碱性成纤维细胞生长因子及相关细胞因子转染对人骨关节炎软骨细胞的作用

目的 探讨腺病毒介导的人碱性成纤维细胞生长因子(bFGF)单独及与白细胞介素-1受体拮抗蛋白(IL-1Ra)和(或)胰岛素样生长因子(IGF)-1共同转染人骨关节炎(OA)软骨细胞后对软骨细胞的影响.方法 采用单独编码人bFGF的重组腺病毒载体或多重组合的重组腺病毒载体转染单层培养的人OA软骨细胞.6 d后分别检测培养上清液中目的 基因表达和糖胺聚糖(GAG)含量.四甲基偶氮唑蓝(MTT)法及流式细胞术分析软骨细胞的增殖及凋亡.甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色观察软骨细胞基质的合成.免疫印迹法检测Ⅱ型胶原、基质金属蛋白酶(MMP)-3及其抑制剂-1(TIMP-1)的表达.采用单因素方差分析,并进行组间两两比较.结果 各基因转染后,细胞上清液日的基因表达与OA对照组相比明显增高(P<0.05 ). bFGF单独转染可促进软骨细胞增殖,增加Ⅱ型胶原和蛋白多糖的合成(P<0.05).与bFGF单独转染相比,联合IL-1Ra和(或)IGF-1共同转染后,可降低软骨细胞的凋亡率[分别为:(26.1±1.6)%、(19.4±1.0)%、(18.4±1.1)%、(13.9±1.8)%,P<0.05],进一步增加了软骨基质的生物合成(P<0.05).同时,抑制了MMP-3的表达,增加了TIMP-1的表达.结论 腺病毒介导的bFGF转染入OA软骨细胞可促进细胞增殖,增加基质的合成.与IL-1Ra和IGF-1共转染后可发挥协同作用,进一步增加基质合成;同时,抑制了基质的降解.     

硫酸氨基葡萄糖对白细胞介素1β诱导人骨关节炎软骨细胞合成前列腺素E2的影响

目的探讨硫酸氨基葡萄糖(glucosamine sulfate,GS)对白细胞介素1β(IL-1β)诱导体外培养的人骨关节炎软骨细胞(human osteoarthritic chondrocyte,HOC)合成前列腺素E_2 (prostaglandin E_2,PGE_2)的影响及其作用机制。方法取10例骨关节炎患者行全膝关节置换术的股骨髁和胫骨平台软骨标本,酶消化法获取软骨细胞进行体外培养。在原代或第二代的HOC培养液中加入IL-1β(5×10~(-3)μg/L)和不同浓度的GS(0.2、2和20 mmol/L)作用24 h(对照组不加IL-1β和GS),首先应用ELISA法检测GS对IL-1β诱导HOC合成PGE_2的影响,然后采用RT-PCR法和Western蛋白印迹法分别检测其对IL-1β诱导HOC表达COX-2 mRNA和蛋白的影响。结果对照组HOC培养液中PGE_2浓度为(109.46±17.48)pg/ml,IL-1β刺激后软骨细胞合成PGE_2为(3607.22±239.34)pg/ml,其差异有统计学意义(P<0.01)。0.2、2和20 mmol/L GS以浓度依赖的方式抑制IL-1β诱导HOC合成PGE_2(P<0.05),其浓度分别为(2594.23±185.18)、(1057.53±126.81)和(565.43±52.79)pg/ml;单独使用20 mmol/L GS时软骨细胞合成PGE_2为(169.94±30.03)pg/ml,与对照差异无统计学意义(P>0.05)。IL-1β刺激后,HOC表达COX-2 mRNA和蛋白上调(P<0.01),GS能以浓度依赖的方式抑制IL-1β诱导HOC表达COX-2 mRNA和蛋白上调(P<0.01)。结论GS抑制HOC在IL-1β诱导下分泌炎性介质PGE_2,其机制是下调调控它们表达的COX-2 mRNA和蛋白的表达;这可能是GS既能缓解症状和又能保护软骨的机制之一。     

Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes

Abstract

Objective. In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism.

Methods. Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry.

Results. We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5’-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066).

Conclusion. Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.     

Histamine stimulates prostaglandin E production by rheumatoid synovial cells and human articular chondrocytes in culture

Histamine stimulates prostaglandin E (PGE) production by cultures of adherent rheumatoid synovial cells and human articular chondrocytes. When subcultured synovial fibroblasts or human articular chondrocytes were "primed" by preincubation with conditioned media from primary adherent rheumatoid synovial cell cultures (synovial factor), each produced even higher PGE levels upon histamine exposure. This histamine stimulation was prevented by histamine H1, but not H2, antagonists and was more marked if serum was absent from the culture media. Thus, histamine-induced PGE production by these cells is mediated via H1 receptor activation and subsequent arachidonic acid liberation.     

