TNF-α Release In Macrophages Research Articles

A66 EFFECTS OF HYMENOLEPIS DIMINUTA EXTRACELLULAR VESICLES ON MOUSE MACROPHAGES AND THEIR MITOCHONDRIA

Abstract Background In recent years, extracellular vesicles (EVs) have emerged as important means of intercellular communication and immune modulation between helminth and host. Earlier work revealed that excretory/secretory products from the rat tapeworm Hymenolepis diminuta (HES) have anti-inflammatory properties when applied to macrophages. In this study, we have investigated the immunomodulatory properties of H. diminuta EVs (HEVs) on macrophages. Aims 1. To isolate and characterize bacterial contaminant-free H. diminuta EVs (Hd-EVs). 2. To determine the effects of Hd-EVs on murine macrophage metabolism and LPS-evoked cytokine production. Methods Adult H. diminuta were cultured for 6h in serum-free RMPI and Hd-EVs isolated from the conditioned medium using an exoEasy kit. Hd-EV protein content was measured using a micro-BCA kit, and size and quantity were measured using Nanosight tracking analysis. Murine bone marrow-derived macrophages or the human macrophage THP-1 cell line were treated with LPS (10 ng/ml) ± a crude H. diminuta extract (HdE), HES, or Hd-EVs for 24h. TNF-α levels in the culture medium was measured by ELISA. Macrophage oxidative phosphorylation (OX.PHOS) and glycolysis were measured in real-time using an Agilent Seahorse XF Analyzer. Results Analysis of 14 Hd-EV samples revealed 4.1 X 109 - 5.48 X 1010 particles/mL, with Hd-EVs predominantly 100-350 nm in diameter and with a protein content of 64.9-181.6 ng/mL. HdE and HES reduced TNF-α production in LPS-activated macrophages by 39-76% (n=11), while Hd-EVs did not lower TNF-α production. Metabolic analysis suggests that HES and Hd-EVs can up-regulate macrophage glycolysis, while only Hd-EVs exposed macrophages showed increased OX.PHOS (n=6). Conclusions We have successfully isolated and begun to characterize EVs from adult H. diminuta. Despite the known abilities of HES, we unexpectedly noted that exposure to Hd-EVs did not result in a statistically significant decrease in macrophage TNF-α release. However, the increase in OX.PHOS macrophages treated with Hd-EVs is something to examine further. The next steps are to determine the contents and immunomodulatory activity of the Hd-EVs, thus advancing knowledge of how helminths can manipulate their hosts. Funding Agencies NSERC Clinical Practice

Sphingomyelin 14:0 – the link between obesity and neuroinflammation

AbstractBackgroundOur laboratory has demonstrated that the consumption of a Western‐type diet (WD), triggers sterile inflammation and long‐term epigenetic reprogramming of myeloid cells, altering their immune response (Christ et al 2018). However, the precise molecular trigger mediating these changes was unknown. We have recently identified that during these WD conditions, there is an increase in circulating sphingomyelin 14:0 (S14), which was associated with atherosclerotic disease progression. As obesity, WD and atherosclerosis are all risk factors for Alzheimer’s disease (AD) and dementia, we set out to determine whether S14 was the signaling factor linking these conditions with AD development.MethodSerum samples were obtained from obese patients for lipidomic analysis. Mice were fed a high‐fat diet mimicking WD for a period of 8 weeks, before samples were obtained for lipidomic analysis. Microglia and macrophages were prepared from wildtype, TLR4−/−, Trif−/− and MyD88−/− mice to assess whether myeloid cells recognize S14, and the molecular pathways triggered by the lipid. Cells were prepared for RNA sequencing, to determine the genes and networks induced by S14. These genes and pathways were confirmed at a protein level by western blot and flow cytometry, and at a functional level using real‐time metabolic, and phagocytic assays. Results were confirmed in human myeloid cells.ResultLipidomic analysis confirmed that obese patients have increased circulating levels of S14, which were associated with inflammatory markers and cardiovascular disease. In vitro, we found using RNAseq that S14 triggers an extensive increase in pro‐inflammatory genes and pathways. We confirmed that S14 binds to TLR4/MD2 triggering NFkB activation and the release of TNFα in macrophages and microglial cells. S14 also reduced metabolic function and altered phagocytic capacity in microglia.ConclusionWe have found that S14, a lipid increased during obesity and WD conditions, is a novel TLR4 ligand that can activate macrophages and microglia triggering sterile inflammation. This pathway is conserved between murine and human cells. Critically, S14 reduced microglial phagocytosis, and rewired cellular metabolism. Together, our data shows for the first time that S14 is an immune stimulus, capable of triggering the innate immune responses and inflammation associated with dementia progression.

