Influence of macrophage products on the release of plasminogen activator, collagenase, beta-glucuronidase and prostaglandin E2 by articular chondrocytes.

We describe the effects of products of mononuclear phagocytes on the secretory activity of chondrocytes. The primary confluent cultures of rabbit articular chondrocytes were exposed to standard medium alone or enriched with conditioned medium obtained from cultures of rabbit peritoneal macrophages, the mouse macrophage cell line P388D1 or human blood mononuclear cells. Four markers of release were assessed, the neutral proteinases plasminogen activator and collagenase, the acid hydrolase beta-glucuronidase and prostaglandin E2, and the kinetics of their changes were monitored. Chondrocytes that were cultured in standard medium secreted large amounts of plasminogen activator, some beta-glucuronidase, but no collagenase, and released only minor amounts of prostaglandin E2. The addition of conditioned medium from rabbit macrophages induced a rapid release of large quantities of prostaglandin E2 and an abundant secretion of collagenase, while abolishing or strongly decreasing plasminogen activator secretion. In addition, beta-glucuronidase secretion was markedly enhanced. The decrease in secretion of plasminogen activator appeared to reflect a diminished production, since no evidence was found for the generation of inhibitors or for an accelerated extracellular breakdown of the enzyme. Conditioned media of the mouse and human mononuclear cells influenced the secretory activities of rabbit articular chondrocytes in a similar way, suggesting that the factor (or factors) acting on chondrocytes is produced by a variety of macrophages, and that its action is not species-restricted. The time course and concentration-dependence of the effects observed indicate that the secretion of plasminogen activator and collagenase are influenced in a strictly reciprocal fashion by the macrophage products. The release of prostaglandin E2 paralleled that of collagenase.     

Stimulation of procollagenase synthesis in human rheumatoid synovial fibroblasts by mononuclear cell factor/interleukin 1

In order to define mechanisms regulating the synthesis of procollagenase in human rheumatoid synovial fibroblasts, the proteins synthesized by cultured cells were labeled with [35S]methionine. Labeled medium proteins were analyzed by SDS-PAGE directly and after immunocomplexing with a specific antibody to human fibroblast collagenase. Labeling of both the predominant form of the enzyme (Mr approximately 55 000) as well as a minor species (Mr approximately 61 000) was increased following incubation with the monokine, mononuclear cell factor/interleukin 1. The approximately 61 kDa form of the procollagenase appears to be a glycosylated form of the approximately 55 kDa precursor based on binding to Con A-Sepharose and decrease in the approximately 61 kDa form after culture in the presence of tunicamycin. Thus, mononuclear cell factor, homologous with interleukin 1, partially purified from monocyte conditioned medium increased incorporation of [35S]methionine into several medium proteins, including those complexed by the anticollagenase antibody. In the presence of mononuclear cell factor/interleukin 1, labeling of the procollagenase was increased 12-14-fold over control cultures incubated with medium alone. Therefore, one of the mechanisms involved in increase of collagenase activity in the medium of cultured synovial fibroblasts in the presence of mononuclear cell factor/interleukin 1 is a stimulation of enzyme protein synthesis.     

Stimulation of prostaglandin synthesis in cultured rat synovial cells by a factor derived from polymorphonuclear leukocytes

Stimulation of synovial cell prostaglandin production by a factor obtained from casein-induced peritoneal polymorphonuclear (PMN) cells has been investigated. Both the extract and short time cultured medium of rat peritoneal PMN cells stimulate prostaglandin (PG)E2 production as well as collagenase production in the culture of rat synovial cells. PGE2 production by the cells in the presence of the PMN factor is much faster (5 to 24 hr) than collagenase production (24 hr or later, Biomedical Res. 3, 506-516, 1982). This stimulating factor is confirmed to be derived from PMN cells, based on the purification of the cells from peritoneal exudate cells by the Ficoll-Urographin method. Elution profile of the factor on gel filtration has indicated that both PGE2 and collagenase productions by synovial cells are stimulated by the same effluent fractions corresponding to molecular weights of 15,000 - 20,000 daltons and 30,000 - 40,000 daltons. These results suggest that PMN cells are involved in PG production as well as collagenase production in the inflamed tissue by stimulating connective tissue cells such as synovial cells.     