Effects of nimesulide and sodium diclofenac on interleukin-6, interleukin-8, proteoglycans and prostaglandin E2 production by human articular chondrocytes in vitro.

OBJECTIVES: The aim of this study was to investigate the effects of two nonsteroidal anti-inflammatory drugs (NSAIDs), nimesulide and sodium diclofenac, on the production of proteoglycans (PG), prostaglandin E2 (PGE2) and cytokines (IL-6 and IL-8) by human articular chondrocytes in vitro. METHODS: Enzymatically isolated chondrocytes were cultured under constant agitation in a well defined culture medium. Specific radioimmunoassays were used to quantify PG and PGE2 production. Cytokine production (IL-6 and IL-8) was assayed by enzyme amplified sensitivity immunoassays (EASIAs). RESULTS: At a concentration of 3 micrograms/ml, nimesulide did not affect the PG production by chondrocytes. This concentration was superior to the highest level of nimesulide found in the synovial fluid of patients with rheumatoid arthritis 3 hours after the last oral administration of nimesulide (100 mg twice daily for 7 days). At 6 micrograms/ml a significant reduction in the PG content was obtained in the cellular phase in 5 out of the 8 cultures investigated. No similar effect was observed in the culture supernatants. Above this concentration nimesulide inhibited PG production in a dose-dependent manner. At concentrations ranging from 0.005 to 1 microgram/ml diclofenac did not significantly alter PG production. At therapeutic concentrations PGE2 production was totally inhibited by nimesulide, thus suggesting that PG inhibition is not linked to PGE2 production. Nimesulide inhibited PGE2 production by unstimulated (IC50 = 6 ng/ml) and IL-1 beta-stimulated (IC50 = 6.9 ng/ml) chondrocytes. At these concentrations, PGE2 production was fully inhibited by diclofenac. Furthermore, both nimesulide and diclofenac at therapeutic concentrations significantly decreased spontaneous and IL-1 beta-stimulated IL-6 production by human chondrocytes, but did not modify IL-8 production. CONCLUSION: From the results of this study we conclude that nimesulide and diclofenac at therapeutic concentrations are potent inhibitors of PGE2 and IL-6 production while they do not modify proteoglycan or IL-8 production.     

Suppressive effects of hyaluronan on MMP-1 and RANTES production from chondrocytes

The purpose of the study was to examine the effects of hyaluronan (HA) on human chondrocytes in terms of production of MMP-1 and RANTES. Chondrocytes were obtained from patients with osteoarthritis (OA) or femoral neck fracture (control). Chondrocytes in monolayer culture were treated with various molecular weights (1.2, 50, 800 and 1,900 kD) of HA and then stimulated with IL-1. Production and expression of MMP-1 and RANTES were quantified by ELISA and real-time polymerase chain reaction (PCR). The response was blocked by anti-CD44 antibody. Production of MMP-1 was significantly suppressed by both 800- and 1,900-kD HA, while production of RANTES was suppressed by 1,900-kD HA. Expression of MMP-1 and RANTES mRNA was inhibited by 1,900-kD HA. Suppressive effects of HA on production of MMP-1 were canceled by treatment of anti-CD44 antibody. Higher CD44 expression was found in OA chondrocytes than in those of control. High-molecular-weight HA suppressed MMP-1 and RANTES production, mediated partly by CD44–HA interaction.     

The effect of tumor necrosis factor alpha and gamma-interferon on the resorption of human articular cartilage and on the production of prostaglandin E and of caseinase activity by human articular chondrocytes

In cultured human articular chondrocytes, addition of tumor necrosis factor alpha (TNF alpha) stimulated caseinase activity over the range of 10(-11) M to 10(-7) M and stimulated prostaglandin E (PGE) production over the range of 10(-10) M to 10(-7) M. Maximal stimulation was observed at 10(-8)M TNF alpha for both activities. Gamma-interferon (gamma-IFN) had a variable effect on PGE production and no significant effect on caseinase activity in articular chondrocyte cultures over a concentration range of 0.1-1,000 units/ml. Co-incubation of TNF alpha with gamma-IFN enhanced PGE production and decreased caseinase activity. Concentrations as low as 1 unit/ml of gamma-IFN had significant effects on TNF-stimulated production of PGE and on caseinase activity. Resorption of human articular cartilage was stimulated by TNF alpha (10(-7) M) and was inhibited by gamma-IFN (1,000 units/ml). It is possible that cartilage breakdown in vivo may be modulated by such interactions between cytokines.     

