Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes

OBJECTIVES: To determine the in vitro effects of oxygen tension on interleukin (IL)-1beta induced nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) production by bovine chondrocytes. DESIGN: Enzymatically isolated bovine chondrocytes were cultured for different periods in suspension in 21 (atmospheric), 5 or 1% (low) oxygen tension and in the absence or in the presence of increased amounts (0.01 to 1nM) of IL-1beta. Nitrite and nitrate concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. PGE(2) production was quantified by a specific radioimmunoassay (RIA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA steady state levels were also quantified by real-time polymerase chain reaction (PCR). RESULTS: In the absence of IL-1beta, ()NO production remained stable whatever the oxygen tension used. IL-1beta dose-dependently increased *NO production in both atmospheric and low oxygen conditions but the effect was more pronounced in low (1 and 5%) than in atmospheric (21%) oxygen tension (P<0.001). Under low and atmospheric oxygen tension, iNOS gene expression was increased by IL-1beta, but to a lesser extent in 21% than in 1 or 5% oxygen (P<0.01). In the basal condition, bovine chondrocytes spontaneously produced PGE(2) whatever the oxygen tension used. At 21% oxygen, IL-1beta dose-dependently increased PGE(2) production while no significant effect was observed at 1 or 5% oxygen. COX-2 gene expression was significantly upregulated by IL-1beta in both low and atmospheric oxygen tension. No significant difference between oxygen tension conditions was observed. CONCLUSIONS: This study demonstrates that a hypoxic environment fully blocks COX-2 activity but favours iNOS gene expression in chondrocytes culture. These findings indicate that O(2) tension modulates cellular behaviour in culture and supports the concept of chondrocyte culture in low oxygen tension to reproduce in vitro the life conditions of chondrocytes.     

Differential effects of SB 242235, a selective p38 mitogen-activated protein kinase inhibitor, on IL-1 treated bovine and human cartilage/chondrocyte cultures

The p38 MAP kinase inhibitor, SB 242235, was evaluated for its effects on the metabolism of bovine and human cartilage and primary chondrocyte cultures. SB 242235 had no effect on proteoglycan synthesis (PG) in bovine articular cartilage explants (BAC), as measured by [(35)S]-sulfate incorporation into glycosaminoglycans (GAGs). In addition, the compound had no effect on IL-1 alpha-induced GAG release from these cultures. However, there was a potent, dose-dependent inhibition of nitric oxide (NO) release from IL-1 alpha-stimulated BAC with an IC(50)of approximately 0.6 microM, with similar effects observed in primary chondrocytes. The effect on BAC was time dependent, and mechanistically did not appear to be the result of inhibition of protein kinase C (PKC), protein kinase A (PKA) or MEK-1. The effect on NO release in bovine chondrocytes was at the level of inducible nitric oxide synthase (iNOS) gene expression, which was inhibited at similar concentrations as nitrite production. In primary human chondrocytes, IL-1 beta induction of p38 MAP kinase was inhibited by SB 242235 with an IC(50)of approximately 1 microM. Surprisingly, however, treatment of IL-beta-stimulated human cartilage or chondrocytes with SB 242235 did not inhibit either NO production or the induction of iNOS. On the other hand, the natural product hymenialdisine (HYM), a protein tyrosine kinase (PTK) inhibitor, inhibited NO production and iNOS in both species. In contrast to the differential control of iNOS, PGE(2)was inhibited by SB 242235 in both IL-1-stimulated bovine and human chondrocyte cultures. These studies indicate that there are species differences in the control of iNOS by p38 inhibitors and also that different pathways may control IL-1-induced proteoglycan breakdown and NO production.     

Effects of human cathelicidin antimicrobial peptide LL-37 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro

BACKGROUND: Lipopolysaccharides (LPS), released by Gram-negative bacteria, cause vascular expression of inducible nitric oxide synthase (iNOS) leading to nitric oxide (NO) production and septic shock. Human cathelicidin antimicrobial peptide (LL-37) can bind and neutralize LPS. We wanted to study whether LL-37 affects LPS or interleukin-1beta (IL-1beta)-induced production, release and function of NO in intact rat aorta rings and cultured rat aorta smooth muscle cells. METHODS: Isolated segments of thoracic aorta and cultured cells were incubated in the presence of LPS, LL-37, LPS + IL-37, IL-1beta, IL-1beta + IL-37 or in medium alone. Smooth muscle contraction in response to phenylephrine and accumulation of the sdegradation products of NO, nitrate and nitrite, were measured on aorta segments. Levels of iNOS were assessed by Western blot and cytotoxic effects were detected by measurement of DNA fragmentation in cultured cells. Number of viable cells were determined after Trypan blue treatment. RESULTS: Both LPS and IL-1beta reduced contractility in response to phenylephrine and increased NO production as well as iNOS expression. LL-37 inhibited the LPS depression of vascular contractility induced only by LPS. LL-37 reduced both the LPS- and IL-1beta-induced NO production and iNOS expression. LL-37 at high concentrations induced DNA fragmentation and decreased the number of living cells. CONCLUSION: IL-37 reduces NO production induced by LPS and IL-1beta. The reduction does not seem to result only from neutralization of LPS but also from a cytotoxic effect, possibly via induction of apoptosis.     

Evaluation of chondrocyte cell-associated matrix metabolism by flow cytometry

OBJECTIVE: To analyze human articular chondrocyte cell-associated matrix aggrecan, hyaluronan (HA) and type II collagen metabolism using flow cytometry, and to compare the results obtained for aggrecan with classic(35)Sulfate incorporation methods and an enzyme linked immunosorbent assay (ELISA). DESIGN: Human articular chondrocytes obtained from five donors were cultured in gelled agarose and tested for their response to different concentrations of interleukin-1beta (IL-1beta). Synthesis and distribution of aggrecan in the cell-associated matrix (CAM), in the interterritorial matrix and in the nutrient medium of the chondrocytes in culture were analyzed using(35)Sulfate incorporation. The results were expressed as pg SO(4)incorporated in aggrecan per 1 x 10(6)cells/h. Flow cytometry with FITC-conjugated monoclonal antibodies against aggrecan and type II collagen, and with the biotinylated hyaluronic acid binding protein (b-HABP), was used to investigate the synthesis and accumulation of aggrecan, type II collagen and HA in the CAM of the cultured cells. The packing of these macromolecules in the CAM of the chondrocytes was assessed by measuring the mean fluorescence intensity (MFI) of the cell sample due to the binding of the specific monoclonal antibodies or b-HABP used. ELISA was used in parallel to quantify CAM aggrecans after these macromolecules were brought into solution with guanidinium chloride. Detection of aggrecan by flow cytometry was compared with(35)S-incorporation in chondrocytes from two subjects and with ELISA in a further two donors. RESULTS: IL-1beta suppressed aggrecan synthesis by chondrocytes in agarose. An IL-1beta dose-dependent suppression of(35)S-aggrecan in the CAM reflected the changes in the interterritorial matrix. IL-1beta-induced aggrecan breakdown was followed by a rise in(35)S-aggrecan metabolites in the incubation media of the cells in culture. Flow cytometry and ELISA confirmed this decreased accumulation of aggrecan in the CAM of the chondrocytes. The results obtained with flow cytometry were closely related to those obtained with ELISA.(35)S-incorporation, on the other hand, indirectly measures the glycosaminoglycan content of the aggrecan and does not necessarily reflect the absolute amount of aggrecan molecules. Therefore, the effects of IL-1beta on cell-associated aggrecan, where assessed with(35)S-incorporation, did not correlate with the results of the flow cytometric assays. Flow cytometry enabled the detection of an impaired synthesis and accumulation of HA and of type II collagen in the CAM of the cultured chondrocytes. IL-1beta-induced changes in CAM aggrecan and hyaluronan closely agreed. CONCLUSIONS: Flow cytometry offers an efficient tool to study the metabolism of the chondrocyte CAM. The MFI has been used as a parameter to quantify the ECM molecules in the CAM.     

Human recombinant erythropoietin inhibits interleukin-1beta-stimulated nitric oxide and cyclic guanosine monophosphate production in cultured rat vascular smooth-muscle cells.