The secretion of the tissue inhibitor of metalloproteinases (TIMP) by human synovial fibroblasts is modulated by all-trans-retinoic acid.

The matrix metalloproteinases are a family of enzymes involved in the turnover of the connective tissues. The regulation of these enzymes is complex, involving the control of synthesis, the activation of proenzyme forms and the presence of specific inhibitors. Retinoids have been reported to inhibit the production of metalloproteinases by human and rabbit synovial fibroblasts and by human skin fibroblasts. The production of the highly specific tissue inhibitor of metalloproteinases (TIMP) by connective tissue cells may be crucial in the regulation of connective tissue breakdown and this present study was undertaken to determine if retinoic acid (RA) could modulate TIMP and collagenase production by synovial fibroblasts. The results show that RA at concentrations from 10(-7) to 10(-5) M significantly stimulated the secretion of TIMP by two of three human synovial cell lines. The effect of mononuclear cell factor (MCF) on TIMP and collagenase levels was also investigated. The apparent reduction of collagenase levels in the presence of RA, could result from a failure to accurately measure this enzyme in the presence of increased levels of TIMP.     

Induction of collagenase mRNA in lapine articular chondrocytes by synovial factors and interleukin-1

The cDNA probe H-9, originally constructed to recognize a portion of the mRNA for lapine synovial collagenase, also hybridized with a RNA of the same size (approximately 2.0 kb) isolated from activated lapine articular chondrocytes. Primary, monolayer cultures of lapine articular chondrocytes did not contain detectable amounts of this RNA, nor did they secrete measurable amounts of collagenase into their culture media. Following exposure to synovial factors, the chondrocytes contained high levels of collagenase mRNA, while their conditioned media had considerable collagenolytic activity. Collagenase mRNA started to appear in chondrocytes 3-5 h after treatment with the synovial material. Maximum levels occurred after 12-24 h. Recombinant human interleukin-1 also induced the appearance of this mRNA. We conclude that chondrocyte collagenase is likely to be the same gene product as synovial collagenase, and that its regulation by lapine articular chondrocytes probably occurs at a pretranslational level.     

Retinoids and synovial factor(s) stimulate the production of plasminogen activator by cultured human chondrocytes. A possible role for plasminogen activator in the resorption of cartilage in vitro

Agents such as retinol, interleukin 1 and catabolin stimulate resorption of cultured cartilage. This process seems to be mediated by chondrocytes, but the mechanism by which breakdown occurs remains unknown. We have found that (10(-6)-10(-8) M) retinoic acid and (1 X 10(-6) M) retinol, in the presence or absence of a factor derived from cultured synovium (synovial factor), stimulate the degradation of fibrin by human chondrocytes in culture. Plasminogen was required for the enhancement of fibrinolysis, suggesting that the breakdown depended upon the production of plasminogen activators and subsequent liberation of plasmin. However, the chondrocytes did not release significant amounts of plasminogen activator, and the effects of the synovial factor and retinoids resulted from augmentation of the production or activity of enzymes which remained bound to the cell layer. The role of plasminogen in the resorption of cultured cartilage was also investigated. In the presence of plasminogen, (1 X 10(-8) M) retinoic acid or synovial factor stimulated the breakdown of cultured bovine nasal cartilage, but in the absence of plasminogen, the effect of synovial factor was abolished and that of retinoic acid reduced. However, in cultures containing both retinoic acid and synovial factor the resorption process was not affected by removal of plasminogen. Thus, the resorption of cartilage matrix in vitro may be partially mediated by plasminogen activators and plasmin.     

Expression of IL-1 genes in human and bovine chondrocytes: a mechanism for autocrine control of cartilage matrix degradation

In this report we describe the presence of interleukin-1 activity in medium conditioned by bovine articular cartilage. Preparations partially purified by Sephacryl S200 chromatography (Mr 18000-25000) stimulate murine thymocyte proliferation in the lymphocyte activation factor assay. Furthermore, the factor(s) activate cartilage tissue to secrete a protease which is essential for the activity of purified synovial collagenase. We also demonstrate the presence of mRNA coding for IL-1 alpha and beta in human articular chondrocytes and conclude that the human monocytic and chondrocytic mRNAs are identical. Our results demonstrating cartilage expression of IL-1 genes suggest the possibility of an autocrine mechanism whereby chondrocyte production of matrix degrading proteases is initiated by chondrocyte derived IL-1.     

