Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)-α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N^G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-α synthesis has been studied in vitro and in vivo. The synthesis of TNF-α in J774 macrophages stimulated with LPS (0.l μg/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 μM) and reduced by either L-arginine (3-30-300 μM) or the NO donor SIN-1 (1-10-1OOμM). The level of TNF-α in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L- arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-α synthesis suggesting an important regulatory link between NO and TNF-α in pathological processes.     

Suppression of Cytokine Production in T Helper Type 2 Cells by Nitric Oxide in Comparison with T Helper Type 1 Cells

We examined the effect of nitric oxide (NO) on cytokine production in T helper (Th) cell subsets, using murine splenic CD4⁺ T cells and two types of Th clones. Interferon-gamma-treated murine peritoneal exudate cells (IFN-PEC) suppressed DNA synthesis to 60% of the control level in CD4⁺ T cells stimulated with the anti-CD3 monoclonal antibody. The production of IL-2 and IL-4 in the CD4⁺ T cells decreased to 63.2% and 9.2%, respectively, of the control value by co-culture with IFN-PEC. The addition of N^G-monomethyl-L -arginine (L-NMMA) partially recovered the suppression of DNA synthesis. In the presence of indomethacin, the suppression of DNA synthesis was partially inhibited and the reduction in the cytokine production caused by IFN-PEC was partially recovered. The simultaneous addition of N^G-monomethyl-L -arginine (L-NMMA) and indomethacin completely inhibited not only the suppression of DNA synthesis but also the reduction in the cytokine production caused by IFN-PEC. Moreover, DNA synthesis in the Th2 clone was suppressed to a greater extent than that in the Th1 clone by co-culture with IFN-PEC. This suppression in the Th1 clone was inhibited by the addition of L-NMMA, whereas the DNA synthesis in the Th2 clone was not recovered by L-NMMA. In addition, sodium nitroprusside (SNP) suppressed IL-4 production in the Th2 clone but had no effect on IL-2 production in the Th1 clone. In the experiment of the co-culture with IFN-PEC, the inhibitory-effect of NO on T cell activation was not clarified by the influence of prostaglandins. However, in conclusion, cytokine production in Th2 cells may be more susceptible to NO than that in Th1 cells.     

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.     

(−)-Nyasol,isolated from Anemarrhena asphodeloides suppresses neuroinflammatory response through the inhibition of I-κBα degradation in LPS-stimulated BV-2 microglial cells

Microglial activation has been associated with neurodegenerative diseases by inducing the neuroinflammatory mediators such as nitric oxide (NO), TNF-α and IL-1β. (?)-Nyasol, a norlignan isolated from a medicinal plant Anemarrhena asphodeloides, showed anti-inflammatory potential in lipopolysaccharide (LPS)-activated BV-2 microglial cells. (?)-Nyasol inhibited the production of NO and prostaglandin E₂ (PGE₂) and also the expression of inducible nitric oxide synthase and cyclooxygenase-2, which are responsible for the respective production of NO and PGE₂. It also suppressed the mRNA levels of TNF-α and IL-1β in activated microglial cells. These effects of (?)-nyasol were correlated with the inactivation of p38 MAPK and the suppression of LPS-induced I-κBα degradation. Taken together, these results suggest that (?)-nyasol can be a modulator in neuroinflammatory conditions induced by microglial activation.     