BACKGROUND: Recently rat vascular smooth-muscle cells (VSMC) have been shown to possess Epo receptor, and respond to various cytokines for producing nitric oxide (NO). In the present study we examined the effect of pharmacological dose of human recombinant erythropoietin (rHuEpo) on the IL-1beta-induced NO and cGMP production as well as inducible nitric oxide synthase (iNOS) in cultured rat VSMC. METHODS: Nitrite, a stable metabolite of NO, and intracellular cGMP contents were assayed by Griess method and enzyme immunoassay. iNOS mRNA expression was analysed by Northern blotting. RESULTS: RHuEpo inhibited IL-1beta-induced nitrite production in a dose- and time-dependent manner with concomitant changes of intracellular cGMP contents. On the other hand, rHuEpo did not inhibit atrial natriuretic peptide- (ANP) or sodium nitroprusside (SNP)-induced nitrite and cGMP production at all. While rHuEpo inhibited IL-1beta-induced iNOS mRNA expression, rHuEpo vehicle did not affect IL-1beta-induced iNOS mRNA expression. CONCLUSIONS: It is suggested that a pharmacological dose of rHuEpo inhibits IL-1beta-induced NO and cGMP production as well as iNOS mRNA expression, presumably via the Epo receptor.     

Expression of TGF-betas and their receptors is differentially modulated by reactive oxygen species and nitric oxide in human articular chondrocytes

OBJECTIVES: To study the effects exerted by two antioxidants, N-monomethyl-L-arginine (L-NMMA), as an inhibitor of nitric oxide (NO) synthesis, and N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, on the expression of the major growth factor involved in cartilage repair, TGF-beta, under the three isoforms beta1, beta2 and beta3, and the receptors I and II of this factor, using lipopolysaccharide (LPS)-treated human chondrocytes in culture. METHODS: Suspension cultures of human chondrocytes derived from the knee of osteoarthritic patients were treated for 48 h with lipopolysaccharide (LPS) (10 microg/ml), L-NMMA (0.5 mM) or NAC (1 mM). Nitrite levels were assayed on the culture media using the Griess spectrophotometric method. After total RNA extraction, the expression of inducible NO synthase (iNOS), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta receptors I and II, was determined by semi-quantitative polymerase chain-reaction (RT-PCR). RESULTS: LPS induced a dramatic increase of both NO production and iNOS mRNA level. The addition of L-NMMA (0.5 mM) abolished NO production without affecting iNOS mRNA levels. In contrast NAC (1 mM) strongly synergized with LPS to stimulate NO synthesis. LPS treatment did not significantly alter TGF-beta1 expression whereas L-NMMA inhibited its production. TGF-beta2 mRNA level was decreased by LPS and was not changed in the presence of L-NMMA. On the other hand, NAC was capable of counteracting the LPS-induced inhibition of TGF-beta2 expression. TGFbeta3 mRNA level was markedly reduced by LPS alone, or with both L-NMMA and NAC. Finally, the expression of TGF-betaRI was slightly increased in the presence of combined LPS and L-NMMA or NAC whereas that of TGFbeta-RII was reduced in the same conditions. CONCLUSIONS: The modulation of TGF-beta system was found to be differentially controlled by NO and ROS productions. Indeed, the control exerted on TGF-beta expression varied according to the isoform: TGF-beta1 mRNA level depends on NO whereas that of TGF-beta2 is regulated by ROS and TGF-beta3 seems to be unaffected by both of them. The expression of TGF-beta receptors appeared to be modulated by NO and ROS levels. The relevance of the present findings to osteoarthritis (OA) physiopathology and the potential use of antioxidant therapy to treat this disease are discussed.     

Bactericidal/permeability-increasing protein inhibits endotoxin-induced vascular nitric oxide synthesis

BACKGROUND: Endotoxin (lipopolysaccharide, LPS) up-regulates inducible nitric oxide synthase (iNOS) in blood vessels during septic shock. This promotes the production of nitric oxide (NO), leading to dilation of the vessels. The aim of the study was to investigate the effects of the LPS-binding endogenous antibiotic bactericidal/permeability-increasing protein (BPI) on the action of LPS on the blood vessels wall and to identify possible influence on underlying NO-related mechanisms. METHODS: Isolated segments of rat thoracic aorta and cultured primary smooth muscle cells were incubated for 5-48 h in the presence of the following combinations of compounds: (a) LPS; (b) interleukin-1beta (IL-1beta); (c) BPI; (d) BPI + LPS; (e) BPI + IL-1beta or (f) neither BPI, LPS nor IL-1beta (control). After incubation of intact segments, we measured smooth muscle contraction in response to phenylephrine and accumulation of the NO end products nitrate and nitrite in surrounding medium. Western blot was used to assess the levels of inducible nitric oxide synthase (iNOS) in cultured cells. RESULTS: Both LPS and IL-1beta decreased contractility and increased NO production, as well as iNOS. Co-incubation with BPI attenuated all the effects of LPS but only the effects of prolonged exposure to IL-1beta in cultured cells. CONCLUSION: We conclude that BPI attenuates the LPS-induced changes in vascular reactivity by inhibiting the expression of iNOS resulting in decreased NO formation and restored responsiveness to vasoconstrictors. The data suggest that BPI can prevent circulatory disturbances during Gram-negative sepsis.     