Extracellular CIRP induces macrophage endotoxin tolerance through IL-6R-mediated STAT3 activation.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern, whose effect on macrophages is not entirely elucidated. Here we identified that eCIRP promotes macrophage endotoxin tolerance. Septic mice had higher serum levels of eCIRP; this was associated with a reduced ex vivo immune response of their splenocytes to LPS. Pretreatment of macrophages with recombinant murine CIRP (rmCIRP) resulted in a tolerance to LPS stimulation as demonstrated by a reduction of TNF-α production. We found that eCIRP increased phosphorylated STAT3 (p-STAT3) in macrophages. A STAT3 inhibitor, Stattic, rescued macrophages from rmCIRP-induced tolerance by restoring the release of TNF-α in response to LPS stimulation. We discovered strong binding affinity between eCIRP and IL-6 receptor (IL-6R) as revealed by Biacore, fluorescence resonance energy transfer (FRET), and their colocalization in macrophages by immunostaining assays. Blockade of IL-6R with its neutralizing Ab inhibited eCIRP-induced p-STAT3 and restored LPS-stimulated TNF-α release in macrophages. Incubation of macrophages with rmCIRP skewed them toward an M2 phenotype, while treatment with anti–IL-6R Ab prevented rmCIRP-induced M2 polarization. Thus, we have demonstrated that eCIRP activates p-STAT3 via a novel receptor, IL-6R, to promote macrophage endotoxin tolerance. Targeting eCIRP appears to be a new therapeutic option to correct immune tolerance in sepsis.

CpG oligonucleotide and α-d-mannose conjugate for efficient delivery into macrophages

For improved intracellular uptake of CpG ODN into macrophages, we developed CpG ODN and mannose conjugate (CpG–Man conjugate) via simple conjugation of α-d-mannose to CpG ODN. CpG–Man conjugate showed greatly enhanced intracellular uptake by 2.1-fold into macrophages, compared to CpG ODN in a TLR-9 receptor-specific manner. In addition, internalized CpG–Man conjugate successfully triggered TNF-α release in macrophages. Taken together, the CpG–Man conjugate can serve as the promising delivery systems of CpG ODN into immune cells as a candidate adjuvant.

Anti-inflammatory and anti-biofilm properties of ent-nemorosone from Brazilian geopropolis

ent-Nemorosone is a benzophenone isolated from geopropolis from Melipona scutellaris whose biological properties remain unexplored. Herein, we evaluated the anti-inflammatory activity of ent-nemorosone by in vivo neutrophil migration and TNF-α quantification, as well as by in vitro TNF-α quantification, ERK 1/2 phosphorylation, NF-kB activation and nuclear translocation of p65 in stimulated macrophages. ent-Nemorosone was also tested for its antimicrobial effects against Streptococcus mutans biofilm adhesion, polysaccharide production, and viability. ent-Nemorosone reduced the influx of neutrophils and TNF-α release into the peritoneal cavity of mice, as well as the in vitro TNF-α levels, ERK 1/2 phosphorylation, NF-kB activation and nuclear p65 translocation. Furthermore, this compound inhibited biofilm adhesion and reduced the amount of extra- and intracellular polysaccharides synthesized by S. mutans. Thus, ent-nemorosone showed promising anti-inflammatory activity by acting on TNF-α release in macrophages, as well as anti-biofilm properties by reducing S. mutans polysaccharide production.

WISP1-αvβ3 integrin signaling positively regulates TLR-triggered inflammation response in sepsis induced lung injury.