Effects of recombinant human osteogenic protein 1 on the production of proteoglycan,prostaglandin E2, and interleukin-1 receptor antagonist by human articular chondrocytes cultured in the presence of interleukin-1β

Objective. Recombinant human osteogenic protein 1 (OP-1) is an effective stimulator of human cartilage ³⁵S-proteoglycan synthesis. The present study was conducted to determine whether stimulation of human articular chondrocytes with OP-1 can help overcome interleukin-1β (IL-1β)-induced suppression of ³⁵S-proteoglycan synthesis. Methods. Human articular chondrocytes in alginate beads were maintained for 3 days in the absence (control) or presence of IL-1β at 0.1–100 pg/ml with or without OP-1 at 50 ng/ml, in medium containing 10% fetal bovine serum (FBS). Incorporation of ³⁵S-sulfate into proteoglycans was quantified during the last 4 hours of culture and reported as counts per minute per μg DNA. Release of interleukin-1 receptor antagonist (IL-1Ra) and prostaglandin E₂ into the medium was monitored by immunoassay. Results. IL-1β at 10 pg/ml caused a 60% decrease in ³⁵S-proteoglycan synthesis. This could be blocked by including 500 ng/ml IL-1Ra in the medium. The presence of 50 ng/ml OP-1 in the IL-1β-containing medium was effective in restoring ³⁵S-proteoglycan synthesis to the level of that found in cultures not treated with IL-1β. The restorative effects of OP-1 and IL-1Ra were cumulative. The rate of release of prostaglandin E₂ and IL-1Ra into the medium was not affected by the presence of OP-1. Conclusion. Treatment of human articular chondrocytes with OP-1 cultured in the presence of FBS is effective in overcoming the down-regulation of proteoglycan synthesis induced by low doses of IL-1β.     

Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases by chondrocytes and synoviocytes in osteoarthritis

OBJECTIVES: To determine the response of glucosamine hydrochloride on chondrocytes and synoviocytes in terms of prostaglandin E2 (PGE2), nitric oxide (NO) and matrix metalloproteases (MMPs). METHODS: Chondrocytes and synoviocytes were prepared from joint specimens of patients who underwent total knee arthroplasty for osteoarthritis (OA). Chondrocytes from patients with femoral neck fracture were served as a normal control. Culture cells were stimulated by 5 ng/ml of IL-1beta and treated with various concentration of glucosamine hydrochloride (from 1 microg/ml to 500 microg/ml). PGE2, NO, MMP-1, MMP-3, and MMP-13 levels were evaluated in the culture supernatant. Further, the expression of COX-2 mRNA was studied by semiquantitative PCR. RESULTS: With IL-1beta stimulation, the levels of these mediators increased dramatically, except for NO from synoviocytes. After stimulation, levels of these mediators in OA chondrocytes were higher than synoviocytes and normal chondrocytes, and the level of MMP-3 was higher than those of MMP-1 and MMP-13. Glucosamine hydrochloride at a concentration of 100 microg/ml suppressed PGE2 production, and partly suppressed NO production. It also suppressed the production of MMPs from normal chondrocytes and synoviocytes but not from OA chondrocytes. CONCLUSION: Glucosamine modulates the metabolism of chondrocytes and synoviocytes and its mode of action differs between cells and conditions.     

Short-chain fatty acids act as antiinflammatory mediators by regulating prostaglandin E2 and cytokines

AIM: To investigate the effect of short-chain fatty acids(SCFAs) on production of prostaglandin E_2 (PGE_2),cytokines and chemokines in human monocytes.METHODS: Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative realtimepolymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE_2, cytokines and chemokines in the supernatant.The effect of SCFAs in vivo was examined by intraplantar injection into rat paws.RESULTS: Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE_2 and that this effect can be enhanced in the presence of lipopolysaccharide(LPS). In addition, we demonstrate that PGE_2 production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1(MCP-1) production and LPS-induced interleukin-10(IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE_2, MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-α and interferon-γ in human PBMC. Finally, we show that SCFAs and LPS can induce PGE_2 production in vivo by intraplantar injectioninto rat paws ( P < 0.01).CONCLUSION: SCFAs can have distinct antiinflammatory activities due to their regulation of PGE_2, cytokine and chemokine release from human immune cells.     