Effects of heme oxygenase‐1 on bacterial antigen‐induced articular chondrocyte catabolism in vitro

This study tested the hypothesis that heme oxygenase‐1 (HO‐1) expression counteracts bacterial antigen‐induced catabolic metabolism in human articular chondrocytes. HO‐1 expression was induced in chondrocytes by the iron‐containing porphoryin, hemin. Anti‐catabolic and anti‐apoptotic effects of HO‐1 expression were evaluated following bacterial antigen (lipopolysaccharides, LPS) activation of chondrocytes by quantification of cytokine and cartilage matrix protein expression. Effects of HO‐1 over‐expression on chondrocyte matrix metabolism were evaluated using plasmid‐driven protein synthesis. Hemin increased HO‐1 expression and LPS increased interleukin‐1beta and interleukin‐6 gene and protein expression in chondrocytes. Hemin‐induced HO‐1 decreased LPS‐induced interleukin‐1beta and interleukin‐6 gene and protein expression. Increased HO‐1 expression partially reversed LPS‐suppression of aggrecan and type II collagen gene expression and suppressed LPS‐induced gene expression of IL‐6, inducible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs), and IL‐1beta. HO‐1 induction was inversely correlated with LPS‐induced chondrocyte apoptosis. HO‐1 over‐expression in chondrocytes decreased matrix protein gene expression. With LPS activation, increased HO‐1 expression decreased chondrocyte catabolism, partially reversed LPS‐dependent inhibition of cartilage matrix protein expression and protected against apoptosis. Without LPS, hemin‐induced HO‐1 and plasmid‐based over‐expression of HO‐1 inhibited cartilage matrix gene expression. The results suggest that elevated HO‐1 expression in chondrocytes is protective of cartilage in inflamed joints but may otherwise suppress matrix turn over. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1943–1949, 2013     

Hyaluronan does not affect cytokine and chemokine expression in osteoarthritic chondrocytes and synoviocytes

OBJECTIVE: Many studies have evidenced the clinical efficacy of hyaluronan (HA) in the treatment of osteoarthritis (OA). However, human and animal studies have described proinflammatory effects of HA on cells not involved in OA. We therefore investigated whether different molecular weight HA preparations can affect proinflammatory cytokine (IL1beta and TNFalpha) or chemokine (IL8, MCP-1 and RANTES) expression in human chondrocytes and synoviocytes isolated from OA patients. DESIGN: Human chondrocytes and synoviocytes were cultured in vitro in the presence or absence of three different purified HA pharmaceutical preparations (1x10(6) Kd, 5x10(5) Kd and 6.5x10(4) Kd) and assessed for the production of proinflammatory cytokines and chemokines and their mRNA expression. RESULTS: basal conditions, both chondrocytes and synoviocytes produce only MCP-1 and IL8, along with low quantities of IL1beta and TNFalpha, but not RANTES. IL8 production was generally about 100 times higher in chondrocytes than in synoviocytes, while MCP-1 was roughly twice as high in synoviocytes than in chondrocytes. At the mRNA level, expression of IL1beta, TNFalpha, IL8, MCP-1 and RANTES did not change in the presence of the three HA preparations either in synoviocytes or in chondrocytes with respect to basal condition. None of the three different HA preparations significantly affected production of IL8 or MCP-1. CONCLUSIONS: These data demonstrate that preparations of HA of the same origin but with different MWs do not induce proinflammatory cytokines and chemokines expressed by chondrocytes and synoviocytes that are either directly or indirectly involved in OA progression.     