We recently noted that the matricellular protein WISP1 contributes to sepsis induced acute lung injury (ALI) via integrin β6. In the current study, we pursued further aspects of WISP1 modulation of TLR signaling in lungs of mice after sepsis and TLR4 mediated release of TNF-α in macrophages. After confirming that TLR4 and CD14 are critical in transducing sepsis mediated ALI, we now demonstrate that intrapulmonary αvβ3 is increased by polymicrobrial sepsis in a TLR4, CD14 dependent fashion. Comparison of cultured macrophages revealed that WISP1 increased release of TNF-α from RAW264.7 cells with baseline expression of αvβ3, but primary cultures of peritoneal macrophages (PMø) required activation of TLR4 to induce de novo synthesis of αvβ3 enabling WISP1 to stimulate release of TNF-α. The specific requirement for β3 integrin was apparent when the effect of WISP1 was lost in PMø isolated from β3−/− mice. WISP1 enhanced TLR4 mediated ERK signaling and U0126 (an ERK inhibitor) blocked LPS induced β3 integrin expression and WISP1 enhanced TNF-α release. Collectively these data suggest that WISP1-αvβ3 integrin signaling is involved in TLR4 pathways in macrophages and may be an important contributor to TLR4/CD14 mediated inflammation in sepsis induced lung injury.

Novel HIV-1 miRNAs stimulate TNFα release in human macrophages via TLR8 signaling pathway.

PurposeTo determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation.MethodsUsing sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA.ResultsHIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis.ConclusionNovel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.

2-Mercaptoethane sulfonate prevents doxorubicin-induced plasma protein oxidation and TNF-α release: Implications for the reactive oxygen species-mediated mechanisms of chemobrain

Doxorubicin (DOX), an anthracycline used to treat a variety of cancers, is known to generate intracellular reactive oxygen species. Moreover, many patients who have undergone chemotherapy complain of cognitive dysfunction often lasting years after cessation of the chemotherapy. Previously, we reported that intraperitoneal administration of DOX led to elevated TNF-α and oxidative stress in the plasma and brain of mice. However, the mechanisms involved in nontargeted tissue damage remain unknown. In this study, we measured plasma oxidative stress and cytokine levels in patients treated with DOX. We observed increased plasma protein carbonylation and elevation of TNF-α 6h after DOX administration in the context of multiagent chemotherapy regimens. Importantly, patients not treated coincidentally with 2-mercaptoethane sulfonate (MESNA) showed statistically significantly increased plasma protein-bound 4-hydroxynonenal, whereas those who had been coincidentally treated with MESNA as part of their multiagent chemotherapy regimen did not, suggesting that concomitant administration of the antioxidant MESNA with DOX prevents intravascular oxidative stress. We demonstrate in a murine model that MESNA suppressed DOX-induced increased plasma oxidative stress indexed by protein carbonyls and protein-bound HNE, and also suppressed DOX-induced increased peripheral TNF-α levels. A direct interaction between DOX and MESNA was demonstrated by MESNA suppression of DOX-induced DCF fluorescence. Using redox proteomics, we identified apolipoprotein A1 (APOA1) in both patients and mice after DOX administration as having increased specific carbonyl levels. Macrophage stimulation studies showed that oxidized APOA1 increased TNF-α levels and augmented TNF-α release by lipopolysaccharide, effects that were prevented by MESNA. This study is the first to demonstrate that DOX oxidizes plasma APOA1, that oxidized APOA1 enhances macrophage TNF-α release and thus could contribute to potential subsequent TNF-α-mediated toxicity, and that MESNA interacts with DOX to block this mechanism and suggests that MESNA could reduce systemic side effects of DOX.

Inhibitory effect of (-)-epigallocatechin gallate on titanium particle-induced TNF-α release andin vivoosteolysis

Tumor necrosis factor-α (TNF-α) and inflammatory cytokines released from activated macrophages in response to particulate debris greatly impact periprosthetic bone loss and consequent implant failure. In the present study, we found that a major polyphenolic component of green tea, (-)-epigallocatechin gallate (EGCG), inhibited Ti particle-induced TNF-α release in macrophages in vitro and calvarial osteolysis in vivo. The Ti stimulation of macrophages released TNF-α in a dose- and time-dependent manner, and EGCG substantially suppressed Ti particle-induced TNF-α release. Analysis of signaling pathway showed that EGCG inhibited the Ti-induced c-Jun N-terminus kinase (JNK) activation and inhibitory κB (IκB) degradation, and consequently the Ti-induced transcriptional activation of AP-1 and NF-κB. In a mouse calvarial osteolysis model, EGCG inhibited Ti particle-induced osteolysis in vivo by suppressing TNF-a expression and osteoclast formation. Therefore, EGCG may be a potential candidate compound for osteolysis prevention and treatment as well as aseptic loosening after total replacement arthroplasty.

