Modulation of human dermal fibroblast extracellular matrix metabolism by the lymphokine leukoregulin.

The effect of leukoregulin, a 50-kD lymphokine with unique antitumor properties, was studied in vitro on several fibroblast functions. Leukoregulin did not inhibit fibroblast proliferation, as measured by cell enumeration and [3H]thymidine incorporation, and had no cytotoxic effect in terms of increased membrane permeability detected by trypan blue exclusion, two of the major leukoregulin actions on tumor cells. Leukoregulin induced a dose-dependent decrease in collagen synthesis, demonstrated by decreased [3H]proline incorporation into collagenase-digestible protein, as early as 6 h after the addition of the lymphokine to human fibroblasts. Leukoregulin inhibited the synthesis of both type I and type III collagen, as measured by SDS-PAGE and by specific radioimmunoassay. Neutralizing antibodies to interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma failed to alter the effect of leukoregulin on collagen synthesis, attesting that leukoregulin action was not due to contamination by these cytokines. Inhibition of collagen synthesis occurred concomitantly with increased secretion of prostaglandin E2 and a transient rise in intracellular cyclic AMP content, peaking at 6 h. However, blocking prostaglandin synthesis with indomethacin did not counteract inhibition of collagen synthesis by leukoregulin, demonstrating independence of this action of leukoregulin from cyclooxygenase metabolites. Leukoregulin also stimulated glycosaminoglycan production in a dose-dependent manner, affecting the synthesis of hyaluronic acid as the major fibroblast-derived extracellular glycosaminoglycan. In addition, secretion of neutral proteases (collagenase, elastase, caseinase) was increased. These observations indicate that leukoregulin is able to regulate synthesis of molecules critical to the deposition of the extracellular matrix by nontransformed nonmalignant fibroblasts.

Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis.

OBJECTIVE: The aim of this study was to determine the effects of pharmacologically relevant concentrations of rhein (1,8-dihydroxy-3-carboxyanthraquinone) on the cell proliferation rate of human chondrocytes and synoviocytes. METHODS: Cultures of human osteoarthritic synoviocytes and chondrocytes were incubated with 10(-6), 10(-5), and 10(-4) M rhein. [3H]thymidine incorporation was used to determine rhein proliferative effects after incubation periods of 24 h, 48 h, and 1 week. The cytotoxicity of the drug was assayed with a nonradioactive assay kit. Nuclear extracts were used to detect variations in cell-cycle proteins (p21, p27, and cyclin D1) by Western blotting. The effect of rhein on apoptosis was investigated by measurement of caspase-3/7 activity and DNA fragmentation. RESULTS: Rhein was found to downregulate the proliferation rate of both chondrocytes and synoviocytes, two-fold for 10(-5) M rhein and five- to six-fold for 10(-4) M rhein. No cytotoxicity of the drug was observed. Rhein (10(-4) M) decreased caspase-3/7 activity and did not induce DNA fragmentation. Western blots showed that 10(-4) M rhein increased the expression of p21 and/or p27, but not that of cyclin D1. CONCLUSIONS: Rhein has previously been shown to reduce the interleukin (IL)-1beta deleterious effects on osteoarthritis (OA) cartilage through inhibition of the expression of degrading enzymes. Here, rhein was also found to inhibit proliferation of both synoviocytes and chondrocytes, suggesting that the drug may decrease the development of the inflammatory synovial tissue that accompanies joint pathologies. Both its anti-catabolic and anti-proliferative effects may explain its beneficial effect in the treatment of joint diseases.

Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture.

OBJECTIVE: The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. METHODS: Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). RESULTS: Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. CONCLUSIONS: Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

17Beta-oestradiol up-regulates the expression of a functional UDP-glucose dehydrogenase in articular chondrocytes: comparison with effects of cytokines and growth factors.

OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

Rhein, a diacerhein-derived metabolite, modulates the expression of matrix degrading enzymes and the cell proliferation of articular chondrocytes by inhibiting ERK and JNK-AP-1 dependent pathways.