Effects of the existence of nitric oxide and the degradation of extracellular matrix on CD44 mRNA expression in cultured chondrocytes

We previously reported that interleukin-1β (IL-1β) promoted CD44 mRNA expression in cultured rabbit articular chondrocytes, together with the reduction of extracellular matrix. It was assumed from this result that the existence of nitric oxide (NO) induced by IL-1β and degradation of the extracellular matrix affects CD44 mRNA expression in the chondrocytes. To confirm this assumption, we first carried out production of NO by IL-1β through quantitative detection of inducible nitric oxide synthase (iNOS) mRNA expression in the chondrocytes and measurement of nitrite plus nitrate in the culture medium. IL-1β evidently promoted NO production in the chondrocytes. A tendency for increased release of chondroitin sulfates (CS), as extracellular matrix, to the culture medium was observed by addition of IL-1β, but release of CS to the culture medium did not increase as the result of addition of S-nitroso-N-acetyl penicillamine (SNAP) as NO donor. We then investigated the effects of SNAP, chondroitinase, and hyaluronidase on CD44 mRNA expression in the cultured chondrocytes. CD44 mRNA expression was increased significantly in chondrocytes cultured with SNAP and chondroitinase but not in chondrocytes cultured with hyaluronidase. These results suggest that the existence of NO and the degradation of CS may be associated with upregulation of CD44 mRNA expression in chondrocytes.. Received: Nov. 19, 1997 / Feb. 2, 1998     

Induction of inducible nitric oxide synthase in hepatocytes isolated from rats with ischemia-reperfusion injury

BACKGROUND: Recent evidence indicates that nitric oxide (NO) has a crucial role in hepatic ischemia-reperfusion (I/R) injury. However, little is known about how I/R influences the gene expression of inducible nitric oxide synthase (iNOS) in hepatocytes. Under inflammatory conditions, we compared the induction of iNOS in hepatocytes isolated from normal and I/R-treated rats. METHODS: Hepatocytes were isolated using the collagenase perfusion method from rats treated with I/R (30-minute ischemia of middle and left lobes, followed by 3-hour reperfusion) or sham operation (control): Primary cultures of rat hepatocytes were incubated with an inflammatory cytokine, interleukin-1beta (IL-1beta), to compare the iNOS induction/NO production between the 2 groups. RESULTS: Both control and I/R groups had no production of nitrite (a stable metabolite of NO) in the absence of IL-1beta. In the control group, IL-1beta stimulated dose- and time-dependent production of NO. The I/R group showed more than 2-fold increased levels of NO production. Western and Northern blot analyses revealed that the I/R group also showed increased levels of iNOS protein and its messenger RNA. CONCLUSION: These results suggest that I/R directly affects the inducibility of the iNOS gene in hepatocytes by IL-1beta. Increased NO may be associated with protective or toxic effects in hepatic I/R injury.     

Human renal epithelial cells express iNOS in response to cytokines but not bacteria

BACKGROUND: Epithelial cells form the mucosal barriers that prevent the entry of mucosal pathogens, and respond to bacterial infections by producing various host defense molecules. In this study, we examined the inducible nitric oxide synthase (iNOS) response of primary human renal tubular epithelial cells (HRTEC) following infection with uropathogenic Escherichia coli Hu734, or stimulation with lipopolysaccharide (LPS) or cytokines. METHODS: Induction of iNOS was examined by RT-PCR, Western blot, immunohistochemistry and nitrite measurements. The effects of endogenously produced nitric oxide (NO), and exogenously applied DETA/NO, SIN-1 and H2O2 on cell viability were analyzed using a respiration assay. RESULTS: HRTEC did not produce NO following infection with E. coli Hu734, LPS alone, or in combination with interferon-gamma (IFN-gamma), even though these agents caused a marked increase in iNOS expression by RAW 264.7, a macrophage cell line. In contrast, iNOS protein and mRNA expression by HRTEC increased after exposure to a cytokine mixture consisting of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. This was due to the combination of IL-1beta and IFN-gamma, but the individual cytokines had no effect. Inducible NOS-expressing cell cultures showed reduced viability, and this effect was inhibited with the NOS inhibitor L-NMMA in RAW 264.7 cells, but not in HRTEC. HRTEC were more sensitive to oxidative stress induced by H2O2 than to nitrogen stress induced by DETA/NO. CONCLUSIONS: We conclude that uropathogenic E. coli that attach to HRTEC fail to directly activate iNOS expression, and that iNOS expression during bacterial infection is more likely to result from stimulation by local cytokines such as IL-1beta and IFN-gamma.     

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. BACKGROUND: High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. CONCLUSION: We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.