 45,000. These antibody preparations variously inhibited RP cell collagenase activity. Intracellular collagenase in RP cells in culture was demonstrated by the indirect immunofluorescence antibody technique using polyclonal anti-fibroblast collagenase antibody. RNA samples from RP cells hybridized with rabbit fibroblast collagenase cDNA (clone H9) and showed a distinct band at 2.7 kb. Both control and PMA-stimulated RP cells and PMA-stimulated reference skin fibroblasts demonstrated strong cytoplasmic hybridization between H9 and collagenase mRNA. The results indicate that RP cell collagenase is identical to rabbit fibroblast collagenase, and that the RP cell line provides a useful in vitro reference system for the study of collagenolysis in the rabbit model.     

Serine proteinase activation of latent human skin collagenase

Latent and active collagenase were demonstrated following direct extraction from normal skin homogenates with 0.1M calcium chloride at 60 degrees C. 83% of the collagenase activity was in latent form and could be maximally activated with trypsin. Partial activation of the latent enzyme could also be demonstrated by incubation of the skin extract without added trypsin. This endogenous activation was inhibited by the addition of soya bean trypsin inhibitor, trasylol, di-isopropylphosphofluoridate and phenylmethanesulphonylfluoride, none of which inhibited collagenase directly. This suggests that the skin extracts contain a collagenase activating enzyme with the inhibition profile of a serine proteinase. A chymotryptic proteinase with a similar inhibition profile was extracted from normal human skin and partially purified. This enzyme activated fibroblast procollagenase derived from tissue culture of normal skin. The procollagenase was also partially activated by plasmin and chymotrypsin. This is the first demonstration of a collagenase activating enzyme in human skin and raises the possibility that collagenase activation by this mechanism may be responsible for collagen degradation in some disease processes.     

Purification of pig synovial collagenase to high specific activity.

1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.     

Collagenase synthesis in clonal rabbit odontoblast-like cells (RP cells)

Post-confluent cultures of cloned rabbit odontoblast-like cells (RP: Rabbit Pulp cells) produced latent collagenase, which was isolated from serum-containing culture media by heparin-Sepharose affinity chromatography. RP collagenase was purified 4,420-fold from serum-containing medium to a specific activity of 1,313 units/mg with a yield of 14% by heparin-Sepharose affinity chromatography, carboxymethyl-cellulose ion-exchange chromatography, and by immunoadsorption chromatography. The RP cell culture appears to be a suitable model to use for studying collagenase synthesis in mineralized tissue-forming cells and for elucidating the mechanism of collagen degradation in pulp tissue and dentin matrix.     

The stimulation of collagenase production in rabbit articular chondrocytes by interleukin-1 is increased by collagens

Osteoarthritis is characterized by a loss of articular cartilage due at least in part to the action of degradative enzymes secreted by chondrocytes. We have investigated the effect of type II collagen from cartilage and interleukin 1 on collagenase production in cultures of rabbit articular chondrocytes. Interleukin 1 alone stimulated the chondrocytes to secrete collagenase but this response was increased as much as fivefold by the addition of rabbit type II collagen. Bovine type II and chick type I collagens were also stimulatory. The native form of the collagens was not required since denatured collagens and purified chick type II alpha chains were effective. The observed effects of collagens and interleukin 1 may contribute to the progressive nature of osteoarthritis.     

Purification, characterization and inhibition of human skin collagenase

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32mug of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5-8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25 degrees C, producing the two characteristic products TC(A)((3/4)) and TC(B)((1/4)). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25 degrees C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37 degrees C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the alpha-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37 degrees C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins alpha(2)-macroglobulin and beta(1)-anti-collagenase both inhibited the enzyme, but alpha(1)-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.     

Collagenase activity in cultures of rat prostate carcinoma.

A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.     

Stimulation of collagenase production by collagen in mammalian cell cultures.

In this study, we investigated the possible regulatory role of collagen in collagenase production by cultured human skin fibroblasts and human and rabbit synovial cells. Addition of types I, II or III collagen in solution to the culture media markedly stimulated trypsin-activable collagenase activity in these cultures. In the human cell cultures the stimulatory effect of collagen was further enhanced by a soluble factor isolated from human monocyte culture media (Dayer, Russell and Krane, 1977). Both native and denatured forms of collagen stimulated enzyme production; their relative efficacy varied among the different types. The native form of both types I and II collagen showed a greater effect on collagenase production than the corresponding denatured form, whereas with type III collagen the denatured form was more effective.     

Fibroblast stimulation in schistosomiasis. IV. Isolated egg granulomas elaborate a fibroblast chemoattractant in vitro

Hepatic fibrosis complicates the chronic granulomatous inflammatory reaction to Schistosoma mansoni eggs, and is the major cause of morbidity and mortality in human schistosomiasis. We previously presented evidence that schistosomal egg granulomas secreted factors that can stimulate fibroblast proliferation and collagen synthesis in vitro. We now report that serum-free supernatants from cultures of hepatic egg granulomas isolated from S. mansoni-infected mice contained activity that stimulated the directional migration of human and guinea pig dermal fibroblasts in modified Boyden chambers. This fibroblast chemotactic activity was also detected in culture supernatants of granuloma adherent cells highly enriched for macrophages (95% latex-ingesting) but not in culture supernatants from resident peritoneal macrophages of uninfected or infected mice. This suggests that granuloma macrophages are a source of the chemotactic activity. The chemoattractant had the properties of large molecular weight (greater than 200,000 daltons; Sephadex G-200 gel filtration), pl approximately 4.5 (preparative flatbed isoelectrofocusing in granular matrix), heat stability (56 degrees C; 45 min), and trypsin sensitivity. Since preincubation of the partially purified granuloma and adherent-cell derived chemoattractants with rabbit anti-human fibronectin antibody abolished their chemotactic activity, it appears that the factor is antigenically similar to fibronectin. We propose that egg granuloma macrophages are activated in vivo to secrete a fibronectin-like molecule with activity that stimulates the directional migration of fibroblasts. This factor may therefore play a role in the local recruitment of fibroblasts and, in concert with other granuloma-derived factors, may play an important role in the pathogenesis of hepatic fibrosis in schistosomiasis.     

Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes.

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.     

‘Gelatinase-like’ activity from articular chondrocytes in monolayer culture

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TC_A and TC_B fragments of human Type II collagen into smaller peptides at 37°C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1,10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.     

Effects of retinol and dexamethasone on cytokine-mediated control of metalloproteinases and their inhibitors by human articular chondrocytes and synovial cells in culture

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60–90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.