IL-1α and TNFα act synergistically to stimulate production of myeloid colony-stimulating factors by cultured human bone marrow stromal cells and cloned stromal cell strains

Human bone marrow stromal cells repond to stimulation by the monokines IL-1 and TNF by producing colony-stimulating factors such as GM-CSF and G-CSF. In this study we show that IL-1α and TNFα act synergistically to stimulate GM-CSF and G-CSF production by cultured marrow stromal cells. We further show that IL-1α and TNFα synergistically stimulate production of GM-CSF and G-CSF by a clonal stroma-derived cell strain. Although IL-1 and TNF share many of the same biological activities, we show that IL-1α and TNFα have an unequal ability to induce myeloid-CSF production by both cultures, with IL-1α being the more potent inducer. We found that induction by IL-1α and TNFα was independent of cell proliferation. The effect of IL-1α and TNFα on production of the two myeloid-CSFs by the clonal cells was significantly greater than the unfractionated passaged stromal cultures, having the greater effect on G-CSF production. The clonally derived stromal cells constitutively produced colony-stimulating activity, in particular GM-CSF, at levels easily detected by ELISA. These findings show that, in addition to the overlapping and additive activities of IL-1α and TNFα, they can interact synergistically. Our findings further suggest that a small subpopulation of stroma cells may be the major producer of G-CSF in the marrow microenvironment during immune response. © 1994 wiley-Liss, Inc.     

Reciprocal induction of tumor necrosis factor-α and interleukin-β activity mediates fibronectin synthesis in coronary artery smooth muscle cells

We previously demonstrated an immune-inflammatory response associated with increased expression of interleukin (IL)-β and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL-β was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co-induction of IL-β and tumor necrosis factor (TNF)-α has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF-α expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [³⁵S]-methionine radiolabeling and gelatin-sepharose extraction. As previously shown with IL-β, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF-α antibodies, and exogenous TNF-α upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF-α-stimulated fibronectin production was downregulated to or below control levels in the presence of IL-β antibodies. Likewise, IL-β-stimulated fibronectin synthesis was downregulated to control levels when TNF-α neutralizing antibodies were added. Combining TNF-α and IL-β enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL-β and TNF-α activity and provide the first demonstration of a ‘cytokine loop’ modulating matrix production. © 1995 Wiley-Liss, Inc.     

Immunostimulatory Effect of Enzyme-Modified <Emphasis Type="Italic">Hizikia fusiforme</Emphasis>in a Mouse Model In Vitro and Ex Vivo

Hizikia fusiforme, a brown seaweed, has been utilized as a health food and in traditional medicine. In this study, we investigated whether enzyme-modified H. fusiforme extracts (EH) have immunological effects compared with normal H. fusiforme extracts (NH). The effects of NH and EH on immune responses were investigated by assessing nitric oxide (NO) production, phagocytosis, and cytokine secretion in RAW 264.7 murine macrophages and mice. Also, fucosterol was evaluated to find the active component of NH and EH by addressing cytotoxicity test and NO production. Both of NH and EH significantly increased cell viability and NO synthesis. Tumor necrosis factor-α (TNF-α) expression was more induced by EH with LPS treatment. Phagocytic activity, as the primary function of macrophages, was markedly induced by EH treatment. Additionally, EH encouraged splenocyte proliferation and recovered the levels of cytokines IL-1β, IL-6, and TNF-α in mice. Finally, fucosterol increased NO production with no cytotoxicity, which means that fucosterol is an active component of EH. In conclusion, EH has the potential to modulate immune function and could offer positive therapeutic effect for immune system diseases.     

Attenuated EAN in TNF-α deficient mice is associated with an altered balance of M1/M2 macrophages

