Anti-apoptotic effect of transforming growth factor-beta1 on human articular chondrocytes: role of protein phosphatase 2A.

OBJECTIVE: To study whether transforming growth factor-beta1 (TGF-beta1) is able to protect human chondrocytes from apoptosis and to analyze the role of phosphatases in the possible anti-apoptotic effect of TGF-beta1. METHODS: Cartilage was obtained from patients with osteoarthritis (OA) who were undergoing joint replacement; normal cartilage was obtained from cadavers who had no history of joint disease. Chondrocytes stimulated with tumor necrosis factor-alpha (TNF-alpha) plus Ro 31-8220 (a specific inhibitor of mitogen-activated kinase phosphatase-1 - MKP-1) were employed as an in vitro model of apoptosis. Apoptosis was assessed by flow cytometry and a cell death immunoassay. Protein phosphatase 2A (PP2A) activity was estimated by measuring the absorbance of a molybdate:malachite green:phosphate reaction complex. MKP-1, bcl-2 and bax expressions were quantified by western blot. RESULTS: In OA cells, TGF-beta1 significantly reduced the percentage of hypo-diploid chondrocytes, as well as the percentage of internucleosomal DNA breakage. However, in normal chondrocytes, TGF-beta1 did not reduce apoptosis, as assessed by both the percentage of hypo-diploid chondrocytes and internucleosomal DNA breakage. MKP-1 expression did not show significant modulation in OA or normal chondrocytes. However, PP2A activity was differentially modulated in normal and OA chondrocytes. In OA chondrocytes, PP2A activity was not altered by TGF-beta1 stimulation; however in normal chondrocytes PP2A activity was significantly activated by TGF-beta1. The preincubation of normal chondrocytes with TGF-beta1 plus the PP2A inhibitor protein, IPP2A, reduced internucleosomal DNA breakage when compared with TGF-beta1 stimulation alone. The bcl-2/bax protein ratio was significantly higher in TGF-beta1 plus IPP2A preincubated normal chondrocytes than in cells stimulated with TGF-beta1 alone. CONCLUSION: By manipulating the degree of PP2A activity, these results show the major role that PP2A plays in the outcome of TGF-beta1 signal transduction. These data suggest that PP2A could be a pivotal regulator of anti-apoptotic TGF-beta1-induced effects.

Differential effects of tumor necrosis factor-alpha and interleukin-1beta on cell death in human articular chondrocytes.

OBJECTIVE: The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1beta on cell death in normal human chondrocytes. METHODS: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 microM), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. RESULTS: At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha+Ro was higher than the level induced by Ro alone. The combination of IL-1beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1beta+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha+Ro. As quantified by flow cytometry, TNF-alpha+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1beta+Ro. Pre-incubation for 2h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha+Ro at 24 h. Indomethacin increased the cell death induced by IL-1beta+Ro; however, apoptosis induced by TNF-alpha+Ro was not modified by indomethacin. CONCLUSIONS: These results confirm that TNF-alpha and IL-1beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction.

Mitochondrial activity is modulated by TNFalpha and IL-1beta in normal human chondrocyte cells.