OBJECTIVE: To determine the effects of rhein on the expression of matrix metalloproteinases (MMP-1, -3, 13) and ADAMTs 4, 5 (a disintegrin and metalloproteinase with thrombospondin type-I repeat)/aggrecanases-1, -2 in interleukin-1-stimulated bovine articular chondrocytes, and to investigate the signalling pathways involved in the effects of the drug on gene expression and cell proliferation. METHODS: Bovine chondrocytes were treated with 10(-4) M rhein for 18 h, followed by 10 ng/ml IL-1Beta for 30 min (cytoplasmic extracts) or 24 h (RNA extraction and EMSA). mRNA was assessed by RT-PCR for the expression of MMPs and aggrecanases, and the phosphorylation of MAP kinases was studied by Western blotting. NF-kappaB and AP-1 DNA binding were determined by gel retardation assay. The effects of inhibitors of these signalling pathways were compared to those of rhein. The proliferation of human chondrocytes and synoviocytes treated with the drug was also investigated. RESULTS: IL-1Beta-induced stimulation of the MMPs and aggrecanase-1 was markedly inhibited by rhein. The drug reduced IL-1Beta-induced NF-kappaB and AP-1 DNA binding, as well as the phosphorylation of ERK and JNK. Similar effects were produced by the specific inhibitors of these signalling pathways. In addition, rhein reduced the proliferation of both human chondrocytes and synoviocytes. CONCLUSION: Our data indicate that rhein may reduce the deleterious effects of IL-1Beta on osteoarthritic cartilage through its effects on the ERK- and JNK-dependent pathways. Both its anti-catabolic and anti-proliferative properties may explain its value in the treatment of joint diseases.

[Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation: a review of 28 cases]. / Greffe de chondrocytes autologues dans le traitement des pertes de substance condylienne du genou: bilan de 28 implantations.

PURPOSE OF THE STUDY: The knee has little capacity for spontaneous regeneration of deep cartilage defects. In 1999, the French Society of Arthroscopy initiated a multicentric clinical trial on autologous chondrocyte transplantation using the technique described by Brittberg and Peterson. The protocol of this prospective study was validated by the ethics committee and all patients provided the informed consent for participation. MATERIAL AND METHODS: Patients underwent surgery in seven hospitals: 28 patients (7 female, 21 male, mean age 28 years, age range 18-45 years). The underlying condition was: osteochondritis (n=14), isolated posttraumatic chondropathy (n=8), chondropathy plus ACL tear (n=6). All patients presented deep condylar cartilage defects (ICRS grades 3 and 4). Mean surface area involved after debridement was 490 mm2 (range 150-1050 mm2). Patients were reviewed two years at least after transplantation for functional assessment and an MRI performed 2 to 3 years after transplantation. Control arthroscopy was also performed in 13 patients with biopsy for histology and immunohistochemistry for 10. RESULTS: Twenty-six patients were reviewed with more than two years follow-up (mean 2 years 9 months). There were no general complications; three patients presented a partial avulsion of the autograft treated arthroscopically and one arthrolysis was performed at six months. Function was improved in all patients but four, but pain persisted in one patient. The mean ICRS score improved from 41 points (19-55 points) to 74 points (54-86 points), for an 80% gain. Follow-up MRI was available for 16 knees: the graft was hypertrophied in 11, at level in 3 and insufficient in 2; marginal integration was good in 10 knees and fair in 6. Items of marginal and subchondral integration had a very high positive predictive value for good clinical outcome. The arthroscopic score was nearly normal (range 8-11) in 8 knees and abnormal (range 4-7) in 5. The Knutsen histological groups according to richness of hyaline cartilage were: group 1 (>60%) (n=1); group 2 (>40%) (n=3), group 3 (<40%) n(=4) and group 4 (bone or fibrous tissue) (n=1). The function scores (r=0.80) and the MRI scores (r=0.76) were correlated with the arthroscopy scores. There was no correlation between the histological findings but the sample size was too small for meaningful analysis. DISCUSSION: The clinical results demonstrate an improvement in more than 80% of knees, findings similar to earlier reports. The arthroscopic and histological results were equivalent to those reported by Knutsen, but less satisfactory than those reported by Bentley or Peterson. Cell injections under a periosteal patch constitute the first generation of autologous chondrocyte grafts. Resorbable matrices loaded with chondrocytes before implantation are under development and have provided promising early results.

Effect of interleukin-1beta on transforming growth factor-beta and bone morphogenetic protein-2 expression in human periodontal ligament and alveolar bone cells in culture: modulation by avocado and soybean unsaponifiables.