Influence of the A and B subunits of cholera toxin (CT) and Escherichia coli toxin (LT) on TNF-α release from macrophages

In this study an in vitro model was developed with the aim of investigating the modulatory effect of cholera toxin (CT) and its counterpart the heat labile toxin of Escherichia coli (LT) on TNF-α release induced by murine macrophages and primary human monocytes. Previous studies have demonstrated that the enzymatic activity of CT and LT molecules can inhibit TNF-α release by macrophages. The results obtained in this study showed that CT and LT are both, in a dose dependent manner, able either to induce or inhibit TNF-α release by murine macrophages and primary human monocytes. The results also showed that recombinant B subunits of CT and LT in the absence of their A subunit induce high levels of TNF-α release by macrophages and, in addition, increase the level of TNF-α release induced by LPS. The ability of both B subunits (CTB and LTB) in inducing TNF-α release by macrophages is not related to the level of LPS contamination, since direct measurements of LPS made in the samples employed in this study showed only traces of LPS (3.4 × 10 −8 EU/ml) which is in our system does not induce TNF-α release by macrophages. In contrast to the results obtained with the B subunits, incubation of cells with the A subunit of CT (CTA) inhibit TNF-α release induced by native CT, native LT, recombinant LTB and LPS. This inhibitory effect must be related to the activity of the A subunit since viability tests performed in terms of metabolic rate demonstrated that high concentrations of CTA are not toxic to the cells. The data presented herein demonstrate that the A subunits of CT and LT have an inhibitory effect on TNF-α release in macrophages, whereas their B subunits have a stimulatory effect on TNF-α. The results also suggest that the dose dependent bi-modal effect of native CT and native LT on TNF-α release by macrophages is a result of the combined effect of their individual A and B subunits.

Stimulated TNF-α release in macrophage and enhanced migration of dermal fibroblast by β-glucan

β-Glucan, a heterogeneous group of glucose polymers, modulates wound healing via macrophages, which are stimulated to release growth factors and cytokines, as well as via glucan receptors on fibroblasts, which are activated to migrate and synthesize collagen. The placement of β-glucan/collagen onto an arm of a young child with a scald burn improved healing of a partial-thickness burn wound. In this study, the effects of (1 → 3), (1 → 6)-β- d -glucan on the cytokine release in a macrophage cell line, RAW 264.7, were investigated. The effects of β-glucan on the proliferation and migration of adult human dermal fibroblasts (aHDFs) were also examined. When RAW 264.7 cells were treated with β-glucan, TNF-α secretion was stimulated in a dose-dependent manner, but IL-6 secretion was not. On the other hand, β-glucan treatment to aHDFs with 1 mg/ml resulted in a significant ( p < 0.05) increase not in cell proliferation, but in cell migration. These results suggest that β-glucan can stimulate TNF-α release in macrophages and enhance fibroblast migration, which may accelerate wound healing.

Requirements for Apoptotic Cell Contact in Regulation of Macrophage Responses

An important consequence of macrophage engulfment of apoptotic cells is suppression of inflammatory responses, which was first defined by assay of TNF-alpha release stimulated by LPS. These effects are apparently mediated in part by paracrine effects of TGF-beta released by the subset of stimulated macrophages that ingest apoptotic cells, which suppresses neighboring cells. However, the apoptotic cell-derived signal that stimulates TGF-beta release, and the nature of any additional signals required for the anti-inflammatory response remain poorly defined. In this study, we investigate the requirements for apoptotic cell engagement of macrophage surface receptors in these responses. We show that the apoptotic cell receptors CD36 and alphavbeta3 contribute to apoptotic cell phagocytosis by mouse macrophages, but are not essential for anti-inflammatory responses, suggesting that the mechanisms of response and phagocytosis are separate. In further defining requirements for response, we confirm the importance of TGF-beta in suppression by apoptotic cells, and identify an additional level of control of these effects. We show that LPS-stimulated mouse macrophage TNF-alpha release is only suppressed if macrophages have first contacted apoptotic cells, and hence, bystander macrophages are refractory to TGF-beta released by phagocytosing macrophages. We conclude that the profound suppression of LPS-driven TNF-alpha release by macrophage populations requires hitherto obscure contact-dependent licensing of macrophage responsiveness to TGF-beta by apoptotic cells.