OBJECTIVE: Pro-inflammatory cytokines play an important role in osteoarthritis (OA). In osteoarthritic cartilage, chondrocytes exhibit an alteration in mitochondrial activity. This study analyzes the effect of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) on the mitochondrial activity of normal human chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase, as well as by mitochondrial membrane potential (Deltapsim) and adenosine triphosphate (ATP) synthesis. Bcl-2 family mRNA expression and protein synthesis were analyzed by RNase protection assay (RPA) and Western-blot, respectively. Cell viability was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and apoptosis by 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) stain. Glycosaminoglycans were quantified in supernatant by a dimethyl-methylene blue binding assay. RESULTS: Compared to basal cells, stimulation with TNFalpha (10 ng/ml) and IL-1beta (5 ng/ml) for 48 h significantly decreased the activity of complex I (TNFalpha=35% and IL-1beta=35%) and the production of ATP (TNFalpha=18% and IL-1beta=19%). Both TNFalpha and IL-1beta caused a definitive time-dependent decrease in the red/green fluorescence ratio in chondrocytes, indicating depolarization of the mitochondria. Both cytokines induced mRNA expression and protein synthesis of the Bcl-2 family. Rotenone, an inhibitor of complex I, caused a significant reduction of the red/green ratio, but it did not reduce the viability of the chondrocytes. Rotenone also increased Bcl-2 mRNA expression and protein synthesis. Finally, rotenone as well as TNFalpha and IL-1beta, reduced the content of proteoglycans in the extracellular matrix of normal cartilage. CONCLUSION: These results show that both TNFalpha and IL-1beta regulate mitochondrial function in human articular chondrocytes. Furthermore, the inhibition of complex I by both cytokines could play a key role in cartilage degradation induced by TNFalpha and IL-1beta. These data could be important for understanding of the OA pathogenesis.

Mitochondrial proteomic characterization of human normal articular chondrocytes.

OBJECTIVE: Mitochondrial dysfunctions have been associated with apoptosis, aging and osteoarthritis (OA). Chondrocyte mitochondrial proteins are attractive targets for the study of the metabolism of cartilage degradation. The copurification of "contaminating" proteins has been the major problem in all phases of mitochondrial proteome research. Therefore, we set up a procedure for the proteomic analysis of human chondrocyte mitochondrial proteins. METHOD: Four types of protein extracts were obtained from primary cultured chondrocytes isolated from healthy donors: (1) initial total chondrocyte extract (CE), (2) cytosol-enriched supernatant fraction (CY), (3) crude mitochondria fraction (CM), and (4) pure mitochondria fraction (PM). Mitochondria were purified by density gradient ultracentrifugation. Mitochondrial proteins were separated by means of two-dimensional gel electrophoresis (2-DE) and silver stained. Protein spots were then identified by mass spectrometry using MALDI-TOF/TOF technology. RESULTS: The best 2-DE reference map of mitochondrial proteome was constructed employing PM fraction. Thirty-nine percent of the identified proteins were functionally distributed in the mitochondria, 14% in the endoplasmic reticulum and 36% in the cytoplasm. Examining their biological function, 22% are involved in protein targeting, 12% in signaling, 12% in glycolysis, 10% in RNA, DNA or protein synthesis, 10% in oxidative phosphorylation and 4% in redox. The analysis of mitochondrial Mn-superoxide dismutase (SODM) revealed an age-dependent decrease of this protein. CONCLUSION: PM fraction allowed the obtention of a high quality proteomic map for the study of mitochondrial proteins in human articular chondrocytes. This proteomic approach may be also efficient to analyze both quantitative and qualitative modulations of the mitochondrial proteome in human chondrocytes during aging and pathological conditions such as OA.

Cytokines, tumor necrosis factor-alpha and interleukin-1beta, differentially regulate apoptosis in osteoarthritis cultured human chondrocytes.