Effects of transforming growth factor beta on the production of prostaglandin E and caseinase activity of unstimulated and interleukin 1-stimulated human articular chondrocytes in culture

Transforming growth factor beta (TGF beta) has previously been shown to have actions on chondrocytes and cartilage both in vitro and in vivo which suggest a role in connective tissue repair. In particular, some of its actions have been shown to be antagonistic to those of interleukin 1 (IL-1). In this study, the effects of TGF beta on prostaglandin E (PGE) production and caseinase activity, in the presence and absence of IL-1, in human articular chondrocytes were investigated. TGF beta 1 and TGF beta 2 were shown to modulate IL-1 beta-stimulated PGE production and caseinase activity. Both TGF beta 1 and beta 2 inhibited IL-1 beta-stimulated PGE production in the absence of serum and augmented it in the presence of serum. TGF beta 1 and TGF beta 2 inhibited IL-1-stimulated caseinase activity with and without serum. In general, the TGF beta s had little or no effect on basal PGE or caseinase levels. TGF beta s may be important modulators of chondrocyte metabolism, their effects on PGE production may depend on cytokine interactions; furthermore, their effects on caseinase activity may help prevent cartilage breakdown.      

Effects of tumor necrosis factor alpha and beta on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

We examined the effects of recombinant human tumor necrosis factor (TNF) on human articular cartilage and chondrocytes in culture. Both TNF alpha and TNF beta stimulated cartilage matrix breakdown during prolonged culture and elevated the levels of plasminogen activator (PA) activity in both the supernatants and cell layers of cultured chondrocytes. Characterization of the PA activities by immunochemistry and by zymography following gel electrophoresis indicated that human chondrocytes produce both urokinase-type PA and tissue-type PA in response to TNF. The addition of both interleukin-1 and TNF alpha or TNF beta to chondrocyte cultures demonstrated a synergism between these cytokines in the generation of PA activity in the culture supernatants and cell layers. Our results suggest that both activated lymphocytes and monocytes may contribute to the cartilage destruction of inflammatory arthritis through their stimulation of chondrocytes with TNF beta and TNF alpha, respectively. Since PA is the only neutral proteinase reported to be elevated in TNF-stimulated chondrocyte cultures, it could have an important role in TNF-mediated cartilage destruction.     

Histamine, histamine receptors (H1 and H2), and histidine decarboxylase expression by chondrocytes of osteoarthritic cartilage: an immunohistochemical study

Degeneration and loss of articular cartilage are the characteristic features of osteoarthritis (OA), with the appearance of fibrillations, cell clusters, matrix depletion, and changes in matrix composition all apparent. Histamine has a recognised role in allergic and inflammatory reactions, and is reported to affect several aspects of chondrocyte behaviour. The immunohistochemical (IHC) studies reported here have demonstrated histamine (H), both H₁ and H₂ receptors, and the histamine-producing enzyme histidine decarboxylase (HDC) in a variable proportion of human articular chondrocytes in OA cartilage specimens. Such observations were especially evident within the degenerative, superficial zone, and more so in late-stage disease. By contrast, normal age-matched cartilage specimens showed relatively little immunopositive staining for histamine and HDC. These findings strongly suggest that histamine and H-receptor expression by HAC in OA cartilage is potentially an important contributor to the atypical, aberrant phenotype of OA chondrocytes.     

Inhibition of matrix metalloproteinases and inducible nitric oxide synthase by andrographolide in human osteoarthritic chondrocytes

Objective

The aim of this study was to investigate the effects of andrographolide on matrix metalloproteinases (MMP) 1, 3, and 13 and inducible nitric oxide synthase (iNOS) in human articular chondrocytes from osteoarthritic cartilage.

Methods

Passaged chondrocytes were pretreated with or without andrographolide for 2 h, followed by coincubation with interleukin-1 beta (IL-1β) 1 ng/ml for 24 h. Expression levels of MMP-1, 3, and 13, tissue inhibitor of metalloproteinase-1 (TIMP-1), and iNOS were evaluated using real-time-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Nitric oxide (NO) was analyzed using the Griess reaction assay. Involvement of nuclear factor kappa B (NF-κB) was assessed by Western blotting, transient transfection, and luciferase reporter assay.

Results

Andrographolide tested in these in vitro studies was found be an effective antiarthritic agent, as evidenced by potent inhibition of MMP-1, 3, and 13 and iNOS expression, as well as upregulation of TIMP-1 in IL-1β-stimulated human articular chondrocytes (p < 0.05). The mechanism of andrographolide’s inhibitory effects was mediated by attenuating the activation of NF-κB in human chondrocytes in the presence of IL-1β.

