Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.     

The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.     

Amnion cell biosynthesis of interleukin-8: regulation by inflammatory cytokines.

The cellular constituents of the placenta are important participants in the recruitment and trafficking of inflammatory cells within the placenta. In infection-induced labor, gestational tissues synthesize and release a variety of inflammatory cytokines whose effects include increased prostaglandin biosynthesis and the initiation of uterine contractions. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, has been recently described as being elevated in the amniotic fluid of mothers with chorioamnionitis. We investigated the biosynthesis of IL-8 by human amnion cells and its regulation by other inflammatory cytokines. Cultured amnion cells obtained from normal term placentae were found to produce IL-8 in response to pathophysiologic concentrations of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha). Treatment of amnion cells stimulated by IL-1 beta with cycloheximide resulted in increased IL-8 production, while incubation of IL-1 beta treated amnion cells with actinomycin D resulted in a concentration-dependent decrease in detectable amounts of IL-8. Northern blot analysis of cultured amnion cells stimulated with IL-1 beta demonstrated a rapid increase in IL-8 mRNA which peaked at 2-4 hr. These in vitro results suggest inflammation of gestational tissues in vivo may result in locally produced IL-8 and, in association with other inflammatory mediators, may be important in the pathophysiology of infection-induced labor.     

Gene therapy of autoimmune diseases with vectors encoding regulatory cytokines or inflammatory cytokine inhibitors

Gene therapy offers advantages for the immunotherapeutic delivery of cytokines or their inhibitors. After gene transfer, these mediators are produced at relatively constant, non-toxic levels and sometimes in a tissue-specific manner, obviating limitations of protein administration. Therapy with viral or nonviral vectors is effective in several animal models of autoimmunity including Type 1 diabetes mellitus (DM), experimental allergic encephalomyelitis (EAE), systemic lupus erythematosus (SLE), colitis, thyroiditis and various forms of arthritis. Genes encoding transforming growth factor beta, interleukin-4 (IL-4) and IL-10 are most frequently protective. Autoimmune/ inflammatory diseases are associated with excessive production of inflammatory cytokines such as IL-1, IL-12, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma). Vectors encoding inhibitors of these cytokines, such as IL-1 receptor antagonist, soluble IL-1 receptors, IL-12p40, soluble TNFalpha receptors or IFNgamma-receptor/IgG-Fc fusion proteins are protective in models of either arthritis, Type 1 DM, SLE or EAE. We use intramuscular injection of naked plasmid DNA for cytokine or anticytokine therapy. Muscle tissue is accessible, expression is usually more persistent than elsewhere, transfection efficiency can be increased by low-voltage in vivo electroporation, vector administration is simple and the method is inexpensive. Plasmids do not induce neutralizing immunity allowing repeated administration, and are suitable for the treatment of chronic immunological diseases.     

Hyaluronan differently modulates TLR-4 and the inflammatory response in mouse chondrocytes

Hyaluronic acid (HA) may exert different action depending on its degree of polymerization. Small HA fragments induce proinflammatory responses, while highly polymerized HA exerts a protective effect in inflammatory pathologies such as rheumatoid arthritis. In both cases the toll-like receptor 4 (TLR-4) seems to be involved in the modulation of the inflammation process. The aim of this study was to investigate the influence of short HA oligosaccharides (HA 4-mers) and high molecular weight HA (HMWHA) in the inflammatory response in normal mouse chondrocytes. Messenger RNA and related protein levels were measured for TLR-4, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interleukin-18 (IL-18) in cells with and without the addition of HA. NF-kB activation was also evaluated. 4-mer HA treatment produced a significant up-regulation of all parameters considered while HMWHA did not exert any activity in untreated cells although it was able to reduce the effects of 4- mers HA significantly. Specific TLR-4 small interference RNA (siRNA) was used to confirm TLR-4 as the target of HA action. This study suggests that HA may modulate proinflammatory cytokines via its different degree of polymerization and inflammatory action may be modulated as a result of the interaction between HA and TLR-4.     