OBJECTIVE: This study addresses the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on cell death in human chondrocytes. METHODS: Osteoarthritis (OA) human chondrocytes stimulated with Actinomycin-D (ActD) were used as a cellular apoptotic model. Caspase family mRNA expression and protein synthesis were analyzed by the ribonuclease protection assay and Western-blot, respectively. Cell viability and apoptosis were evaluated using the 3-[4,5-dimethylthiazol-2yl] 2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively. RESULTS: TNF-alpha and IL-1beta differentially affected the pattern of caspase mRNA expression by human chondrocytes. TNF-alpha induced a gradual increase in caspase-1 and -8 mRNA levels that was not seen with IL-1beta. The time sequence of caspase-3 and -7 inductions by TNF-alpha differs from that induced by IL-1beta. Cell viability was not modified by TNF-alpha or IL-1beta in cultured chondrocytes. Then, we employed ActD as a model to facilitate cell death. Treatment with TNF-alpha and ActD (TNF-alpha/ActD) increased cell death induced by ActD (23%). Treatment with IL-1beta and ActD (IL-1beta/ActD) did not modulate ActD-induced cell death. Similarly, IL-1beta/ActD did not induce an increase in the activation of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP) cleavage observed by the incubation with TNF-alpha/ActD. These different effects were not due to bcl-2 or mcl-1 levels. Inhibition of PGE2 synthesis by indomethacin increased the cell death induced by IL-1beta/Act-D (59%). An inhibitor of caspase-8 significantly reduced only the TNF-alpha/ActD-induced cell death (58%). CONCLUSION: TNF-alpha and IL-1beta differentially regulate the apoptotic pathway in human chondrocytes. This difference is dependent on PGE2 and caspase-8 levels.

Phosphatase-1 and -2A inhibition modulates apoptosis in human osteoarthritis chondrocytes independently of nitric oxide production.

OBJECTIVE: To characterise the role of phosphatase-1 and -2A (PP1/2A) in the modulation of apoptosis in human osteoarthritis (OA) chondrocytes. METHODS: Human OA chondrocytes were isolated from cartilage obtained from the femoral heads of patients undergoing joint replacement surgery. Cell viability was evaluated by MTT assay. Apoptosis was quantified by ELISA, which measures DNA fragmentation. Nitric oxide (NO) production was evaluated by the Greiss method, and inducible nitric oxide synthase (iNOS) protein synthesis was studied by western blotting. RESULTS: Inhibition of PP1/2A by the specific inhibitor okadaic acid (OKA) dose and time dependently caused a reduction of cell viability (OKA at 50 nmol/l: a reduction to 60% and 43% at 48 and 72 hours, respectively). Genomic DNA from chondrocytes treated with OKA at 50 and 100 nmol/l for 48 hours displayed increased internucleosomal DNA fragmentation by 11 and 13 fields, respectively. Light microscopy and DAPI studies showed that OKA induced DNA condensation and fragmentation, typical of death by apoptosis. The caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK increased cell viability, reduced by OKA at 50 nmol/l to 87% and 73%, respectively. OKA did not increase iNOS protein synthesis or NO production. CONCLUSION: PP1/2A modulate apoptosis in human OA chondrocytes; this is independent of NO production but dependent on caspases.

Effect of nitric oxide on mitochondrial respiratory activity of human articular chondrocytes.

OBJECTIVE: To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Delta psi m). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ(1) reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Delta psi m was measured by 5,5',6,6'-tetracholoro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP) was used as an NO compound donor. RESULTS: SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Delta psi m and induced apoptosis. CONCLUSIONS: This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC.

Papel de la mitocondria en la artrosis / The role of mitochondria in osteoarthritis

No disponible

EP2/EP4 signalling inhibits monocyte chemoattractant protein-1 production induced by interleukin 1beta in synovial fibroblasts.

