Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) is induced in rabbit articular chondrocytes by cotreatment with interleukin 1β and a protein kinase C activator

The synthesis of an 88-kDa gelatinolytic enzyme, identified as a zymogen of matrix metalloproteinase (proMMP)-9, was induced in the primary culture of rabbit articular chondrocytes by cotreatment with recombinant interleukin 1β (rIL-1β) and the protein kinase C (PKC) agonists, phorbol 12,13-dibutyrate (PDBu) or mezerein. Negligible 88-kDa gelatinolytic activity was produced by unstimulated cells or cells treated with a PKC activator alone at concentrations up to 100 ng/ml, and only a modest induction occurred with rIL-1β alone at concentrations of 1–100 ng/ml. However, when these cells were treated with a PKC activator in the presence of IL-1β (1 ng/ml), induction was striking, with enzymic activity detectable at a concentration as low as 1 ng/ml of mezerein or 10 ng/ml of PDBu. Rabbit chondrocytes in culture constitutively produced the zymogen of MMP-2 (proMMP-2) and its production was not altered by treatment with IL-1β or PKC agonists alone or in combination. Recombinant tumor necrosis factor α (rTNFα) did not substitute for IL-1β in inducing proMMP-9 in the presence of PKC activators, nor was the combination of IL-1β or TNFα alone effective. These data indicate that rabbit articular chondrocytes have a potential to synthesize and secrete proMMP-9 under certain biological and pathological conditions but that the expression of proMMP-9 is differently regulated from that of other MMPs.     

Effects of human recombinant tumor necrosis factor-alpha and interleukin 1 on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes

We examined effects of human rTNF alpha on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes. The effects of human recombinant IL-1 alpha and IL-1 beta were also given attention. rTNF alpha, as well as rIL-1 alpha and rIL-1 beta, decreased the incorporation of [35S]sulfate into glycosaminoglycan to about 10% of the levels in the control. The half-maximal doses of rTNF alpha, rIL-1 alpha or rIL-1 beta required for the suppression of glycosaminoglycan synthesis (by rTNF alpha, rIL-1 alpha, and rIL-1 beta) were 2 ng/ml, 30 ng/ml, or 5 ng/ml, respectively. rTNF alpha stimulated incorporation of [3H]thymidine in the chondrocytes in a dose- and time-dependent manner. DNA synthesis was increased to about threefold over the control cultures in the presence of 1 microgram/ml rTNF alpha for 72 hr. The stimulatory effect of rTNF alpha on DNA synthesis was observed in both subconfluent and confluent cultures, whereas rIL-1 alpha and rIL-1 beta had no stimulatory activity on DNA synthesis. The addition of rTNF alpha to the cultures of chondrocytes stimulated DNA synthesis, even in medium containing no fetal calf serum. The fetal calf serum acted synergistically with rTNF alpha in increasing DNA synthesis. We propose that both TNF and IL-1 may be involved in inflammatory diseases of cartilage, and that TNF alpha, but not IL-1, may have some physiologic growth factor function for chondrocytes.     

Tumor necrosis factor-alpha enhances inhibitory effects of interleukin-1 beta on Leydig cell steroidogenesis

Human recombinant tumor necrosis factor-alpha (rTNF alpha) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNF alpha with human recombinant interleukin-1 beta (rIL-1 beta) enhanced the inhibitory effects of rIL-1 beta. The rIL-1 beta dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNF alpha had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNF alpha in a dose dependent manner. In the presence of both rTNF alpha and rIL-1 beta, hCG-induced cyclic AMP formation and binding of [125I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNF alpha and IL-1 may be produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.     

Matrix metalloproteinase 3 (stromelysin) activates the precursor for the human matrix metalloproteinase 9.

Matrix metalloproteinase 9 (MMP-9), also known as 92-kDa gelatinase/type IV collagenase, is secreted from neutrophils, macrophages, and a number of transformed cells in zymogen form. Here we report that matrix metalloproteinase 3 (MMP-3/stromelysin) is an activator of the precursor of matrix metalloproteinase 9 (proMMP-9). MMP-3 initially cleaves proMMP-9 at the Glu40-Met41 bond located in the middle of the propeptide to generate an 86-kDa intermediate. Cleavage of this bond triggers a change in proMMP-9 that renders the Arg87-Phe88 bond susceptible to the second cleavage by MMP-3, resulting in conversion to an 82-kDa form. alpha 2-Macroglobulin binding studies of partially activated MMP-9 demonstrate that the 82-kDa species is proteolytically active, but not the initial intermediate of 86 kDa. This stepwise activation mechanism of proMMP-9 is analogous to those of other members of the MMP family, but the action of MMP-3 on proMMP-9 is the first example of zymogen activation that can be triggered by another member of the MMP family. The results imply that MMP-3 may be an effective activator of proMMP-9 in vivo.     