Hyaluronan in part mediates IL-1beta-induced inflammation in mouse chondrocytes by up-regulating CD44 receptors

Interleukin-1beta (IL-1beta) elicits the expression of inflammatory mediators through a mechanism involving the CD44 receptor. Hyaluronan (HA) depolymerization also contributes to CD44 activation. This study investigated the potential of HA fragments, obtained by hyaluronidase (HYAL) treatment, as mediators of CD44 activation on IL-1beta-induced inflammation in mouse chondrocytes.mRNA and related protein levels were measured for CD44, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in chondrocytes, treated or untreated with IL-1beta, either with or without the addition of HYAL. The level of NF-kB activation was also assayed.CD44 mRNA expression was higher than controls in chondrocytes treated with IL-1beta. IL-1beta also induced NF-kB up-regulation and increased TNF-alpha, IL-6, MMP-13 and iNOS expression. Different effects resulted from HYAL treatment. Treatment of chondrocytes exposed to IL-1beta with HYAL synergistically increased the same parameters up-regulated by IL-1beta, while the same parameters were increased by HYAL in chondrocytes not exposed to IL-1beta but to a lesser extent. Specific CD44 blocking antibody and hyaluronan binding protein (HABP), which inhibit HA activity, were used to confirm CD44 to be the target of IL-1beta action through HA mediation. HA levels and molecular size further confirm the role of degraded HA.These findings suggest that IL-1beta exerts inflammatory activity via CD44 by the mediation of HA fragments derived from HA depolymerization.     

NF-kappa B mediates the stimulation of cytokine and chemokine expression by human articular chondrocytes in response to fibronectin fragments

Fibronectin fragments (FN-f) that bind to the alpha(5)beta(1) integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene beta (GRO-beta). Constitutive and FN-f-inducible expression of GRO-alpha and GRO-gamma were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1beta expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-kappaB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.     

Synergistic induction of osteopontin by aldosterone and inflammatory cytokines in mesangial cells

Hypertensive nephrosclerosis is characterized by activation of the renin-angiotensin-aldosterone system in combination with an inflammatory response characterized by an infiltration of T-cells and mononuclear cells, which release proinflammatory cytokines like IL-1beta/TNFalpha. In various models of experimental hypertensive disease the chemokine osteopontin (OPN) enhances further leukocyte infiltration. Therefore, we investigated the induction of OPN expression in renal mesangial cells (MCs) by aldosterone and the inflammatory cytokines IL-1beta/TNFalpha. Incubation with aldosterone resulted in a time- and concentration-dependent increase in OPN mRNA and protein. OPN mRNA expression followed a biphasic time course with an early increase between 4 and 8 h and the second phase starting at 14 h. The early phase was independent of protein synthesis, indicating a direct effect of aldosterone. Aldosterone-mediated induction of OPN was prevented by spironolactone, indicative of a receptor-mediated aldosterone effect. The mineralocorticoid receptor (MR) was identified in MCs by RT-PCR and immunoprecipitation, and shown to interact with a putative aldosterone-response element of the OPN promoter. The proinflammatory cytokines IL-1beta and TNFalpha only marginally affected OPN expression in MCs. However, coincubation of aldosterone and the cytokines synergistically increased OPN mRNA and protein levels. Since the synergistic effect on OPN mRNA was inhibited by diphenyleneiodonium, we assume an involvement of reactive oxygen species (ROS). We conclude that the chemokine OPN is a target gene of aldosterone in renal MCs, which is activated via the MR, and that proinflammatory cytokines enhance aldosterone-dependent OPN expression. In vivo, this may result in further leukocyte infiltration aggravating hypertensive nephrosclerosis.     

ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation

We have investigated gene and protein expression of ST2/ST2L in a murine alveolar macrophage (AM) cell line, MH-S, reacting to inflammatory stimuli in vitro and in the lung tissue of an acute lung injury model in vivo. We have also analyzed the effect of soluble ST2 protein on inflammatory response of MH-S cells. Lipopolysaccharide (LPS) and proinflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha induced ST2 mRNA expression in MH-S cells. In an acute lung injury model, protein and mRNA expression levels of ST2 increased to the maximal level at 24-72h after the LPS challenge. Furthermore, pretreatment with ST2 protein significantly reduced the protein production and gene expression of IL-1alpha, IL-6, and TNF-alpha in LPS-stimulated MH-S cells in vitro. These results suggest that increases in endogenous ST2 protein in AM, which is induced by inflammatory stimuli, such as LPS and proinflammatory cytokines, may modulate acute lung inflammation.     