BACKGROUND: Besides its proinflammatory properties, prostaglandin E(2) (PGE(2)) acts as a regulator of the expression of inducible genes. Inhibition of PGE(2) synthesis might thus result in a paradoxical deleterious effect on inflammation. OBJECTIVE: To examine the effect of PGE(2) on monocyte chemoattractant protein-1 (MCP-1) expression in cultured synovial fibroblasts (SF) stimulated with interleukin (IL)1beta. METHODS: MCP-1 expression was assessed in SF stimulated with IL1beta in the presence of PGE(2) or different NSAIDs by RT-PCR or northern blot and immunocytochemistry. Expression of cyclo-oxygenase (COX) isoforms was studied by western blot techniques. The role of PGE(2) receptors (EP) in PGE(2) action was assessed employing EP receptor subtype-specific agonists. RESULTS: PGE(2) significantly inhibited IL1beta induced MCP-1 expression in SF in a dose dependent manner. IL1beta increased COX-2 and did not alter COX-1 synthesis in SF. 11-Deoxy-PGE(1), an EP(2)/EP(4) agonist, reproduced PGE(2) action on MCP-1 expression. Butaprost, a selective EP(2) agonist, was less potent than PGE(2). Sulprostone, an EP(1)/EP(3) agonist, had no effect on IL1beta induced MCP-1 expression. Inhibition of endogenous PGE(2) synthesis by NSAIDs further enhanced MCP-1 mRNA expression in IL1beta stimulated SF, an effect prevented by addition of exogenous PGE(2). CONCLUSION: Activation of EP(2)/EP(4) receptors down regulates the expression of MCP-1 in IL1beta stimulated SF, while PGE(2) pharmacological inhibition cuts off this signalling pathway and results in a superinduction of MCP-1 expression. The data suggest that NSAIDs may intercept a natural regulatory circuit controlling the magnitude of inflammation, which questions their continuous administration in inflammatory joint diseases.

Fisiopatología de la artrosis: ¿cuál es la actualidad? / Physiopathology of arthrosis: what is the state of the art?

La artrosis (OA) es una patología degenerativa de las articulaciones que se caracteriza por la degradación del cartílago articular hialino. Su progresión es lenta y tiene una etiología múltiple que implica el envejecimiento, la obesidad y la influencia genética como algunos de los factores que favorecen el desarrollo de la OA. En su fase final refleja una insuficiencia de los procesos de reparación del cartílago, resultando en la degradación de la matriz extracellular, muerte del condrocito (por apoptosis) y pérdida total de la integridad del cartílago. El condrocito es el único tipo celular presente en el cartílago maduro y causante de la reparación del tejido dañado. Sin embargo, el desarrollo de esta patología no sólo afecta al cartílago, sino a toda la estructura articular, incluyendo el hueso subcondral y el tejido sinovial. En esta revisión se evaluaran estos cambios desde el punto de vista molecular y celular y se revelará la complejidad de esta patología que incluye múltiples cambios en la estructura articular (AU)

Fibrin generated in the synovial fluid activates intimal cells from their apical surface: a sequential morphological study in antigen-induced arthritis.

OBJECTIVE: Fibrin deposits adhered to the synovial surface are typical of rheumatoid joints. Since fibrin appears to have a role in arthritis perpetuation our aim was to investigate how these deposits are formed and the consequences of their adhesion to the tissue. METHODS: The appearance of fibrin aggregates either free in the synovial fluid or attached to the membrane was studied in rabbits with antigen-induced arthritis by histological techniques at different time points from challenge. In the fixed synovial membranes areas of fibrin-bound synovium were evaluated by qualitative variables to obtain a sequential profile of morphological changes. RESULTS: Fibrin aggregates appeared from the initial stages of the disease in the synovial effusion. Later on, they were localized on the synovial surface and progressive changes were noted at the fibrin-tissue interface, ending with the invasion of the aggregates by synovial cells and their incorporation into the tissue. CONCLUSION: Fibrin aggregates generated inside the joint cavity may constitute a source of activation and acquisition of invasiveness of the synovial fibroblasts, a process to explore within the perpetuating mechanisms of rheumatoid arthritis.

Modulation of cell recruitment by anti-inflammatory agents in antigen-induced arthritis.

