Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.     

Hydrocortisone inhibits prostaglandin production but not arachidonic acid release from cultured macrophages

We have investigated the action of hydrocortisosone on arachidonic acid mobilisation in cultures of mouse peritoneal macrophages, mouse L929 cells and the mouse macrophage-like cell line RAW264. Hydrocortisone inhibits both arachidonic acid release and prostaglandin production by L929 cells. However, prostaglandin production by macrophages or RAW264 cells is inhibited with a concomitant stimulation rather than inhibition of arachidonic acid release. These data suggest that hydrocortisone acts at the level of phospholipase activity in fibroblasts but at a later stage of prostanoid production in macrophages.     

The active metabolite of leflunomide,A77 1726, increases the production of IL-1 receptor antagonist in human synovial fibroblasts and articular chondrocytes

Leflunomide is an immunomodulatory agent used for the treatment of rheumatoid arthritis. In this study, we investigated the effect of A77 1726 – the active metabolite of leflunomide – on the production of IL-1 receptor antagonist (IL-1Ra) by human synovial fibroblasts and articular chondrocytes. Cells were incubated with A77 1726 alone or in combination with proinflammatory cytokines. IL-1Ra production was determined by ELISA. A77 1726 alone had no effect, but in the presence of IL-1β or tumour necrosis factor-α it markedly enhanced the secretion of IL-1Ra in synovial fibroblasts and chondrocytes. The effect of A77 1726 was greatest at 100 μmol/l. In synovial fibroblasts and de-differentiated chondrocytes, A77 1726 also increased IL-1β-induced IL-1Ra production in cell lysates. Freshly isolated chondrocytes contained no significant amounts of intracellular IL-1Ra. A77 1726 is a known inhibitor of pyrimidine synthesis and cyclo-oxygenase (COX)-2 activity. Addition of exogenous uridine did not significantly modify the effect of A77 1726 on IL-1Ra production, suggesting that it was not mediated by inhibition of pyrimidine synthesis. Indomethacin increased IL-1β-induced IL-1Ra secretion in synovial fibroblasts and de-differentiated chondrocytes, suggesting that inhibition of COX-2 may indeed enhance IL-1β-induced IL-1Ra production. However, the stimulatory effect of indomethacin was consistently less effective than that of A77 1726. A77 1726 increases IL-1Ra production by synovial fibroblasts and chondrocytes in the presence of proinflammatory cytokines, and thus it may possess chondroprotective effects. The effect of A77 1726 may be partially mediated by inhibition of COX-2, but other mechanisms likely concur to stimulate IL-1Ra production.     

Necrotic tumor cells oppositely affect nitric oxide production in tumor cell lines and macrophages

Nitric oxide (NO) is a highly reactive free radical with profound tumoricidal activity, produced by both macrophages and tumor cells. While it has been postulated that necrotic tumor cells can augment macrophage anti-tumor action, we investigated the effect of tumor cell necrosis on NO synthesis and viability of L929 fibrosarcoma and C6 astrocytoma cell lines. The presence of necrotic tumor cells dose-dependently reduced NO production in IFN-gamma stimulated L929 cells, and rescued them from NO-dependent autotoxicity. This effect was mediated through soluble products, since it was completely preserved after blocking the contact between the necrotic and live cells. On the other hand, apoptotic tumor cells were unable to suppress IFN-gamma-triggered NO release and subsequent decrease of cell respiration in L929 cultures. Similar results were obtained with C6 astrocytoma cell line. This down-regulation of NO synthesis in response to necrotic cell products was not specific for tumor cell lines, since necrotic tumor cells markedly suppressed NO production in cytokine-stimulated primary fibroblasts and astrocytes. In contrast, both murine and rat peritoneal macrophages readily increased their basal or IFN-gamma-induced NO production when incubated with necrotic tumor cells. Taken together, these results suggest that tumor cell necrosis might promote or restrict tumor growth through suppression or enhancement of NO synthesis in tumor cells and macrophages, respectively, with net effect presumably depending on the extent of macrophage infiltration.     