Stimulation of human chondrocyte prostaglandin E2 production by recombinant human interleukin-1 and tumour necrosis factor

In this study we have examined the effects of recombinant cytokine preparations on the production of prostaglandin E2 (PGE2) by human articular chondrocytes in both chondrocyte monolayer and cartilage organ cultures. The cytokines chosen for this study included only those reported to be present in rheumatoid synovial fluids and which therefore could conceivably play a role in chondrocyte activation in inflammatory arthritis. Of the cytokines tested, interleukin-1 (IL-1; alpha and beta forms) consistently induced the highest levels of PGE2 production followed, to a lesser extent, by tumour necrosis factor (TNF; alpha and beta forms). The IL-1s were effective at concentrations 2-3 orders of magnitude less than the TNFs, with each cytokine demonstrating a dose-dependent increase in PGE2 synthesis for the two culture procedures. The increased PGE2 production by the chondrocytes exhibited a lag phase of 4-8 h following the addition of the IL-1 or TNF and was inhibited by actinomycin D and cycloheximide, indicating a requirement for de novo RNA and protein synthesis, respectively. Our results suggest that IL-1 may be the key cytokine involved in modulating chondrocyte PGE2 production in inflammatory arthritis; they further extend the list of human chondrocyte responses which are affected by both IL-1 and TNF.     

Adenosine A2A receptor activation and hyaluronan fragment inhibition reduce inflammation in mouse articular chondrocytes stimulated with interleukin-1β

Small hyaluronan (HA) fragments produced from native HA during inflammation contribute greatly to cell injury in many pathologies. HA oligosaccharides increase proinflammatory cytokine levels by activating both CD44 and toll-like receptor (TLR)-4. Stimulation of CD44 and TLR-4 then activates nuclear factor-κB, which induces the production of proinflammatory cytokines. The adenosine 2A receptor (A(2A)R) is also involved in several inflammation pathologies, and the nucleoside adenosine acts as a potent endogenous inhibitor of inflammation in various tissues by interacting with this receptor. The aim of this study was to investigate the effects of an HA-blocking peptide that inhibits the proinflammatory action of HA oligosaccharides produced during inflammation, together with a specific A(2A)R agonist in a model of normal mouse articular chondrocytes stimulated with interleukin (IL)-1β. IL-1β stimulation significantly increased mRNA expression and the related protein production of TLR-4, TLR-2, CD44 and A(2A)R in articular chondrocytes. The induced nuclear factor-κB activation was also associated with increased levels of inflammatory cytokines, including tumor necrosis factor-α and IL-6, and other inflammatory mediators, such as matrix metalloprotease-13 and inducible nitric oxide synthase. Treatment of chondrocytes with the HA-blocking peptide Pep-1 and/or a specific A(2A)R agonist (CGS-21680) significantly reduced all of the inflammatory parameters upregulated by IL-1β. These results suggest that the inflammatory response may be reduced either by blocking oligosaccharides from HA degradation or by A(2A)R stimulation.     

Reduction of inflammatory cytokines and prostaglandin E2 by IL-13 gene therapy in rheumatoid arthritis synovium

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. IL-13 is a cytokine that inhibits activated monocytes/macrophages from secreting a variety of proinflammatory molecules. The aim of this study was to examine whether gene therapy-delivered IL-13 could reduce the production of key proinflammatory mediators in RA synovial tissue (ST) explants. Adenoviral vectors encoding the genes for human IL-13 (AxCAIL-13) and bacterial beta-galactosidase were generated and examined for protein production. Vectors were used to infect RA ST explants and RA synovial fibroblasts, and conditioned medium (CM) was collected at various times for analysis by ELISA and competitive immunoassay. AxCAIL-13 decreased the production of RA ST explant proinflammatory IL-1beta by 85% after 24 h. Likewise, TNF-alpha levels were decreased by 82 and 75% whereas IL-8 levels were reduced 54 and 82% after 24 and 48 h, respectively, in RA ST explant CM. Monocyte chemotactic protein-1 concentrations were decreased by 88% after 72 h in RA ST explant CM. RA ST explant epithelial neutrophil-activating peptide-78 concentrations were decreased 85 and 94% whereas growth-related gene product-alpha levels were decreased by 77 and 85% at 24 and 48 h, respectively, by AxCAIL-13. Further, IL-13 significantly decreased PGE2 and macrophage inflammatory protein-1alpha production. These results demonstrate that increased expression of IL-13 via gene therapy may decrease RA-associated inflammation by reducing secretion of proinflammatory cytokines and PGE2.     