OBJECTIVE: To study the effect of meloxicam (MXC) and diclofenac (DCF) on the recruitment of leucocytes during acute experimental arthritis. METHODS: Rabbits with antigen-induced arthritis were treated with MXC, DCF, or not treated. After 48 hours, synovial fluid (SF) leucocyte influx and prostaglandin E(2) (PGE(2)) levels were evaluated. Interleukin 8 (IL8) and monocyte chemotactic peptide-1 (MCP-1) expression and synthesis were studied in the inflamed tissues. RESULTS: Arthritic knees showed synovial effusion with a high leucocyte count and PGE(2) concentration, and an increased expression of IL8 and MCP-1. Both non-steroidal anti-inflammatory drugs (NSAIDs) reduced PGE(2) levels and the polymorphonuclear cell (PMN) concentration in SF, while the mononuclear cell (MN) concentration was unchanged in the treated groups in comparison with controls. A definite reduction of IL8 levels was obtained with the treatments, but the drugs did not prevent the up regulation of MCP-1. CONCLUSION: The effect of these NSAIDs in acute arthritis may be related to the down regulation of IL8 production. The results suggest a differential effect of anti-inflammatory drugs on PMN and MN recruitment to the joint.

Aceclofenac increases the synthesis of interleukin 1 receptor antagonist and decreases the production of nitric oxide in human articular chondrocytes.

OBJECTIVE: Interleukin 1 receptor antagonist (IL-1Ra) may play an important role in cartilage degradation by inhibiting IL-1 activity and therefore blocking IL-1 stimulation of prostaglandin E2 (PGE2) synthesis. Nitric oxide (NO) formation is increased during inflammation. High concentrations of NO exert negative effects on chondrocyte functions. We investigated the possible effects of 3 different nonsteroidal antiinflammatory drugs (NSAID; aceclofenac, piroxicam, aspirin) on IL-1Ra and NO production in human articular chondrocytes. METHODS: Normal and osteoarthritic (OA) cartilage samples were obtained from autopsy and prosthetic joint surgery, respectively. Chondrocytes were isolated and stimulated with 4 different stimuli: IL-1, tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and insulin-like growth factor (IGF). The 3 NSAID were added simultaneously to each different concentration of stimulus. IL-1Ra was measured in supernatant by ELISA; nitrites were quantified by the Griess reaction; PGE2 level was measured by enzyme immunoassay. RESULTS: OA samples spontaneously produced higher levels of IL-1Ra than normal samples (130+/-2.3 vs 30+/-3.1 pg/mI). IL-1, TNF-alpha, and LPS produced dose dependent increases in synthesis of IL-1Ra. In their presence, IL-1Ra was detected in supernatant at 48 h, but its highest level was measured at 144 h. The most potent stimulus was IL-1, followed by TNF-alpha. Fetal bovine serum and IGF in turn did not modify the basal levels of IL-1Ra. In contrast to piroxicam and aspirin, aceclofenac 10 microg/ml and TNF-alpha 10 ng/ml increased almost 46 times the basal amount of IL-1Ra produced by OA chondrocytes. Additionally, aceclofenac and aspirin inhibited NO synthesis. Finally, the 3 NSAID reduced the levels of PGE2 detected after stimulation with IL-1. CONCLUSION: Proinflammatory stimuli induce IL-IRa synthesis in human articular chondrocytes. Aceclofenac may modulate PGE2 production by increasing IL-IRa production and decreasing NO synthesis. Some NSAID exert diverse prostaglandin independent effects.

Cyclosporin A prevents the histologic damage of antigen arthritis without inducing fibrosis.

OBJECTIVE: To study the effects of cyclosporin A (CSA) in a model of rheumatoid arthritis (RA) and the activation of growth factors related to pannus development at the site of injury. METHODS: Antigen arthritis was induced in the knees of 14 New Zealand rabbits, and the animals were randomized into 2 therapeutic groups: CSA at 10 mg/kg/day and CSA solvent. After 3 weeks of treatment, the rabbits were killed, and synovial tissues were obtained and compared with healthy specimens with regard to histopathologic lesions, deposition of transforming growth factor beta (TGFbeta) and collagens, and messenger RNA expression of platelet-derived growth factor B (PDGF-B) and TGFbeta. The effect of CSA on the expression of TGFbeta and PDGF-B was also examined in cultured synovial cells. RESULTS: CSA administration alleviated the histologic damage and avoided the overdeposition of matrix elements in the injured tissue. It was also able to normalize the enhanced expression of TGFbeta and PDGF-B observed in the untreated rabbits. Despite this modulation found in vivo, CSA up-regulated in a dose-dependent manner the gene expression of both trophic factors by healthy cultured synovial cells. CONCLUSION: The present study shows that continuous administration of CSA prevents the development of chronic synovitis in an experimental model of RA. As reported in other cell types, CSA promoted TGFbeta transcription by synovial cells in vitro, but failed to display a profibrogenic effect in the inflamed environment.