Interleukin 1 production by the human monocyte cell line U937 requires a lymphokine induction signal distinct from interleukin 2 or interferons

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.     

Macrophage factor that induces neutral protease secretion by normal rabbit chondrocytes. Studies of some properties and effects on metabolism of chondrocytes

Normal rabbit-articular chondrocytes secrete very small amounts of degradative enzymes in culture. Rabbit peritoneal macrophages, when activated with lipopolysaccharides, release a factor in the medium which stimulates the chondrocytes to produce significantly high levels of collagenase and other neutral protease for 2-3 days. The soluble mediator from macrophages appears to be a polypeptide with a molecular weight of 13000-15000 and can be inactivated by short-term treatment with trypsin or pronase. The enzyme-synthesis by chondrocytes can be stimulated to the same extent by repeated addition of the macrophage-medium. The metabolism of chondrocytes is altered due to the presence of this mediator. The cellular proliferation is diminished, while the rates of degradation as well as biosynthesis of the matrix are increased. These studies suggest the possibility that in the conditions such as osteoarthritis, where the synovial cells may not play an active role in cartilage degradation, the proteases can be produced by the cartilage cells themselves after the stimulation by macrophage-derived mediators. These intrinsic enzymes may be responsible for the slow, but progressive degeneration of cartilage tissue.     

Inhibitor of human collagenase from cultures of human tendon.

A potent inhibitor of human collagenases, released from human tendon explants in culture, has been purified and partially characterized. The tendon inhibitor has an estimated molecular weight of 25,000. It is relatively heat-stable but undergoes loss of activity following exposure to trypsin. It inhibits trypsin-activated rheumatoid synovial collagenase as well as the enzyme obtained from polymorphonuclear leukocytes. No inhibition of collagenase from Clostridium histolyticum (clostridiopeptidase A, EC 3.4.24.3) was noted. This collagenase inhibitor may be a factor in the regulation of extracellular connective tissue catabolism.     

Single-turnover and steady-state kinetics of hydrolysis of cephalosporins by beta-lactamase I from Bacillus cereus.

Purified mast cells derived from rat peritoneal fluids and dog mastocytomas were extracted with 1 M-NaCl and sonication techniques. The mast-cell products increased the production of mononuclear cell factor from human peripheral blood mononuclear cells in culture, as judged by the enhanced stimulation of prostaglandin E (2-5 fold) and collagenase (3-11-fold) production by cultured adherent synovial cells. Heparin alone (1-10 micrograms/ml) induced a similar stimulation of mononuclear-cell-factor production by monocyte cultures, whereas histamine (1-10 micrograms/ml) had no effect. The stimulatory effect of mast-cell products and heparin represented a direct effect on mononuclear cells; they did not potentiate the effect of monokine on the synovial cells. These results suggest that mast-cell-macrophage interactions may play a significant role in the pathogenesis of inflammation and connective-tissue degradation.     

Identification of TIMP-2 in human alveolar macrophages. Regulation of biosynthesis is opposite to that of metalloproteinases and TIMP-1.

We have identified the metalloproteinase inhibitor TIMP-2 as a secreted product of human alveolar macrophages. In contrast to human fibroblasts, TIMP-2 was released from macrophages free of any apparent complexed metalloproteinases. Also in marked distinction to fibroblasts, TIMP-2 secretion from mononuclear phagocytes was subject to modulation by a variety of agents. TIMP-2 was synthesized by macrophages placed in culture under basal conditions in amounts approximately 30% of those secreted by fibroblasts on a per cell basis. The additions of lipopolysaccharide, denatured type I collagen, and zymosan to culture medium each resulted in a dose-dependent and profound decrease in macrophage TIMP-2 protein production and steady-state mRNA levels. In contrast, all of these agents markedly enhanced the biosynthesis of macrophage interstitial collagenase and TIMP-1 as assessed by analysis of identical cell and conditioned media samples. In human fibroblasts, TIMP-2 biosynthesis was unaffected by interleukin-1, tumor necrosis factor-alpha, platelet-derived growth factor, and phorbol ester despite the massive collagenase stimulation induced by each of these agents. We conclude that TIMP-2 is a potentially important mononuclear phagocyte product whose biosynthesis is regulated in a distinct and completely opposite manner to that of collagenase and TIMP-1.     