Functional genomic analysis in arthritis-affected cartilage: yin-yang regulation of inflammatory mediators by alpha 5 beta 1 and alpha V beta 3 integrins

Osteoarthritis-affected cartilage exhibits enhanced expression of fibronectin (FN) and osteopontin (OPN) mRNA in differential display and bioinformatics screen. Functional genomic analysis shows that the engagement of the integrin receptors alpha 5 beta 1 and alpha v beta 3 of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of alpha 5 beta 1 using activating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory mediators such as NO and PGE2 as well as the cytokines, IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by alpha 5 beta1 integrin ligation is mediated via induction and autocrine production of IL-1 beta, because type II soluble IL-1 decoy receptor inhibits their production. In contrast, alpha v beta 3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1 beta, NO, and PGE2 (triggered by alpha 5 beta 1, IL-1 beta, IL-18, or IL-1 beta, TNF-alpha, plus LPS) in a dominant negative fashion by osteoarthritis-affected cartilage and activated bovine chondrocytes. These data demonstrate a cross-talk in signaling mechanisms among integrins and show that integrin-mediated "outside in" and "inside out" signaling very likely influences cartilage homeostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis.     

Interleukin-1beta-induced expression of the urokinase-type plasminogen activator receptor and its co-localization with MMPs in human articular chondrocytes

The urokinase-type plasminogen activator receptor (uPAR) plays a critical role in cartilage degradation during osteoarthritis as it regulates pericellular proteolysis mediated by serine proteinases. Another important family of proteinases responsible for ECM destruction in arthritis are the matrix metalloproteinases (MMPs). MMPs are regulated by IL-1beta, a cytokine that plays a pivotal role in pathogenesis of osteoarthritis. This study was undertaken to address two questions: 1. Is uPAR-expression regulated by proinflammatory cytokines such as IL-1beta? 2. Does a functional co-localization exist between uPAR and MMPs? By immunohistochemical analysis we observed enhanced expression of uPAR on chondrocytes derived from osteoarthritic human cartilage compared to non-osteoarthritic controls. We found an IL-1beta-mediated expression of uPAR by immunoelectron microscopy. Western blot analysis demonstrated that IL-1beta-stimulated expression of uPAR on chondrocytes in vitro increased in a dose-dependent manner. Furthermore, we found a functional co-localization between uPAR and MMP-9 on IL-1beta-stimulated chondrocytes by means of a co-immunoprecipitation assay. Expression of uPAR in osteoarthritic cartilage but not in healthy cartilage suggests that uPAR plays a role in cartilage breakdown. We propose that uPAR-mediated effects e.g. pericellular proteolysis are one of other cytokine (IL-1beta)-mediated events that contribute to the pathogenesis of osteoarthritis. Furthermore, we found that MMPs and uPAR were part of the same cell surface complexes in chondrocytes. This finding underlines a functional interaction between MMPs and the serine proteinase system in the fine regulation of pericellular proteolysis. Interfering with uPAR signaling may present a novel target in arthritis therapy to prevent excessive proteolytic degradation.     

A dietary colorant crocin mitigates arthritis and associated secondary complications by modulating cartilage deteriorating enzymes,inflammatory mediators and antioxidant status