Production of gelatin-degrading matrix metalloproteinases ('type IV collagenases') and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha.

The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. Interleukin-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to 35 kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chondrocyte activation by interleukin-1: analysis of the synergistic properties of fibroblast growth factor and phorbol myristate acetate

Following activation, monolayers of lapine articular chondrocytes secreted into their culture media large amounts of prostaglandin E2 (PGE2) and the neutral metalloproteinases collagenase and gelatinase. Partially purified preparations of synovial "chondrocyte activating factors" (CAF), which contain interleukin-1 (IL-1), generally proved stronger activators of chondrocytes than recombinant, human, IL-1 alpha (rHIL-1 alpha) or IL-1 beta (rHIL-1 beta). The presence of synergistic cytokines within the synovial material provides one possible explanation of this discrepancy. As first reported by K. Phadke (1987, Biochem. Biophys. Res. Commun. 142, 448-453) fibroblast growth factor (FGF) synergized with rHIL-1 in promoting the synthesis of neutral metalloproteinases. In our hands FGF alone did not induce neutral metalloproteinases and increased PGE2 synthesis only modestly. However, at doses from 1 ng/ml to 1 microgram/ml, FGF progressively enhanced the synthesis of PGE2, collagenase, and gelatinase by chondrocytes responding to rHIL-1. Acidic and basic FGF synergized equally well with both rHIL-1 alpha and rHIL-1 beta. Phorbol myristate acetate (PMA), but not the Ca2+-ionophore A23187, could substitute for FGF as a synergist. PMA alone was a poor inducer of collagenase or gelatinase but, unlike FGF, it greatly enhanced the synthesis of PGE2 by chondrocytes. Dot-blot analyses with a cDNA probe to collagenase mRNA confirmed that partially purified synovial CAF induced collagenase mRNA more effectively than rHIL-1, with rHIL-1 alpha being superior to rHIL-1 beta in this regard. The synergistic effects of both FGF and PMA upon IL-1-mediated collagenase induction were associated with increased abundance of collagenase mRNA.     

Effects of IL-6 and its soluble receptor on proteoglycan synthesis and NO release by human articular chondrocytes: comparison with IL-1. Modulation by dexamethasone.

Contradictory results have been reported on the effects and role of IL-6 on proteoglycan (PG) synthesis. Having shown recently that in vitro IL-6 depends on the presence of soluble IL-6 receptor alpha (sIL-6Ralpha) to fully exert its effects on chondrocytes, we conducted the present study to analyse the effects of IL-6 on PG synthesis by human articular chondrocytes in the presence of sIL-6Ralpha. PG synthesis was quantified by specific ELISA using a monoclonal antibody (MAB) raised against the keratan sulphate region of PG as a capture antibody, and a MAB to the acid binding region as a detector. It proved specific for PG from primary (differentiated) chondrocytes. In the absence of sIL-6Ralpha, IL-6 had a slight inhibitory effect on PG synthesis by articular chondrocytes. sIL-6Ralpha alone also had slight but consistent inhibitory effects. When adding sIL-6Ralpha at concentrations of 50 ng/ml corresponding to levels found in synovial fluid, the effects of IL-6 increased consistently. However, even at optimal concentrations (30-100 ng/ml of IL-6sR per 100 ng/ml of IL-6), maximal inhibition (48%) did not equal the degree of inhibition achieved by IL-1 at 1 ng/ml (66%). Similar effects, although slightly weaker, were observed on osteoarthritic cells. Dexamethasone, over a wide range of concentrations, markedly enhanced proteoglycan synthesis and completely reversed the downregulatory effects of IL-1 and IL-6 + sIL-6Ralpha. The effects of IL-1 were partially inhibited by an anti-IL-6 antibody. Finally, unlike IL-1, IL-6 + sIL-6Ralpha only weakly stimulated nitric oxide (NO) synthesis. In conclusion, sIL-6Ralpha potentiates the inhibitory effect of IL-6 on PG synthesis by articular chondrocytes, but the overall effect of IL-6 + IL-6sR is moderate compared to the effects of IL-1.     