Conclusions

Andrographolide was a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes, suggesting that this natural compound may merit consideration as a therapeutic agent for treating and preventing osteoarthritis.     

Effects of growth factors and cytokines on proteoglycan and collagen synthesis by chondrocytes in guinea pigs with spontaneous osteoarthritis

We examined the effects of various growth factors and cytokines on proteoglycan (PG) and collagen synthesis by chondrocytes isolated from osteoarthritic and normal articular cartilage of Hartley strain guinea pigs. The guinea pig represents a useful animal model of spontaneous osteoarthritis (OA). Cartilage tissue samples were obtained from the knee joints of under-3-month-old guinea pigs (control group) as well as 5- to 8-month-old guinea pigs with OA changes (OA group). Chondrocytes were isolated enzymatically and maintained in suspension culture. Growth factor addition groups were then prepared from both the OA group and the control group, using the factors TGF-β, bFGF, and IGF-1 (1.25 ng/ml each). Cytokine addition groups were also prepared using IL-1α and IL-1β (10 ng/ml each). An addition group was also prepared for sodium hyaluronate (HA) (500 μg/ml). In each group, ³⁵S was added as a PG metabolic marker, ³H-proline was added as a collagen metabolic marker, and the groups were cultured. Next, ³⁵S and ³H-proline uptake was measured by a liquid scintillation counter. The results revealed that (1) both PG synthesis and collagen synthesis were promoted significantly more in OA chondrocytes than in normal chondrocytes; (2) with the addition of growth factors, PG and collagen synthesis was enhanced in OA chondrocytes; and (3) PG synthesis and collagen synthesis were inhibited in both normal and OA chondrocytes with the addition of IL-1α and -β. This result suggests that the repair function is activated more in OA chondrocytes than in normal chondrocytes, thereby promoting the synthesis of the cartilage matrix by chondrocytes. This synthesizing capability is enhanced and acts to effectively repair degenerative articular cartilage further through the addition of growth factors. Received September 20, 1999 / Accepted December 6, 1999     

Down regulation by iron of prostaglandin E2 production by human synovial fibroblasts

OBJECTIVE: To examine the effect of iron on the prostaglandin (PG) E2 production by human synovial fibroblasts in vitro. METHODS: Human synovial fibroblasts were isolated from synovial tissue of rheumatoid arthritis (RA) and osteoarthritis (OA) patients and cultured in medium. Synovial fibroblasts were stimulated by human recombinant interleukin (IL) 1 beta (0.1-10 ng/ml) with or without ferric citrate (Fe-citrate, 0.01-1 mM). The amount of PGE2 in the culture medium was measured by an enzyme linked immunosorbent assay. RESULTS: The production of PGE2 by the synovial fibroblasts was increased by stimulation with IL1 beta at all concentrations tested. Fe-citrate but not sodium citrate (Na-citrate) down regulated the production of PGE2 by the synovial fibroblasts, both with and without stimulation by IL1 beta. Fe-citrate inhibited the spontaneous PGE2 production by the cells in a dose dependent manner, and a maximum inhibition by Fe-citrate was observed at the concentration of 0.1 mM with IL1 beta stimulation. The down regulation by iron was reversed by the co-addition of desferrioxamine (100 micrograms/ml), an iron chelator. CONCLUSION: Iron down regulates the PGE2 production by synovial fibroblasts in vitro.     

Effects of tumor necrosis factor α and β on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

We examined the effects of recombinant human tumor necrosis factor (TNF) on human articular cartilage and chondrocytes in culture. Both TNF α and TNFβ stimulated cartilage matrix breakdown during prolonged culture and elevated the levels of plasminogen activator (PA) activity in both the supernatants and cell layers of cultured chondrocytes. Characterization of the PA activities by immunochemistry and by zymography following gel electrophoresis indicated that human chondrocytes produce both urokinase-type PA and tissuetype PA in response to TNF. The addition of both interleukin-1 and TNFα or TNFβ to chondrocyte cultures demonstrated a synergism between these cytokines in the generation of PA activity in the culture supernatants and cell layers. Our results suggest that both activated lymphocytes and monocytes may contribute to the cartilage destruction of inflammatory arthritis through their stimulation of chondrocytes with TNFβ and TNFα, respectively. Since PA is the only neutral proteinase reported to be elevated in TNF-stimulated chondrocyte cultures, it could have an important role in TNF-mediated cartilage destruction.