BACKGROUND: In periodontal disease, interleukin-1beta (IL-1beta) is responsible for the matrix breakdown through excessive production of degrading enzymes by periodontal ligament fibroblasts and osteoblasts. Transforming growth factor-beta (TGF-beta) plays an important role in tissue regeneration as one of the factors capable of counteracting IL-1beta effects. In this study, we investigated the in vitro effect of avocado and soya unsaponifiables (ASU) on the expression of TGF-beta1, TGF-beta2, and bone morphogenetic protein-2 (BMP-2) by human periodontal ligament (HPL) and human alveolar bone (HAB) cells in the presence of IL-1beta. METHODS: HPL and HAB cells were incubated for 48 hours with ASU (10 microg/ml) in the presence or absence of IL-1beta (10 ng/ml). The steady-state levels of TGF-beta1, TGF-beta2, and BMP-2 mRNAs were determined by Northern blot or reverse transcription-polymerase chain reaction (RT-PCR). The amounts of TGF-beta1 and TGF-beta2 proteins were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The data indicated that IL-1beta strongly decreases the expression of TGF-beta1 and TGF-beta2 by HPL cells. ASU were capable of opposing the cytokine effect. In HAB cells, TGF-beta1 and BMP-2 mRNA levels were downregulated by the cytokine. ASU were found to reverse the IL-1beta-inhibiting effect. In contrast, the cytokine stimulated the production of TGF-beta2 in alveolar bone cells, with no significant effect of ASU. CONCLUSIONS: The results indicate that the IL-1beta-driven erosive effect in periodontitis could be enhanced by a decreased expression of members of the TGF-beta family. The ASU stimulation of TGF-beta1, TGF-beta2, and BMP-2 expression may explain their promoting effects in the treatment of periodontal disorders, at least partly. These findings support the hypothesis that ASU could exert a preventive action on the deleterious effects exerted by IL-1beta in periodontal diseases.

Inhibition of interleukin-1beta-induced activation of MEK/ERK pathway and DNA binding of NF-kappaB and AP-1: potential mechanism for Diacerein effects in osteoarthritis.

In the present report we have shown that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-l transcription factors. Incubation of the cells with 10(-5) M Rhein, the active metabolite of Diacerhein, for 24 h was found to reduce this activity particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to a 1 h treatment with IL-1beta. This effect was greater in hypoxia (3% O2) than in normoxia (21% O2). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M Rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extra cellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by Rhein under the same conditions. In conclusion, Rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of Rhein and its disease-modifying effects on OA cartilage, in spite of the absence of inhibition at prostaglandin level.

Differential expression of membrane-anchored proteoglycans in rabbit articular chondrocytes cultured in monolayers and in alginate beads. Effect of transforming growth factor-beta 1.

Cell-surface proteoglycans (PGs) were extracted with Triton X-100 from rabbit articular chondrocytes cultured in monolayers and in alginate beads. They were first purified on DEAE-Trisacryl columns and the proportion of hydrophobic PGs was determined by both Octyl-Sepharose chromatography and partitioning in Triton X-114. These two methods revealed that the proportion of hydrophobic PGs was higher in monolayer culture system as compared to alginate beads (24 and 15%, respectively). Characterization of the PGs by Sepharose CL 6B gel filtration followed by electrophoresis indicated that the PGs isolated from monolayers were composed of three chondroitin sulfate (CS) PGs (core proteins of 180, 100 and 50 kDa) and a heparan sulfate (HS) PG (core protein of 60 kDa). In the alginate system. CSPGs with core proteins of 180, 45 and 32 kDa were observed, but no HSPG was present. In parallel, the effect of TGF-beta on the distribution of membrane-associated PGs was studied. The results showed that the synthesis of cell-surface PGs was stimulated by TGF-beta in monolayers whereas it was inhibited in alginate beads, but the amount of hydrophobic PGs was not altered by the growth factor. These data clearly indicate that TGF-beta induces a differential expression of the PG families present at the cell surface. Taken together, the results reveal the complex regulation of cell-surface PG distribution, which obviously depends on the culture method used and suggest that rabbit articular chondrocytes may differentially respond to extracellular ligands according to their morphological state and environment.

Expression of decorin and biglycan by rabbit articular chondrocytes. Effects of cytokines and phenotypic modulation.