Effect of Platelet-activating Factor on in vitro and in vivo Interleukin-6 Production

The aim of the present study was to investigate the possible effect of platelet-activating factor (PAF), by comparison with interleukin-1beta and polyriboinositic/polyribocytidylic (poly I-C) acid, on IL-6 production by L 929 mouse fibroblasts. At concentrations above 1 muM PAF, the production of IL-6 by mouse fibroblasts was enhanced in a dose dependent fashion. At 5 muM PAF, the peak increase (60.1 +/- 19.4 U/ml) was similar to that induced by 50 mug/ml poly I-C (60.0 +/- 35.0 U/ml) and higher than the one evoked by 100 U/ml IL-1beta (3.8 +/- 1.8 U/ml). The increase of 11-6 activity induced by 5 muM PAF was maximal after a 22 h incubation period with L 929 cells. Lyso-PAF (5 muM) also increased IL-6 activity from fibroblasts to a similar extent compared with 5 muM PAF. In addition, the IL-6 activity induced by 5 muM PAF was still observed when the specific PAF antagonist, BN 52021 (10 muM), was added to the incubation medium of L 929 cells. The result suggests that the production of IL-6 by L 929 cells evoked by PAF in vitro is not receptor mediated. The in vivo effect of PAF on IL-6 production was also investigated in the rat. Two hours after intravenous injection of PAF (2 to 4 mug/kg), a dramatic increase of IL-6 activity in rat serum was observed, this effect being dose dependent. The increase of IL-6 induced by 3 mug/kg PAF was not observed when the animals were treated with the PAF antagonist, BN 52021 (1 to 60 mg/kg0. These results demonstrate that PAF modulates IL-6 production and that the in vivo effect is receptor mediated.     

Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target.

Inflammatory mouse peritoneal macrophages were activated by IFN-gamma in synergy with IL-2 or Lipid A to mediate TNF production for autocrine generation of cytotoxic nitric oxide (NO) to kill P815 or L1210 tumor targets. It was determined that for IL-2, but not Lipid A, to effectively trigger activation of IFN-gamma-primed macrophages, the tumor targets must be also present for interaction with effector macrophages to mediate the production of TNF and NO. IFN-gamma- and IL-2-activated macrophages from syngeneic DBA/2 and allogeneic C3H mice had identical MHC-unrestricted requirements for interaction with DBA/2 mouse-derived P815 and L1210 targets to mediate production of TNF and NO for tumor cytotoxicity. To further define the mechanistic requirements for macrophage-tumor target interaction, IFN-gamma- and IL-2-activated macrophages were separated from P815 targets in culture by a semipermeable membrane. Under these conditions, both TNF and NO were produced by the macrophage, which indicated that the requirement for tumor target-macrophage interaction may be due to a soluble factor produced by the target rather than to direct physical contact. This was confirmed by experiments in which 24-h cell-free culture fluids, derived from either P815 or L1210 tumor targets, substituted for the intact tumor cells in the stimulation of TNF mRNA synthesis and secretion with NO generation of TNF mRNA synthesis and secretion with NO generation by IFN-gamma- and IL-2-activated C3H or DBA/2 macrophages. The activity in 24-h culture fluids derived from P815 and L1210 tumor targets was tentatively designated as tumor-derived recognition factor(s) (TDRF) since it was produced constitutively by the tumor targets and synergized with IFN-gamma and IL-2 to induce macrophage production of TNF and NO for death of the same targets. A variety of nontransformed human and mouse fibroblasts, mouse spleen lymphocytes, and two adherent mouse fibrosarcomas did not produce detectable TDRF activity, whereas two mouse T lymphomas, EL4 and EL4.IL-2, produced TDRF activity similar to L1210 mouse leukemia and P815 mastocytoma. The C3H/MCA, a TDRF-nonproducing mouse fibrosarcoma, was susceptible to cytotoxicity mediated by macrophages activated by IFN-gamma and Lipid A, but not by IL-2 triggering. Exogenous TDRF derived from L1210 targets reconstituted the cytotoxic activity for C3H/MCA MCA targets mediated by IFN-gamma- and IL-2-activated macrophages accompanied by the production of TNF and cytotoxic NO.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitric oxide production in murine spleen cells: role of interferons and prostaglandin E(2) in the generation of cytotoxic activity