Design of Potent and Selective 2-Aminobenzimidazole-Based p38α MAP Kinase Inhibitors with Excellent in Vivo Efficacy

We report the design and discovery of a 2-aminobenzimidazole-based series of potent and highly selective p38alphainhibitors. The lead compound 1 had low-nanomolar activity in both ATP competitive enzyme binding and inhibition of TNFalpha release in macrophages. Compound 18 showed excellent pharmacokinetics properties and oral activity in the rat collagen induced arthritis model compared with other p38 reference compounds. A SAR strategy to address CyP3A4 liability is also described.

Effect of pamidronate on the stimulation of macrophage TNF-α release by ultra-high-molecular-weight polyethylene particles: a role for apoptosis

The demonstration that one of the mechanisms of action of bisphosphonates (BPs) is the induction of osteoclast and macrophage apoptosis, suggests a potent therapeutic role for the BPs and other apoptosis-modulating agents in the management of periprosthetic osteolysis. The purpose of this study was to improve our understanding of the basic underlying molecular events leading to the inhibitory effect of pamidronate on the macrophage response to ultra-high-molecular-weight polyethylene (UHMWPE) particles. Murine J774 macrophages were incubated for 0–72 h in the presence of UHMWPE particles and/or pamidronate. TNF-α release was measured by ELISA while poly(ADP-ribose)polymerase (PARP) expression was measured by Western blot. DNA was analyzed on agarose. The appearance of PARP fragment and the fragmentation of DNA were used as markers of apoptosis. We observed a dose-dependent response to UHMWPE particles with TNF-α release reaching 4, 10, and 19 times control with 10, 25, and 125 particles/macrophage, respectively. UHMWPE particles (25 particles/macrophage) stimulate TNF-α release by a factor of 10, 7, and 6 after 24, 48, and 72 h, respectively, indicating a rapid stimulating effect of UHMWPE particles on TNF-α release. Our results also showed that at 10 particles/macrophage, pamidronate inhibits UHMWPE-induced TNF-α release by 12%, 14%, and 23% respectively after 24, 48, and 72 h ( p<0.05 vs. 24 and 48 h). With 25 particles/macrophage, the inhibition of TNF-α reached 9%, 12%, and 15% after 24, 48, and 72 h ( p<0.05 vs. 24 h), respectively. There is no significant difference between the inhibition by pamidronate of TNF-α release induced by 125 particles/macrophages at 24, 48, and 72 h. When cells are pre-incubated for 48 h with pamidronate prior to addition of UHMWPE particles for 24 h, we observed an increased inhibition of TNF-α compared to the co-incubation protocol. The inhibiting effect of pamidronate reaches 56% when pre-incubated with macrophages prior to incubation with 10 particles of UHMWPE/macrophage ( p<0.05 vs. co-incubation). Co-incubation of pamidronate with UHMWPE particles also led to the appearance of the proteolytic PARP fragment after 24 h incubation. We also demonstrated the stimulation of DNA fragmentation (DNA laddering) after 48–72 h with pamidronate. The proteolytic cleavage of PARP, an early event in the induction of apoptosis, precedes the inhibition of UHMWPE particle-induced TNF-α release by pamidronate whereas the fragmentation of DNA, a late apoptotic event, parallels this inhibition. Our results suggest the induction of macrophage apoptosis is associated with the inhibitory effect of pamidronate on TNF-α release. There is a need for the development of medical management of periprosthetic osteolysis. The demonstration that drugs such as pamidronate induce specific apoptosis-related pathways in macrophages contributes data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis.