Articular cartilage degeneration and inflammation are the hallmark of progressive arthritis and is the leading cause of disability in 10–15% of middle aged individuals across the world. Cartilage and synovium are mainly degraded by either enzymatic or non-enzymatic ways. Matrix metalloproteinases (MMPs), hyaluronidases (HAases) and aggrecanases are the enzymatic mediators and inflammatory cytokines and reactive oxygen species being non-enzymatic mediators. In addition, MMPs and HAases generated end-products act as inflammation inducers via CD44 and TLR-4 receptors involved NF-κB pathway. Although several drugs have been used to treat arthritis, numerous reports describe the side effects of these drugs that may turn fatal. On this account several medicinal plants and their isolated molecules have been involved in modern medicine strategies to fight against arthritis. In view of this, the present study investigated the antiarthritic potentiality of Crocin, a dietary colorant carotenoid isolated from stigma of Crocus sativus. Crocin effectively neutralized the augmented serum levels of enzymatic (MMP-13, MMP-3 and MMP-9 and HAases) and non-enzymatic (TNF-α, IL-1β, NF-κB, IL-6, COX-2, PGE₂ and ROS) inflammatory mediators. Further, Crocin re-established the arthritis altered antioxidant status of the system (GSH, SOD, CAT and GST). It also protected the bone resorption by inhibiting the elevated levels of bone joint exoglycosidases, cathepsin-D and tartrate resistant acid phosphatases. Taken together, Crocin revitalized the arthritis induced cartilage and bone deterioration along with inflammation and oxidative damage that could be accredited to its antioxidant nature. Thus, Crocin could be an effective antiarthritic agent which can equally nullify the arthritis associated secondary complication.     

The inflammatory side of human chondrocytes unveiled by antibody microarrays

Although being largely used for pathobiological models of cartilage diseases such as osteoarthritis (OA), human chondrocytes are still enigmatic cells, in as much as a large part of their secretome is unknown. We took advantage of the recent development of antibody-based microarrays to study multiple protein expression by human chondrocytes obtained from one healthy and five osteoarthritic joints, in unstimulated conditions or after stimulation by the proinflammatory cytokines interleukin-1 (IL-1) or tumour necrosis factor (TNF). The secretion media of chondrocytes were incubated with array membranes consisting of 79 antibodies directed against cytokines, chemokines, and angiogenic or growth factors. Several proteins were identified as new secretion products of chondrocytes, including the growth or angiogenic factors EGF, thrombopoietin, GDNF, NT-3 and -4, and PlGF, the chemokines ENA-78, MCP-2, IP-10, MIP-3alpha, NAP-2, PARC, and the cytokines MIF, IL-12, and IL-16. Most of the newly identified chemokines were increased intensely after stimulation by IL-1 or TNF, as for other proteins of the array, including GRO proteins, GM-CSF, IL-6, IL-8, MIP-1beta, GCP-2, and osteoprotegerin. The up-regulation by cytokines suggested that these proteins may participate in the destruction of cartilage and/or in the initiation of chemotactic events within the joint during OA. In conclusion, the microarray approach enabled to unveil part of an as yet unexplored chondrocyte secretome. Our findings demonstrated that chondrocytes were equipped with a proinflammatory arsenal of proteins which may play an important part in the pathogenesis of OA and/or its drift towards an inflammatory, rheumatoid phenotype.     

Effects of a tetramethoxyhydroxyflavone p7f on the expression of inflammatory mediators in LPS-treated human synovial fibroblast and macrophage cells

The inhibitory effects of 5,6,3',5'-tetramethoxy 7,4'-hydroxyflavone (labeled as p7F) were elucidated on the productions of proinflammatory cytokines as well as inflammatory mediators in human synovial fibroblasts and macrophage cells. p7F inhibited IL-1beta or TNF-alpha induced expressions of inflammatory mediators (ICAM-1, COX-2, and iNOS). p7F also inhibited LPS-induced productions of nitric oxide and prostaglandin E2 in RAW 264.7 cells. In order to investigate whether p7F would inhibit IL-1 signaling, p7F was added to the D10S Th2 cell line (which is responsive to only IL-1beta and thus proliferates), revealing that p7F inhibited IL-1beta-induced proliferation of D10S Th2 cells in a doseresponse manner. A flow cytometric analysis revealed that p7F reduced the intracellular level of free radical oxygen species in RAW 264.7 cells treated with hydrogen peroxide. p7F inhibited IkappaB degradation and NF-kappaB activation in macrophage cells treated with LPS, supporting that p7F could inhibit signaling mediated via toll-like receptor. Taken together, p7F has inhibitory effects on LPS-induced productions of inflammatory mediators on human synovial fibroblasts and macrophage cells and thus has the potential to be an antiinflammatory agent for inhibiting inflammatory responses.