IL-1beta synthesis by chondrocyte analyzed by 3D microscopy and flow cytometry: effect of Rhein

Several factors are known to be involved in the destruction of the articular cartilage. Interleukin-1 (IL-1) plays an important role in the pathogenesis of osteoarthritis (OA) either directly or through the stimulation of catabolic factors. The action of IL-1 on articular cartilage is multifaceted and it most likely plays an important role in the mechanism of cartilage destruction. IL-1 suppresses the synthesis of the cartilage matrix components and promotes the degradation of cartilage matrix macromolecules. Diacerein is an anthraquinone molecule that has been shown to reduce the severity of OA, both in man and in animal models. The present study was designed to evaluate in vitro effects of diacerein on IL-1beta expression in LPS or IL-1alpha stimulated chondrocytes. Intracellular IL-1beta production was analysed in articular chondrocytes cultured in monolayer or in alginate 3D-biosystems in the presence of lipopolysaccharide (LPS) or IL-1alpha, with or without diacerein. The results show that LPS and IL-1alpha increase intracellular IL-1beta and Diacerein inhibited LPS-induced and IL-1alpha induced IL-1beta production by articular chondrocytes. Moreover, the effect of mechanical stimulation was analysed. An inhibitory effect of DAR at therapeutic concentrations on IL-1beta production in articular chondrocytes is suggested.     

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.     

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties.

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).     

Opiate regulation of IL-1beta and TNF-alpha in cultured human articular chondrocytes

In order to investigate if beta-endorphins anti-inflammatory effect in cartilage-damaging states is mediated via tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we examined its influence on these two cytokines in vitro. Human articular chondrocytes were obtained from patients undergoing total knee arthroplasty and stimulated with beta-endorphin (60-6000 ng/ml). Protein levels of TNF-alpha and IL-1 beta were measured by ELISA in supernatants from articular chondrocyte cultures. beta-Endorphin significantly increased the levels of IL-1 beta for all concentrations used after 15 min incubation, and when stimulated with 600 and 6000 ng/ml after 24 h incubation. The opioid-induced increase in IL-1 beta was blocked by naltrexone in the group tested. TNF-alpha expression was also significantly stimulated by 60 and 600 ng/ml beta-endorphin after 15 min, an effect blocked by naltrexone in the group tested. These findings indicate that the mechanism of beta-endorphins anti-inflammatory influence in cartilage-damaging states is not apparently mediated via these two cytokines modulation.     

Regulation of interleukin-2 and interleukin-6 production from T-cells: involvement of interleukin-1 beta and transforming growth factor-beta

The effect of recombinant (r) interleukin-1 beta (rIL-1 beta) and transforming growth factor-beta (TGF-beta) on the production of interleukin-2 (IL-2) and interleukin-6 (IL-6) from an antigen-specific (LBRM-33-1A5) and an antigen-nonspecific (EL-4-NOB-1) T-cell line was investigated. rIL-1 beta induced the production of IL-2 and IL-6 from EL-4-NOB-1 cells in a dose-related manner. The LBRM-33-1A5 cells required phytohemagglutinin (PHA) in addition to rIL-1 beta in order to produce IL-2 and IL-6. IL-2 production was found to precede IL-6 production in both cell lines. No IL-2 or IL-6 production was observed by adding r murine tumor necrosis factor-alpha or r murine interferon gamma to the cells. The presence of 1 ng/ml TGF-beta reduced IL-2 and IL-6 production from both T-cell lines by more than 80%. The inhibition of IL-2 and IL-6 production was still evident by a concentration as low as 10 pg/ml of TGF-beta. rIL-1 beta and PHA also stimulated murine thymocytes to produce IL-6 which was inhibited up to 85% in the presence of 1 ng/ml TGF-beta. Taken together these results suggest that TGF-beta may suppress immune responses by inhibiting the endogenous production of IL-2 and IL-6.     

Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system

The objective of this study was to determine the presence of autocrine/paracrine regulation of matrix metalloproteinase-9 (MMP-9) expression mediated by proinflammatory cytokines in human fetal membranes. Fetal membranes obtained from women who underwent cesarean delivery before labor were manually separated into amnion and chorion layers and maintained in culture. These explants were stimulated with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and either lipopolysaccharide (LPS) alone or LPS with anti-TNFalpha or anti-IL-1beta-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1beta, TNFalpha, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNFalpha, and IL-1beta. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNFalpha neutralizing antibody but not with anti-IL-1beta. No significant effect of LPS, TNFalpha, or IL-1beta on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNFalpha-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNFalpha appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNFalpha and IL-1beta produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.     