Tenidap decreases IL-8 and monocyte chemotactic peptide-1 (MCP-1) mRNA expression in the synovial tissue of rabbits with antigen arthritis and in cultured synovial cells.

Since IL-8 and MCP-1 are chemoattractant proteins that participate in the recruitment of inflammatory cells into the arthritic joint, we examined the effects of tenidap, a new anti-inflammatory drug of the oxindole family, on IL-8 and MCP-1 expression in the joints of rabbits with acute antigen arthritis. The model was induced by injecting 5 mg/ml ovalbumin into the knees of 20 preimmunized rabbits. Animals were randomized into two groups: treated with tenidap (15 mg/kg per 12 h), or untreated. The effect of tenidap treatment was evaluated on chemokine production in synovial membranes of rabbits with arthritis and in cultured monocytic and synovial cells (SC). By immunoperoxidase staining, chemokines were localized in the synovial tissue. Chemokine messenger RNA levels in the synovial membranes and in cultured cells were analysed by reverse transcription-polymerase chain reaction (RT-PCR). At the end of the study, tenidap significantly reduced neutrophil infiltration into the joint cavity (27+/-4 x 10(6) cells/ml versus 45+/-6 x 10(6) cells/ml in untreated; P<0.05), and synovial effusion (134+/-15 microl versus 236+/-19 microl in untreated; P<0.005). Untreated rabbits showed synovial membrane up-regulation in mRNA expression of IL-8 and MCP-1 (11- and seven-fold versus healthy rabbits, respectively) that was markedly decreased by tenidap (two- and three-fold versus healthy rabbits, respectively). IL-8 and MCP-1 were localized in the synovial tissue in a perivascular pattern and areas of the interstitium and lining, mostly coinciding with cell infiltration. Tenidap also reduced the accumulation of IL-8 and MCP-1 proteins. In cultured synovial and monocytic cells, tumour necrosis factor-alpha (TNF-alpha) elicited an increase in gene expression of IL-8 (four- and nine-fold, respectively) and MCP-1 (nine- and four-fold, respectively) that was significantly reversed in both cell types by 10 microM tenidap. These results suggest that the beneficial effect of tenidap in acute antigen arthritis could be related to the down-regulation in gene expression and synthesis of IL-8 and MCP-1, two key chemokines involved in the recruitment of inflammatory cells.

The 80-kD fibronectin fragment increases the production of fibronectin and tumour necrosis factor-alpha (TNF-alpha) in cultured mesangial cells.

The presence of fibronectin (FN) fragments has been demonstrated in several inflammatory processes, and they have been implicated in the recruitment of mononuclear cells. However, the interaction of these FN fragments with resident cells has hardly been studied. We have hypothesized that the 80-kD FN fragment, which includes the RGD cell binding domain of FN, could contribute to the pathogenesis of glomerular damage through the interaction with mesangial cells (MC) via alpha5beta1 integrin. Since an increase in the glomerular deposit of matrix components, particularly FN, is frequently observed in progressive glomerulonephritis, we studied whether its synthesis is modulated by the 80-kD FN fragment and the native FN molecule. While the 80-kD FN fragment stimulated FN in a dose-dependent manner, both at the mRNA and protein level, the whole FN molecule exerted a dual effect. High doses produced FN inhibition, while low doses elicited a certain increase. This stimulation was abrogated by the presence of Sam-1, a MoAb against the alpha-subunit of the alpha5beta1 integrin. Since cytokines play a fundamental role in glomerular injury, we studied the production of TNF-alpha, one of the most powerful mediators of inflammation. TNF-alpha synthesis was induced by the 80-kD FN fragment, in a dose-dependent manner, but not by native FN. When MC were incubated with the 29- and 31-kD FN fragments, which lack the RGD cell binding domain, TNF-alpha secretion was not detected. These results strongly suggest that in cultured MC, the 80-kD FN fragment induces the synthesis of matrix proteins such as FN, and cytokines such as TNF-alpha, via alpha5beta1 integrin. This mechanism could contribute to the perpetuation of renal injury.