The production of nitric oxide (NO) was measured in cultures of spleen cells stimulated by lipopolysaccharide (LPS), IL-2 or LPS + IL-2. We observed that NO synthesis is increased by IFN-gamma but inhibited by IFN-alpha/beta. This is not the case when IL-2 is present in the cultures, since interferons play a minor role in the regulation of the NO production. When IL-2 and LPS were associated in the cultures, the IFN-alpha/beta role seems more important than that of IFN-gamma. PGE(2) inhibits NO production in LPS supplemented cultures but has a slight effect in the presence of IL-2 and no effect with IL-2 + LPS. 3-isoButyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, induces a decrease of IFN production. In the presence of H-7, an inhibitor of protein kinase C (PKC), NO production is reduced when the cultures are supplemented by LPS or IL-2 but not when IL-2 and LPS are both added. H-7 also reduced IFN production. In the presence of N(G)-monomethyl-L-arginine (N-MMA), an inhibitor of NO synthesis, IFN production was increased, with no change in the cytotoxic activity. Hence, interferons regulate NO production by mouse spleen cells and, in return, NO modulates the generation of IFN.     

Mechanism of interferon action. Kinetics of decay of the antiviral state and protein phosphorylation in mouse fibroblasts treated with natural and cloned interferons

The kinetics of decay of the antiviral state and protein phosphorylation induced with natural mouse interferon (IFN) and with cloned human IFN were examined in monolayer cultures of mouse Ll929 fibroblast cells. The antiviral state measured by single cycle virus yield reduction with either vesicular stomatitis virus or reovirus decayed significantly within 2 to 3 days following removal of IFN and by 5 to 8 days virus yields had returned to the level of untreated control cells. Trypsinization of IFN-treated cells did not detectably alter the rate of decay of the antiviral state; however, the decay occurred slightly more rapidly in actively growing as compared to stationary cell cultures. The decay of the IFN-induced protein kinase which catalyzes the phosphorylation of endogenous protein P1 and purified initiation factor eIF-2 alpha correlated with the decay of the antiviral state. The decay rates of the antiviral state and protein kinase observed in mouse L929 cells that had been treated with natural mouse IFN synthesized in Newcastle disease virus-induced L929 cells were comparable to the decay rates observed in L929 cells that had been treated with recombinant human IFN-alpha A/D synthesized in Escherichia coli. The induction and decay of the antiviral state and protein kinase following treatment with a single dose of IFN did not significantly affect the sensitivity of the cell population to a subsequent treatment with a single dose of IFN. However, continuous treatment of L929 cells with natural mouse IFN or recombinant human IFN prevented the decay of both the antiviral state and protein kinase but also ultimately lead to cell death. The results suggest that protein phosphorylation may play an important role in the mechanism of IFN action in mouse L929 fibroblasts.     

Effects of the active metabolite of leflunomide, A77 1726, on cytokine release and the MAPK signalling pathway in human rheumatoid arthritis synoviocytes

Inflammatory cytokines or soluble factors are essential in the pathogenesis of rheumatoid arthritis (RA). Leflunomide is an effective disease modifying antirheumatic drug (DMARD) in RA. The objective of the present study was to evaluate for the first time the effects of A77 1726 on cytokine (interleukin (IL)-8, IL-10, IL-11 secretion and tumor necrosis factor-alpha soluble receptor I (sTNFRI)) shedding in human RA fibroblast-like synoviocytes (FLS). At 100 microM, we observed an increase in IL-10 secretion, a decrease in IL-11 release and no effect on sTNFRI shedding and IL-8 secretion in IL-1beta-stimulated human RA FLS. Furthermore, at this dose, our results also confirmed that A77 1726 decreased IL-6 and prostaglandin E2 (PGE2) synthesis while it increased IL-1 receptor antagonist secretion (IL-1Ra). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate cytokine expression. At 100 microM, the effect of A77 1726 on IL-10 and IL-11 secretion seemed to be associated with the status of p38 MAPK activation. Our results confirmed the immunoregulatory action of leflunomide in the cytokine network involved in RA pathogenesis. It could shift the balance from cytokine mediated inflammation to cytokine directed inhibition of the inflammatory process.     

Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis

 Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1α (IL-1α) and interferon γ (IFNγ) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy. Received: 28 June 1998 / Accepted: 23 August 1996     

Interferon Regulatory Factor (IRF)-1 Is a Master Regulator of the Cross Talk between Macrophages and L929 Fibrosarcoma Cells for Nitric Oxide Dependent Tumoricidal Activity

Macrophage tumoricidal activity relies, mainly, on the release of Tumor Necrosis Factor alpha (TNFα) and/or on reactive oxygen or nitrogen intermediates. In the present work, we investigated the cytotoxic activity of resident peritoneal macrophages against L929 fibrosarcoma cell line in vitro and in vivo. Resident macrophages lysed L929 cells in a mechanism independent of TNFα and cell-to-cell contact. The cytotoxic activity was largely dependent on nitric oxide (NO) release since treatment with L-NAME (NOS inhibitor) inhibited L929 cells killing. Macrophages from mice with targeted deletion of inducible NO synthase (iNOS) together with L929 cells produced less NO and displayed lower, but still significant, tumoricidal activity. Notably, NO production and tumor lysis were abolished in co-cultures with macrophages deficient in Interferon Regulatory Factor, IRF-1. Importantly, the in vitro findings were reproduced in vivo as IRF-1 deficient animals inoculated i.p with L929 cells were extremely susceptible to tumor growth and their macrophages did not produce NO, while WT mice killed L929 tumor cells and their macrophages produced high levels of NO. Our results indicate that IRF-1 is a master regulator of bi-directional interaction between macrophages and tumor cells. Overall, IRF-1 was essential for NO production by co-cultures and macrophage tumoricidal activity in vitro as well as for the control of tumor growth in vivo.     

Involvement of prostaglandin-producing pathway in the cytotoxic action of tumor necrosis factor.

To elucidate the cytotoxic mechanism of tumor necrosis factor (TNF), we isolated TNF-resistant sublines of L929 cells. As compared with L929 cells, TNF-resistant cells retained similar number and affinity of TNF-binding sites, and showed a similar growth rate. TNF stimulated arachidonate release from L929 cells, while no stimulation was observed at all in TNF-resistant cells tested. The cytotoxic action of TNF on L929 cells was inhibited by indomethacin, suggesting that prostaglandin may be involved in the action. Therefore, TNF-stimulated prostaglandin production was examined in L929 and TNF-resistant sublines. The amount of PGE2 produced by L929 cells was increased more than 5-fold after the addition of TNF, whereas the amount of PGE2 did not change in the resistant sublines following addition of the factor. TNF-stimulated arachidonate release and PGE2 production were reversed by islet-activating protein (IAP)-treatment of L929 cells. These results suggest that arachidonate release and subsequent prostaglandin production are important for the cytotoxic action of TNF and that these processes are mediated by GTP-binding protein (G protein) that is coupled to the TNF-receptor.     

Lipid A-associated proteins from periodontopathogenic bacteria induce interleukin-6 production by human gingival fibroblasts and monocytes

Abstract The aim of this study was to determine whether lipid A-associated proteins (LAP) from two periodontopathogenic species of bacteria were able to stimulate interleukin-6 (IL-6) release from human gingival fibroblasts and myelomonocytic cells. LAP and lipopolysaccharide (LPS) were extracted from Porphyromonas gingivalis and Prevotella intermedia and added to cultures of human gingival fibroblasts and mono-mac-6 monocytic cells. Release of IL-6 into the culture supermatants was determined by ELISA. LAP and LPS from Por. gingivalis , but not from Prev. intermedia , stimulated IL-6 release from both cell types in a dose-dependent manner although LPS was less potent than LAP in inducing IL-6 release from the fibroblasts. IL-6 was detectable in cultures of both cell types following stimulation with LAP from Por. gingivalis at a concentration as low as 10 ng/ml. In response to LAP from Prev. intermedia , IL-6 was produced by mono-mac-6 cells but not by fibroblasts. Our results show that bacterial cell wall components other than LPS can induce IL-6 release from cells of the periodontium in vitro. The production of such potent immunomodulatory agents in vivo may contribute to the connective tissue breakdown characteristic of chronic periodontitis.     