Cytokine regulation of interleukin 6 production by human endothelial cells

The influence of recombinant (r) human tumor necrosis factor alpha (rTNF-alpha), r human interleukin 1 beta (rIL-1 beta), and r human interferon gamma (rIFN-gamma) on the production of interleukin 6 (IL-6) by human endothelial cells (HEC) was investigated. The addition of 1-100 U/ml of either rTNF-alpha or rIL-1 beta to cultures of HEC monolayers caused a dose-related increase in IL-6 production as detected after 24 hr of incubation. In contrast to rIL-1 beta and rTNF-alpha, the use of up to 1000 U/ml of rIFN-gamma caused only a moderate increase in IL-6 production. However, significantly greater quantities of IL-6 were produced by HEC monolayers subjected to 1000 U/ml of rIFN-gamma in combination with 1-100 U/ml of rTNF-alpha. Furthermore, the addition of graded concentrations of human transforming growth factor beta (TGF-beta) to cultures resulted in a dose-related inhibition of rIL-1 beta- and rTNF-alpha-induced IL-6 production by HEC. The results demonstrate that rIL-1 beta and rTNF-alpha share the ability to stimulate HEC for production of IL-6 and indicate that TGF-beta may act as an immunosuppressive agent, at least partially, through its ability to inhibit the action of TNF-alpha and IL-1 on endothelial cells.     

Interleukin 1 induces interleukin 1. II. Recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells

Interleukin 1 (IL-1) alters several potentially pathogenic endothelial cell (EC) functions. The authors report here that recombinant human IL-1 (rIL-1) alpha (0.1 to 10 ng/ml) or IL-1-beta (1 to 100 ng/ml) induce concentration- and time-dependent increases in IL-1-beta mRNA levels in EC derived from adult human saphenous vein. rIL-1 induced IL-1-alpha mRNA only in EC treated concomitantly with cycloheximide (2 micrograms/ml). IL-1-beta mRNA production began within 1 hr of exposure to rIL-1, peaked after 24 hr, and declined thereafter. Actinomycin D prevented the appearance of IL-1 mRNA in rIL-1-treated EC. rIL-1 also induced the release of biologically active IL-1 from EC, which was inhibited by cycloheximide (1 microgram/ml). When compared on the basis of their activity in the thymocyte costimulation assay, rIL-1-alpha and rIL-1-beta were equipotent as inducers of IL-1 production by EC. EC stimulated with rIL-1 produced prostaglandin E2, which inhibits IL-1 production by other cell types and also decreases the responsiveness of thymocytes to IL-1. When EC were exposed to rIL-1 in the presence of indomethacin (1 microgram/ml), which blocked prostaglandin E2 production, greater amounts of rIL-1-induced IL-1 release were detected, although the inhibitor did not affect IL-1-beta mRNA levels. IL-1-induced IL-1 production was unlikely to be caused by endotoxin contamination of tissue culture media or IL-1 preparations, because the lipopolysaccharide (LPS) antagonist polymyxin B (10 micrograms/ml) blocked LPS-induced IL-1 production by EC but did not affect IL-1 release in response to rIL-1-beta (100 ng/ml). The IL-1-inducing property of rIL-1-beta was heat-labile, whereas heated LPS stimulated EC IL-1 production. The source of IL-1 in our cultures was not monocyte/macrophages, as treatment of EC with monoclonal antibody to the monocyte antigen Mo2 under conditions that lysed adherent peripheral blood monocytes did not affect production of IL-1 by EC in response to LPS (1 microgram/ml) or rIL-1-beta (100 ng/ml). IL-1 elicits a coordinated program of altered endothelial function that increases adhesiveness for leukocytes and coagulability. IL-1-induced IL-1 gene expression in human adult EC could thus provide a positive feedback mechanism in the pathogenesis of vascular disease including atherosclerosis, vasculitis, and allograft rejection.     

Articular chondrocytes secrete IL-1, express membrane IL-1, and have IL-1 inhibitory activity.