Isoproterenol Inhibits Bacterial Lipopolysaccharide-Stimulated Release of Tumor Necrosis Factor-α from Human Heart Tissue

Recent evidence suggests that inflammatory cytokines, particularly tumor necrosis factor a (TNF-α), may play a role in heart disease. Elevated plasma levels of the cytokine have been reported in congestive heart failure and severe angina and after myocardial infarction. The exact role of TNF-α in heart disease and how production is stimulated and regulated in the heart are current areas of investigation. Regarding regulation of production, isoproterenol elevates cyclic AMP and inhibits TNF-α release in macrophages. Therefore we hypothesized that stimulation of β-adrenergic receptors of the sympathetic nervous system would inhibit release of the cytokine from heart tissue. With Institutional Review Board approval and patient consent atrial tissue was obtained during preparation for cardiac bypass. The tissue was divided into segments, placed in culture medium, and incubated for various times in the presence or absence of lipopolysaccharide (LPS) (20 μg/mL) and/or isoproterenol (1 μM). The medium was removed and analyzed for biologically active TNF-α by the L929 cell cytotoxicity assay. Tissue samples were weighed and TNF-α release was expressed as pg TNF-α/mg tissue. Initially, to determine the time course of release, measurements were made at 2, 5, 10, 15, 30, 60, 120, 180, and 360 minutes after the addition of LPS. Elevated TNF-α levels in the culture medium were reliably detected at 360 minutes after exposure to LPS. In atrial tissue obtained from seven patients TNF-α released into the culture medium at 360 minutes was 6 ± 3 pg/mg tissue. In the presence of LPS, levels of the cytokine in the culture medium increased to 604 ± 233 pg/mg tissue ( P &lt; 0.05 vs LPS alone). When isoproterenol and LPS were simultaneously added to the culture medium release of TNF-α was reduced by 87 per cent to 82 ± 40 pg/mg tissue ( P &lt; 0.05 vs LPS alone). Our results show that activation of the β-adrenergic receptor inhibits myocardial production of TNF-α. This finding suggests that the sympathetic nervous system inhibits production of the cytokine and that impaired sympathetic function in heart failure may play a role in the elevated levels of TNF-α.

Wood smoke particles generate free radicals and cause lipid peroxidation, DNA damage, NFκB activation and TNF-α release in macrophages

The present study investigated the generation of free radicals by wood smoke and cellular injuries caused by these radicals. Wood smoke was collected after thermolysis of western bark. Electron spin resonance (ESR) techniques were used to measure both carbon-centered radicals and generation of reactive oxygen species (ROS) by wood smoke. Wood smoke, in the presence of H 2O 2, was found to be able to generate hydroxyl radical ( OH). DNA strand breakage was measured by exposing wood smoke to λ Hind III fragments using gel electrophoresis. Wood smoke combined with H 2O 2 caused DNA damage. Sodium formate, an OH radical scavenger, or deferoxamine, a metal chelator, inhibited the DNA damage. Cellular DNA damage was also measured in cultured RAW 264.7 mouse macrophage cells by the single cell gel (SCG) electrophoresis assay. Cells were exposed to wood smoke samples for various times and significant DNA damage was observed. Elemental analysis was performed on the filter samples and the presence of Fe was noteworthy. Wood smoke is also able to cause lipid peroxidation, activate nuclear transcription factor, NFκB, and enhance the release of TNF-α from RAW 264.7 cells. The results indicate that the free radicals generated by wood smoke through the reaction of Fe with H 2O 2 are able to cause DNA and cellular damage and may act as a fibrogenic agent.

Vesnarinone is a selective inhibitor of macrophage TNFα release

Vesnarinone is an experimental drug that has been used successfully in the treatment of congestive heart failure patients. In this report we investigate the effect of vesnarinone on the cytokine secretory products of mononuclear phagocytes. In a concentration-dependent manner, the drug inhibits the endotoxin(LPS)stimulated release of tumor necrosis factor (TNF)α and suppresses interleukin(IL)-6 release, but does not affect the release of IL-lα, IL-10 and leukemia inhibitory factor (LIF) by mouse peritoneal macrophages. Using competitive polymerase chain reaction (PCR) analyses, we find that vesnarinone significantly reduces TNFα, but not IL-10 mRNA. In addition to LPS, the drug inhibits TNFα release induced by several other stimuli. The inhibitory effect of the drug on the TNFα biosynthesis can be observed in differentiated human monocytes, in macrophage cell lines, and in synovial adherent cells from rheumatoid arthritis patients. Although the precise mode of action of vesnarinone in the signal transduction pathway leading to the selective inhibition of TNFα is not known, the drug might be useful in the treatment of diseases involving that cytokine.