Antifibroproliferative effect of tenidap in chronic antigen-induced arthritis.

OBJECTIVE: To determine whether tenidap regulates extracellular matrix metabolism in chronic arthritis. METHODS: Antigen arthritis was induced in the knees of 30 rabbits. Animals were distributed into 3 groups: untreated, tenidap-treated, and diclofenac-treated rabbits. Three weeks after disease induction, synovial membranes were extracted and processed for histopathologic examination and detection of type I collagen (CI) and fibronectin (FN) by immunoperoxidase. Simultaneously, we analyzed the in vitro effect of tenidap on healthy synovial cell (SC) proliferation, FN expression and synthesis, and expression of transforming growth factor beta1 (TGFbeta1) messenger RNA. RESULTS: Untreated animals showed synovial lining hyperplasia, cellular infiltration at the sublining, and increased deposition of matrix proteins. These findings were not apparent in tenidap-treated rabbits, where CI and FN had the same distribution as in healthy synovial membranes. In vitro, tenidap inhibited SC proliferation (> or =25 microM) and down-regulated the expression and synthesis of FN in a dose-dependent manner (> or =1 microM). This antifibrotic effect was associated with a reduction of TGFbeta1 message. CONCLUSION: Tenidap down-regulates the fibroproliferative changes typical of chronic arthritis, an effect that fits the profile of a disease-modifying agent for rheumatoid arthritis.

Glomerular up-regulation of EIIIA and V120 fibronectin isoforms in proliferative immune complex nephritis.

Fibronectin: (FNs) comprise a family of adhesive glycoproteins that are prominent components of mesangial extracellular matrix and accumulate during glomerular injury. By alternative splicing of an unique mRNA precursor, various FN isoforms can be originated. In rat, three regions of the molecule are involved: EIIIA, EIIIB and V. Because specific FN isoforms are expressed in embryogenesis and wound healing, conditions characterized by cell migration and adhesion, we examined the pattern of FN isoforms in the mild and severe phases of a progressive immune complex proliferative nephritis in rats. We constructed specific probes to analyze the splicing pattern of FN pre-mRNAs by ribonuclease protection assays. FN mRNAs containing EIIIA, EIIIB and V regions increased along, the progression of nephritis, though the increment of EIIIB-FN mRNA was modest. However, different regulation of all these isoforms was observed. The percentage of FN mRNA containing the EIIIA exon versus total FN increased with the severity of the disease, while the percentage of FN mRNA containing the EIIIB exon decreased. Relative V-FN mRNA expression versus total FN mRNA increased only in the severe phase. By means of specific antibodies we also studied the presence of EIIIA, EIIIB and V-FN proteins in the kidney. In the normal glomerutus, EIIIA-FN protein was barely detectable in the mesangium, increasing in the mild phase of nephritis. In the severe phase of nephritis, increased EIIIA-FN was localized in the mesangium, in Bowman's capsule and in crescents. By contrast, EIIIB-FN protein in the glomerulus was absent even in the severe phase. V120-FN protein, an isoform that mediates the attachment of leukocytes through the VLA-4 integrin, was present in the mesangium and glomerular capillary loops in control animals, and increased in the severe phase of nephritis, coinciding with a strong leukocyte infiltration. In conclusion, our results show that during immune glomerular injury there were marked changes in the pattern of FN isoforms expression. Since those isoforms, particularly V120 isoform, are important in cell adhesion and migration, their up-regulation may facilitate the recruitment of cells into the injured glomeruli. The blockade of the interaction between V120-FN and infiltrating leukocytes may represent a new approach to the treatment of nephritis.