The role of tumor necrosis factor (TNF)-α and its receptors in neuroautoimmune and neuroinflammatory diseases has been controversial. On the basis of our previous studies, we hereby aimed to further clarify TNF-α's mechanism of action and to explore the potential role of TNF-α receptor (TNFR)1 as a therapeutic target in experimental autoimmune neuritis (EAN). EAN was induced by immunization with P0 peptide 180-199 in TNF-α knockout (KO) mice and anti-TNFR1 antibodies were used to treat EAN. Particularly, the effects of TNF-α deficiency and TNFR1 blockade on macrophage functions were investigated. The onset of EAN in TNF-α KO mice was markedly later than that in wild type (WT) mice. From day 14 post immunization, the clinical signs of TNF-α KO mice were significantly milder than those of their WT counterparts. Further, we showed that the clinical severity of WT mice treated with anti-TNFR1 antibodies was less severe than that of the control WT mice receiving PBS. Nevertheless, no difference with regard to the clinical signs of EAN or inflammatory infiltration in cauda equina was seen between TNF-α KO and WT mice with EAN after blockade of TNFR1. Although TNF-α deficiency did not alter the proliferation of lymphocytes in response to either antigenic or mitogenic stimuli, it down-regulated the production of interleukin (IL)-12 and nitric oxide (NO), and enhanced the production of IL-10 in macrophages. Increased ratio of regulatory T cells (Tregs) and reduced production of interferon (IFN)-γ in cauda equina infiltrating cells, and elevated levels of IgG2b antibodies against P0 peptide 180-199 in sera were found in TNF-α KO mice with EAN. In conclusion, TNF-α deficiency attenuates EAN via altering the M1/M2 balance of macrophages.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Calreticulin transacetylase catalyzed modification of the TNF-α mediated pathway in the human peripheral blood mononuclear cells by polyphenolic acetates

Polyphenols, coumarin (1,2-benzopyrone) and chromone (1,4-benzopyrone), are naturally occurring constituent of variety of plant species. They have attracted immense interest because of their diverse pharmacological activities. Not much was known about biological activities of acetyl derivative (polyphenolic acetates) of parent polyphenols. In previous investigations, we have conclusively established calreticulin transacetylase catalyzed activation of endothelial nitric oxide synthase (eNOS) by polyphenolic acetates. In the present work, calreticulin transacetylase of human peripheral blood mononuclear cells was characterized with respect to specificity for various polyphenolic acetates and its role in the activation of TNF-α induced nitric oxide synthase (iNOS). Peripheral blood mononuclear cells incubated with a model polyphenolic acetate, 7,8-diacetoxy-4-methylcoumarin (DAMC), along with l-arginine caused activation of NOS. The incubation of peripheral blood mononuclear cells with TNF-α and DAMC resulted in increased production of NO as compared to TNF-α alone. This increased NO production was attenuated by l-Nω-nitro-l-arginine methyl ester (l-NAME), a well known non-specific inhibitor of NOS, and 1400W (N-[3-(aminomethyl) benzyl] acetamidine), a specific inhibitor of human iNOS. These results substantiate the CRTAase catalyzed activation of iNOS. Further, expression of NOS isoforms by semi-quantitative PCR and real-time RT-PCR confirms the preponderance of iNOS in TNF-α treated peripheral blood mononuclear cells over the untreated one. It was also observed that polyphenolic acetates inhibit TNF-α mediated release of IL-6 from peripheral blood mononuclear cells.     

The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-α and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-α and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-κB-dependent inflammatory responses by directly blocking the phosphorylation and degradation of IκBα and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-κB. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.     

Nitric oxide mediates interleukin-1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage

Interleek-1beta (IL-1) is a key mediator of cartilage matrix degradation in osteoarthritis and rheumatoid arthritis. It was found that the IL-1-induced suppression of glycosaminoglycan (GAG) synthesis in rat articular cartilage occurred simultaneously with the accumulation of nitrite (a metabolite of nitric oxide (NO) in aqueous milieu) in the culture medium. NO-synthase inhibitors, L-NMMA and L-NIO, inhibited both these IL-1 effects. Dexamethasone suppressed GAG synthesis additively to IL-1, but did not alter nitrite accumulation. Three NO-donors (GEA 3175, SNAP and SIN-1) also had an inhibitory effect on cartilage GAG synthesis. Therefore, it is concluded that IL-1 induced suppression of GAG synthesis in rat articular cartilage is mediated by the production of NO.     