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.     

Release of neutral proteinases from mononuclear phagocytes and synovial cells in response to cartilaginous wear particles in vitro

Cartilaginous wear particles were retrieved from synovial fluid aspirates of human diarthrodial joints and added to cultures of human or murine mononuclear phagocytes or human synovial cells. In each case, addition of the wear particles elevated the production of proteinases active at neutral pH against collagen, gelatin, azocasein and the synthetic pentapeptide phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg. Synovial cells secreted more than five times as much collagenase as the same number of the other cells. All types of cell secreted significant quantities of enzymes active against the noncollagenous substrates. Mild treatment of the spent media with trypsin stimulated all of these enzymic activities. The spent culture media of synovial cells which had been exposed to cartilaginous wear particles released hydroxyproline and glycosaminoglycan from powdered cartilage, indicating the production of enzymes which degrade both the collagen and proteoglycan of the cartilaginous matrix. Cultures of mononuclear phagocytes, in contrast, while solubilizing chondroitin sulphate from cartilage, released very little hydroxyproline. The ability of wear particles to elicit these effects suggests a role for them in the pathogenesis of oesteoarthritis and other types of joint deterioration.     

Autocrine control of collagenase synthesis by synovial fibroblasts

Fibroblasts respond to exogenous stimuli, such as Interleukin 1, phorbol esters, or crystals of monosodium urate monohydrate, by synthesizing and secreting large quantities of collagenase. Here we show that addition of exogenous stimuli results in the production of an autologous protein that is, itself, capable of inducing collagenase. This autocrine has been partially purified. Activity resides in a protein(s) with a pl of 5 or 8 and with Mr of approximately 15K. Conversely, conditioned medium taken from unstimulated cultures contains an inhibitor of collagenase synthesis. This protein, which has a Mr approximately 20-25k by HPLC gel filtration antagonizes collagenase synthesis induced by phorbol esters, exogenous parallel 1, and the autologous inducer. We conclude that synovial fibroblasts regulate collagenase synthesis via an autocrine mechanism that includes the synthesis of both an inducer and inhibitor. Both proteins are active at nanomolar amounts and may function as polypeptide hormones in regulating collagenase synthesis and, hence, connective tissue remodeling.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

Mononuclear cell-mediated modulation of synovial cell metabolism. I. Collagen synthesis

Human PHA-stimulated mononuclear cells produce a factor which inhibits synovial cell collagen and non-collagen protein synthesis, whereas it enhances hyaluronic acid (HA) production. Indomethacin (10(-4)-10(-6) M), a cyclo-oxygenase inhibitor, suppresses this effect, suggesting that the mechanism is prostaglandin-mediated. The active material, of apparent molecular weight 12 000-20 000, also displays the properties of the mononuclear cell factor (MCF) previously described by others, since its stimulates collagenase and PGE2 release by the cultured synovial cells. Furthermore, it co-purifies with interleukin 1 (IL 1) as shown by lymphocyte-activating factor activity. This strongly suggests that IL 1 could be responsible for some (or all) the effects observed on MCF-exposed synovial cells. From these data, we deduce the possibility that mononuclear cells may participate in limiting synovial collagen deposition in rheumatoid arthritis.     

Partial purification of a factor from human synovium that possesses interleukin 1, chondrocyte stimulating and catabolin-like activities

Human synovial explants in culture release material that stimulates the production of prostaglandin E2 (PGE2) and several extracellular enzymes by human chondrocytes. Fractionation of conditioned medium by gel filtration revealed a protein of approx. 15 kDa, which in addition to stimulating production of PGE2 and plasminogen activator by human articular chondrocytes, possessed interleukin 1 activity and induced cartilage degradation. Further purification using iso-electric focussing again showed co-elution of these activities with a major pI of 6.9 and a minor pI of 5.1-5.3. This study indicated that human synovium releases a factor that is closely related to or identical with interleukin 1 and suggests that this protein may participate in cellular interactions that occur within the rheumatoid joint.     

A specific collagenase from rabbit fibroblasts in monolayer culture

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.