An orally active ETA/ETB receptor antagonist ameliorates proteinuria and glomerular lesions in rats with proliferative nephritis.

The proliferation of mesangial cells and the extracellular matrix expansion constitute the most outstanding morphological aspects of the majority of progressive glomerular diseases. In vitro, endothelin-1 (ET-1) is mitogenic for mesangial cells and induces matrix protein synthesis. We studied the possible participation of ET-1 in the pathogenesis of renal damage in a normotensive model of proliferative nephritis. Coincidentally with maximal proteinuria and glomerular lesions, an increase was found in the glomerular mRNA expression of preproET-1 and the ETA receptor (10 and 6 times compared to controls, respectively), but not of the ETB receptor, and in ET-1 urinary excretion (217 +/- 33 vs. 84 +/- 4 pg ET-1/24 hr, N = 4 to 5, P < 0.05). By in situ hybridization, an increase in preproET-1 mRNA expression in glomerular endothelial, epithelial and mesangial cells, and in come tubular cells was observed. The administration of bosentan, an ETA/ETB receptor antagonist, had a beneficial effect on the evolution of nephritis preventing the appearance of intense proteinuria (76 +/- 35 vs. 380 +/- 77 mg/24 hr, N = 4 to 5, P < 0.05), the morphological lesions and the renal function impairment (creatinine clearance 367 +/- 46 vs. 268 +/- 33 microliters/min/100 g, N = 4 to 5). Simultaneously, there was a decrease in ET-1 urinary excretion (88 +/- 14 vs. 217 +/- 33 pgET-1/24 hr, N = 4,5, P < 0.05) and in the renal preproET-1 mRNA expression. The mean systolic blood pressures remained in the normal range in all animals. These data indicate that ET-1 participates in the pathogenesis of proteinuria and glomerular injury in a model of proliferative nephritis. The nonpeptidic orally active ETA/ETB receptor antagonists could be useful in the treatment of some human nephritis.

Receptors for immune complexes activate gene expression and synthesis of matrix proteins in cultured rat and human mesangial cells: role of TGF-beta.

Most human glomerulonephritis are induced by the deposition and/or formation of immune complexes in the glomerular region. Recently, it has been demonstrated that cultured glomerular mesangial cells (MC) express Fc receptors for IgA and IgG (Fc-alpha and Fc-gamma receptors). In this work, we studied whether the interaction of IgA and IgG complexes with MC induces accumulation of mesangial matrix, the histologic hallmark of progressive glomerular diseases. The exposure of MC to IgA and IgG complexes increased extracellular matrix components, such as fibronectin (FN) and collagens, at both the mRNA and protein levels in a time- and dose-dependent manner. Monomeric IgA or F(ab')2 fragments did not increase FN production, indicating that a constant region of IgA and cross-linking of Fc-alpha receptors are required. We also explored the role of TGF-beta, a profibrogenic cytokine, in the regulation of matrix synthesis. Both IgA and IgG complexes caused in MC an augmentation in TGF-beta1 mRNA and TGF-beta activity and the conversion of latent TGF-beta to the biologically active form. The coincubation of cells with complexes and a neutralizing Ab to TGF-beta significantly reduced the FN synthesis. These results indicate that the Fc receptor occupancy of MC increases the production of extracellular matrix proteins. The autocrine TGF-beta synthesis appears to be largely responsible for this effect. These findings could have implications for a better understanding of the glomerulosclerosis process in immune complex nephritis.