Inhibition of LPS-induced TNF-α production by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

The purpose of this study was to examine whether rCGRP has effects on TNF-α produced by mouse resident peritoneal macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5 × 10⁵ cells per well and allowed to adhere for 2 hr. Pretreatment with rCGRP (10 nM − 1 μM) for 24 hr, the macrophages were cultured with LPS 1 μg/ml for another 24 h. The medium was harvested for measuring TNF-α by ELISA kits. The results showed that rCGRP had no direct effects on TNF-α production, but it inhibited LPS-induced TNF-α production in a concentration-dependent manner. When rCGRP was at a concentration of 1 μM, the LPS-induced TNF-α production was inhibited by 39%. The effect of rCGRP was reversed by hCGRP8-37 (10 μM), an antagonist of CGRP₁ receptor. The LPS-induced TNF-α production from macrophages was also inhibited by forskolin 3 μM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 μM or Rp-cAMPS 100 μM, the inhibitors of cAMP-dependent protein kinase, the effect of rCGRP was abolished. These data suggest that the LPS-induced TNF-α production is inhibited by rCGRP via activation of cAMP responses in mouse resident peritoneal macrophages.     

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

Phenelzine (Monoamine Oxidase Inhibitor) Increases Production of Nitric Oxide and Proinflammatory Cytokines via the NF-κB Pathway in Lipopolysaccharide-Activated Microglia Cells

Phenelzine is a potent monoamine oxidase inhibitor that is used in patients with depression. It is also well known that nitric oxide (NO) synthase inhibitors show preclinical antidepressant-like properties, which suggests that NO is involved in the pathogenesis of depression. The purpose of this study was to determine if phenelzine affects the production of NO and tumor necrosis factor-alpha (TNF-α) in activated microglia cells. BV-2 microglia cells and primary microglia cells were cultured in DMEM and DMEM/F12 and then cells were treated with LPS or LPS plus phenelzine for 24?h. The culture medium was collected for determination of NO, TNF-α, and IL-6 and cells were harvested by lysis buffer for Western blot analysis. Phenelzine increased the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS), as well as the release of TNF-α and IL-6 in BV-2 microglia cells. It is also confirmed that phenelzine increased the levels of NO, TNF-α and IL-6 in LPS-activated primary microglia cells. Phenelzine increased nuclear translocation of NF-κB by phosphorylation of IκB-α in LPS-activated microglia cells. These findings suggest that high doses of phenelzine could aggravate inflammatory responses in microglia cells that are mediated by NO and TNF-α.     

Chondrocyte response to growth factors is modulated by p38 mitogen-activated protein kinase inhibition

Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.Basal and growth factor-stimulated proliferation and proteoglycan synthesis were determined in primary cultures of rabbit articular chondrocytes, first-passage synovial fibroblasts, and cartilage organ cultures. Studies were performed with or without p38 MAPK inhibitors, in IL-1-activated and control cultures. Media nitric oxide and prostaglandin E2 were assayed.p38 MAPK inhibitors blunt chondrocyte and cartilage proteoglycan synthesis in response to transforming growth factor beta; responses to insulin-like growth factor 1 (IGF-1) and fetal calf serum (FCS) are unaffected. p38 MAPK inhibitors significantly reverse inhibition of cartilage organ culture proteoglycan synthesis by IL-1. p38 MAPK inhibition potentiated basal, IGF-1-stimulated and FCS-stimulated chondrocyte proliferation, and reversed IL-1 inhibition of IGF-1-stimulated and FCS-stimulated DNA synthesis. Decreases in nitric oxide but not prostaglandin E2 synthesis in IL-1-activated chondrocytes treated with p38 MAPK inhibitors are partly responsible for this restoration of response. Synovial fibroblast proliferation is minimally affected by p38 MAPK inhibition.p38 MAPK activity modulates chondrocyte proliferation under basal and IL-1-activated conditions. Inhibition of p38 MAPK enhances the ability of growth factors to overcome the inhibitory actions of IL-1 on proliferation, and thus could facilitate restoration and repair of diseased and damaged cartilage.