In this study, the levels of mRNAs coding for aggrecan, decorin and biglycan in rabbit articular chondrocytes were investigated, using both monolayer and 3D-alginate cultures treated with TGF-beta 1 and IL-1 beta. The cells were shown to express higher amounts of proteoglycan messages, specially the aggrecan, in gels than in monolayers. TGF-beta 1 increased aggrecan mRNA in both systems, whereas biglycan message was elevated only in alginate. It markedly decreased decorin expression in monolayer, either in primary or passaged cultures. In contrast, IL-1 beta had a weak inhibitory effect on both decorin and biglycan expression. Subculturing induced a dramatic decrease of aggrecan mRNA, while that of decorin augmented. Biglycan expression transiently increased after two passages, whereas it declined in further subcultures. Passaged chondrocytes transferred to alginate re-expressed high levels of aggrecan, decorin and biglycan. The data point to the influence of morphology, proliferative state and environment of the articular chondrocytes on their biosynthetic responses to cytokines. Although these immature cells do not fully reflect the adult chondrocytes present in the cartilage, this study may help in understanding the behaviour of these cells in osteoarticular diseases, where the surrounding extracellular matrix is profoundly altered.

Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-beta (TGF-beta).

The effect of transforming growth factor-beta (TGF-beta, 1 ng/ml) on proteoglycan synthesis by rabbit articular chondrocytes in culture was studied in the presence of fetal bovine serum. Exposure of confluent cells for 24 h to the factor resulted in a marked increase of 35S-labeled sulfate incorporation in the newly synthesized proteoglycans (PG), as estimated by glycosaminoglycan (GAG) radioactivity (+58%). The onset was observed 6 h after addition of the factor but was significant after 12 h. TGF-beta also enhanced the uptake of [35S]sulfate by chondrocytes, but had no effect on the release of PG by these cells. The effect of TGF-beta on the distribution of PG between the medium and the cell layer was shown to be dependent on the serum concentration in the medium: the relative proportion of cell-layer associated GAG of TGF-beta-treated cells decreased with increasing concentration of fetal bovine serum. The proportion of aggregated PG, the hydrodynamic size of PG monomers and GAG chains were not modified by TGF-beta, but the relative distribution of disaccharides 6- and 4-sulfate in GAG chains was altered by the factor: the proportion of chondroitin 6-sulfate (C6S) was decreased while that of chondroitin 4-sulfate (C4S) was augmented in presence of TGF-beta, leading to a decrease of the ratio C6S/C4S (-11 to -22%, P less than 0.01). The present study indicates that TGF-beta promotes the synthesis of a modified extracellular matrix in cultured articular chondrocytes. This mechanism could be relevant to some aspects of cartilage repair in osteoarticular diseases.

An inositolphosphate glycan released by TGF-beta mimics the proliferative but not the transcriptional effects of the factor and requires functional receptors.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates a number of cellular processes including cell growth and deposition of extracellular matrix protein. Despite the fact that the signal transduction by TGF-beta has been intensively studied, the molecular mechanisms of that pathway are not clear. We have studied the possibility that an inositolphosphate glycan (IPG) is involved in transmission of the TGF-beta 1 signal. We show that TGF-beta 1 induces IPG release in both rabbit articular chondrocytes (RAC), which are growth stimulated by the factor and Mv1Lu cell line, which is growth inhibited. This release requires functional TGF-beta heteromeric receptors in these two cell types. We also demonstrate that IPG mimics TGF-beta 1-induced growth stimulation in mesenchymal cells (+100%) and growth inhibition in epithelial cells (-80%). Moreover TGF-beta receptor I (T beta R-I) is not required for inhibition of proliferation induced by IPG since derivated mutants of the Mv1Lu cell line lacking T beta R-I intracellular domain (R-1B) are significantly inhibited (-65%). Additionally, we show that IPG does not take part in the signalling pathway that leads to activation of matrix gene transcription. These results suggest that TGF-beta effects on growth regulation and extracellular matrix synthesis implicate two different signalling pathways, IPG being only involved in growth regulation.

Different phosphorylated forms of inositolphosphate glycan could be involved in the transforming growth factor-beta 1 (TGF-beta 1) signalling pathway.