Inhibition of apoptosis signal-regulating kinase 1 by nitric oxide through a thiol redox mechanism

Nitric oxide is an endogenous thiol-reactive molecule that modulates the functions of many regulatory proteins by a thiol-redox mechanism. NO has now been shown to inhibit the activation of apoptosis signal-regulating kinase 1 (ASK1) in murine fibrosarcoma L929 cells through such a mechanism. Exposure of L929 cells to interferon-gamma resulted in the endogenous production of NO and in inhibition of the activation of ASK1 by hydrogen peroxide. The interferon-gamma-induced inhibition of ASK1 activity was blocked by N(G)-nitro-l-arginine, an inhibitor of NO synthase. Furthermore, the NO donor S-nitro-N-acetyl-dl-penicillamine (SNAP) inhibited ASK1 activity in vitro, and this inhibition was reversed by thiol-reducing agents such as dithiothreitol and beta-mercaptoethanol. SNAP did not inhibit the kinase activities of MKK3, MKK6, or p38 in vitro. The inhibition of ASK1 by interferon-gamma was not changed by 1H- (1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one, an inhibitor of guanylyl cyclase nor was it mimicked by 8-bromo-cyclic GMP. Site-directed mutagenesis revealed that replacement of cysteine 869 of ASK1 by serine rendered this protein resistant to the inhibitory effects both of interferon-gamma in intact cells and of SNAP in vitro. Co-immunoprecipitation data showed that NO production inhibited a binding of ASK1, but not ASK1(C869S), to MKK3 or MKK6. Moreover, interferon-gamma induced the S-nitrosylation of endogenous ASK1 in L929 cells. Together, these results suggest that NO mediates the interferon-gamma-induced inhibition of ASK1 in L929 cells through a thiolredox mechanism.     

Endothelin-1 induces proliferation of human lung fibroblasts and IL-11 secretion through an ET(A) receptor-dependent activation of MAP kinases

Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ET(A) or ET(B) selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ET(A) antagonist BQ-123, but not by the specific ET(B) antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ET(A) receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation.     

Mode of action of monoclonal-nonspecific suppressor factor (MNSF) produced by murine hybridoma

Monoclonal-nonspecific suppressor factor (MNSF), a product of murine T cell hybridoma, suppresses antibody response to lipopolysaccharide. In an attempt to clarify the functional mechanisms in vitro, we investigated the mode of action of MNSF. This factor inhibited the antibody response by B cells (depleting T cells and M?), thereby indicating that the lymphokine acts directly on B cells, without interaction between B and T cells or M?. MNSF activity was absorbed by mitogen-stimulated T or B cells, but not by resting lymphocytes. Proliferative responses to T cell and B cell mitogens were inhibited dose dependently by the addition of MNSF. Kinetic studies showed that MNSF suppressed the antibody response, in all culture periods, thereby indicating that immunoglobulin secretion and proliferation were inhibited. The effect of growth factor on MNSF-mediated suppression was investigated to search for a possible suppression of MNSF action. Interleukin 2 (IL-2) remarkably inhibited MNSF activity, and the effect of IL-1 or IL-4 was less. IL-2 was most effective when added on the fourth day of culture. MNSF also inhibits division in the plasmacytoma line MOPC-31C or in thymoma EL4, but not in L929 fibroblasts. Tumor necrosis factor (TNF) inhibits cell division of various tumor cells and suppresses the pokeweed mitogen-induced antibody response, without cytotoxic action, as does MNSF. While MNSF and TNF have similar biochemical and physiochemical properties, the cross-reaction tests showed that both are antigenically discrete lymphokines. Although MNSF lacks TNF activity, the concomitant addition of both factors to L929 increases the cytotoxic action, a finding indicative of a synergistic effect.