Previous work from our laboratory has shown that rabbit articular chondrocytes, like macrophages, produce reactive oxygen intermediates, express Ia antigen, and can mediate immunologic functions such as antigen presentation and induction of mixed and autologous lymphocyte reactions. We were interested in seeing if these cells could secrete interleukin-1 (IL-1) or express membrane form of IL-1 (mIL-1). Using the standard C3H/HeJ thymocyte assay, neither secreted IL-1 nor mIL-1 activity was detected in untreated or LPS-treated chondrocytes. However, the D10.G4.1 proliferation assay showed that chondrocytes, stimulated with LPS, secrete IL-1 and express the mIL-1 in a dose- and time-dependent manner. The IL-1 activity in LPS-stimulated chondrocyte supernatant and on fixed cells could be inhibited by anti-IL-1 antibodies. Sephadex G-75 chromatography of pooled, concentrated LPS culture supernatant resolved into two peaks of IL-1 activity at 13-17 and at 45-70 kDa, respectively. The bioactivity of chromatographic fractions were similar using both the thymocyte and D10.G4.1 bioassays. Western blot analysis of chondrocyte supernatant detects 17-kDa IL-1 beta; no processed 17-kDa IL-1 alpha was seen but IL-1 alpha-specific reactivity was observed at 64 kDa. Immunoblot analysis of chondrocyte lysates shows that cell-associated IL-1 is IL-1 alpha and is 37 kDa in size. PCR analysis shows the presence of mRNA for IL-1 beta and IL-1 alpha in LPS-treated cells; IL-1 beta mRNA was detected in untreated chondrocytes. The inability to detect IL-1 by the thymocyte assay is due to the presence of a chondrocyte inhibitor of IL-1 that can be demonstrated in cell sonicates, supernatants, and on paraformaldehyde-fixed chondrocytes. Chromatography of LPS-stimulated supernatant showed a peak of IL-1 inhibitory activity at 21-45 kDa. Chondrocytes which secrete IL-1 and express mIL-1 could play a critical role in maintaining chronic inflammation in rheumatoid arthritis. Therefore, the ability of chondrocytes to produce both IL-1 and an inhibitor to IL-1 is important in interpreting the mechanism of cartilage matrix maintenance and degradation.     

Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor

Regulation of the fibrinolytic system of cultured human umbilical vein endothelial cells (HUVECs) by recombinant interleukin 1 beta (rIL-1 beta) and tumor necrosis factor alpha (rTNF alpha) was investigated. Functional and immunologic assays indicated that both cytokines decreased HUVEC tissue-type plasminogen activator (tPA) and increased type 1 plasminogen activator inhibitor (PAI-1) in a dose- and time-dependent manner. Maximal effects (50% decrease in tPA antigen; 300-400% increase in PAI-1 activity) were achieved with 2.5 units/ml rIL-1 beta and 200 units/ml rTNF alpha. Combinations of rIL-1 beta and rTNF alpha were not additive at these maximal concentrations. After a 24-h pretreatment with rIL-1 beta, HUVECs secreted tPA at one-quarter of the rate of control cells and released PAI-1 at a rate that was 5-fold higher than controls. Neither the basal rate of PAI-1 release nor the increased rate of release of PAI-1 in response to rIL-1 beta was affected by subsequently treating the cells with secretagogues (e.g. phorbol myristate acetate) suggesting that PAI-1 is not contained within a rapidly releasable, intracellular storage pool. Northern blot analysis using a PAI-1 cDNA probe indicated that the cytokines increased the steady-state levels of the 3.2- and 2.3-kb PAI-1 mRNA species, but with a preferential increase in the larger mRNA form. The fact that both rIL-1 beta and rTNF alpha act in a similar manner strengthens the hypothesis that the local development of inflammatory/immune processes could reduce endothelial fibrinolytic activity.     

Recombinant human interleukin-1 stimulates human articular cartilage to undergo resorption and human chondrocytes to produce both tissue- and urokinase-type plasminogen activator

Cytokines capable of stimulating cartilage resorption have frequently been identified as 'interleukin-1 (IL-1)-like' peptides. In this study for the first time we have employed homogeneous recombinant IL-1 alpha and IL-1 beta in an all-human culture system to define the effects of IL-1 on articular cartilage and chondrocytes in culture. Recombinant IL-1 (10-100 U/ml) could stimulate cartilage resorption, although the maximum degree of tissue breakdown rarely reached the levels obtained when cartilage was treated with crude mononuclear-cell conditioned medium or all-trans retinoic acid (1 microM) over a similar time course. Levels of plasminogen activator (PA) activity, a neutral proteinase which may contribute to cartilage destruction in arthritis, increased markedly in the cartilage/chondrocyte culture supernatants and in the chondrocyte cell layers in response to the stimulation of cultures with recombinant IL-1 (1-100 U/ml). Elevated levels of PA activity were detectable after 4-8 h stimulation of the chondrocytes with IL-1 while characterization of the PA activities indicated that both types of PA activity were expressed, viz. urokinase-type PA (u-PA) and tissue-type PA (t-PA). Both IL-1 alpha and IL-1 beta could elicit these responses and their effects were comparable for a given dose. These studies show definitively that pure IL-1, free from contaminating cytokines, is capable of inducing human cartilage resorption and stimulating the expression of two types of PA activity by chondrocytes. In contrast to IL-1, retinoic acid increased the detectable levels of only u-PA in the chondrocyte cell layers. Chondrocyte u-PA may have an important role in cartilage degradative processes since it is one of the few neutral proteinases now known to be increased in activity in retinoid-stimulated cartilage.