Labelling with [3H]glucosamine was used to prepare a transforming growth factor-beta 1 (TGF-beta 1)-sensitive glycosylphosphatidylinositol (GPI) from monolayer cultures of rabbit articular chondrocytes (RAC), which may be involved in control of the cell cycle. The polar headgroup of this glycosylphosphatidylinositol was generated by both phosphatidylinositol-specific phospholipase C (PI-PLC) and pronase E digestion. The molecule emerged in only one peak on a Dowex AG1-X8 chromatogram, eluted at 0.1 N ammonium formate. In contrast, similar experiments performed on cellular extract from cultures previously labelled with [3H]glucosamine displayed four radioactive peaks eluting at 0.1, 0.2, 0.5 and 1 N ammonium formate, respectively. Evidence that the eluting position of these peaks was dependent on the number of phosphate residues present in each fraction was demonstrated by both [32P]phosphorus labelling and change in the position of alkaline phosphatase-induced shift in elution volume. We also demonstrated that the GPI-derived inositolphosphate glycan (IPG) could be hyperphosphorylated into the cell under the action of a kinase whose activity was enhanced by TGF-beta 1 itself. We have also shown that all of these IPG forms could mimic the TGF-beta-induced increase of DNA replication rate of RAC, with a higher activity for peaks III and IV than peaks I and II.

Differential response of cultured rabbit articular chondrocytes (RAC) to transforming growth factor beta (TGF-beta)-evidence for a role of serum factors.

We show that addition of TGF-beta (0.01-10 ng/ml) to proliferating rabbit articular chondrocytes in presence of low level of fetal calf serum (FCS, 2%) results in a sustained decrease of cell number and DNA synthesis up to 72 h. In contrast, incubation with high serum concentration (10% FCS) induces a transient increase of cell number after 48 h without elevation of DNA synthesis. Moreover, when the factor is added in 10% FCS-containing medium, a differential effect is observed at 48 h (either increase or decrease of cell number) depending on the serum level (2 or 10%) present between 24 and 48 h. Recruitment of cells in late S-phase occurred under TGF-beta-treatment in both 2 and 10% FCS. These arrested cells may then be released by further exposure to 10% FCS-containing medium. The data show that factor(s) from the serum modulate(s) the action of TGF-beta on chondrocyte proliferation. Addition of epidermal growth factor (EGF) to the cultures in presence of 2% FCS mimicks the effects observed with 10% serum, suggesting that the serum component(s) involved in the mechanism could be of EGF type.

Comparative effects of interleukin-1 and tumor necrosis factor-alpha on collagen production and corresponding procollagen mRNA levels in human dermal fibroblasts.

The effects of recombinant human Interleukin-1 alpha (IL-1 alpha), Interleukin-1 beta (IL-1 beta), and Tumor Necrosis Factor-alpha (TNF-alpha) on collagen biosynthesis were studied in vitro using dermal fibroblast cultures. Both forms of IL-1 and TNF-alpha induced a dose-dependent inhibition of both types I and III collagen synthesis, as measured by radioimmunoassay, gel electrophoresis, or collagenase-sensitive material. This effect was accompanied by a significant release of prostaglandin E2 into the culture medium. However, indomethacin, a potent inhibitor of prostaglandin synthesis, could not prevent the inhibitory effect of the three cytokines on collagen synthesis. Measurement of type I and type III procollagen mRNA levels in IL-1 treated cells revealed that both IL-1 alpha and IL-1 beta were potent enhancers of procollagen gene expression at pretranslational level. On the other hand, TNF-alpha was found to reduce the steady-state levels of type I and III procollagen mRNA in a dose-dependent manner. Quantitation of IL-1 beta and TNF-alpha transcripts following TNF-alpha treatment of fibroblasts indicated that this cytokine can induce IL-1 beta gene expression in these cells. By contrast, TNF-alpha mRNA remained at a constant level after TNF-alpha exposure. These data suggest that IL-1 and TNF-alpha, two cytokines that share several biologic activities, modulate collagen deposition in dermal fibroblasts by mechanisms that are clearly different: TNF-alpha appears to act at a transcriptional level to inhibit collagen synthesis, whereas IL-1 inhibitory action involves important translational regulation, still unknown, that counterbalances its stimulatory effect on procollagen mRNA levels. Moreover, our data suggest the existence of local fibroblastic cytokine production that may be involved in the modulation of extracellular matrix deposition.

Modulation of rabbit articular chondrocyte (RAC) proliferation by TGF-beta isoforms.

We have previously shown that TGF-beta 1 exerts a bifunctional effect on RAC proliferation. Added to quiescent cultures, it inhibits the entry of G0/G1 cells into S phase whereas in S phase synchronized populations, it stimulates the DNA replication rate with a delayed G2 + M phase and a subsequent transient increase of cell number. As TGF-beta 2 and beta 3 isoforms are also expressed in bone and cartilage tissues, it was of interest to study their effect on RAC proliferation, in comparison to that of TGF-beta 1. Using cell counting and tritiated thymidine incorporation, we found that all the TGF-beta s used here induced an increase of RAC proliferation rate occurring between 24 and 48 h of exposure. TGF-beta 2 appeared as the most efficient form as judged from the maximum of thymidine labelling. However, TGF-beta 3 induced an increase of cell number slightly higher than both TGF-beta 1 and TGF-beta 2 (+30% versus 20% for TGF-beta 1 and beta 2). TGF-beta 2 and beta 3 were able to stimulate the DNA replication rate as previously demonstrated for TGF-beta 1. However, the effect occurred later for TGF-beta 2 and beta 3 (12 h) than for TGF-beta 1 (6 h). This was confirmed by flow cytometric analysis of DNA content. In addition, immunodetection by flow cytometry demonstrated that all TGF-beta isoforms enhanced endogenous expression of TGF-beta-related peptides. The effect was shown to be associated with the cell cycle S phase and was greater for TGF-beta 3 than for TGF-beta 1 and beta 2. These findings suggest that TGF-beta s could act on RAC functions via autocrine and paracrine ways. Taken together, these data indicate that TGF-beta s may modulate proliferation of articular chondrocytes and therefore could play a role in the activation of these cells in the early stages of osteoarthritis.

Effect of ascorbic acid on secreted proteoglycans from rabbit articular chondrocytes.

Addition of ascorbic acid (25, 50 100 micrograms/ml) to cultures of rabbit articular chondrocytes did not change the total amount of proteoglycans produced. However, it induced an increased retention of these macromolecules in the pericellular fraction. The size of the proteoglycan subunits and the length of glycosaminoglycan chains, released in the medium, were not modified on exposure to ascorbic acid (25 micrograms/ml). On the other hand, the rate of non-sulfated chondroitin was increased 2.5-fold, whereas chondroitin-4-sulfate was depressed 1.5-fold.

Transforming growth factor beta stimulates collagen and glycosaminoglycan biosynthesis in cultured rabbit articular chondrocytes.

The effect of transforming growth factor beta (TGF-beta) on the production of matrix macromolecules was studied in cultures of rabbit articular chondrocytes. A 24 h exposure to TGF-beta at concentrations of 0.1, 1 and 10 ng/ml markedly stimulated the synthesis of collagen and non-collagen protein. Similar increases of glycosaminoglycan production was observed in the same experimental conditions. The distribution of these newly synthesized macromolecules between cell layer and medium was not altered by treatment with TGF-beta. The factor slightly enhanced the proliferation of chondrocytes in these experiments but its potent effect on matrix synthesis was independent of this growth stimulation. These results indicate that articular chondrocytes are target cells for TGF-beta and suggest that this growth factor could play a role in the repair process of cartilage during osteoarticular diseases.

Tumor necrosis factor inhibits collagen and fibronectin synthesis in human dermal fibroblasts.

Tumor necrosis factor (TNF) caused inhibition of collagen production by confluent cultures of human dermal fibroblasts in a dose-dependent manner. Concomitant increase of prostaglandin E2 release was observed as a result of TNF-induced cell activation. However, a blockade of the cyclooxygenase pathway of arachidonate metabolism by indomethacin did not abrogate the inhibitory effect of TNF on collagen synthesis, suggesting that this effect could be independent of prostaglandin metabolism. Gel electrophoresis of the newly synthesized macromolecules from the culture media showed that both type I and type III collagens as well as fibronectin were affected by the inhibition. Electrophoresis of cell layer-associated proteins demonstrated that the reduction in amounts of collagen and fibronectin in the medium did not result from an intracellular accumulation of these macromolecules. Production of procollagens was reduced in parallel to that of collagens, suggesting that the effect of TNF is exerted before the processing steps of procollagens. These results clearly show that TNF could play a role in modulation of matrix deposition by fibroblasts during inflammatory processes.