Monocyte Cell Cultures Research Articles

Stem-bark of Terminalia arjuna attenuates human monocytic (THP-1) and aortic endothelial cell activation

Ethnopharmacological relevanceTerminalia arjuna – stem bark extract is traditionally used as cardiotonic in Ayurvedic medicine. Aim of the studyThe present study was aimed to evaluate the molecular basis for cardioprotective potential of Terminalia arjuna (TA) stem bark, using cell cultures of human monocytic (THP-1) and human aortic endothelial cells (HAECs). Materials and methodsInhibitory effect of alcoholic (TAAE) and aqueous (TAWE) extracts of TA-stem bark was assessed on human 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lipoprotein lipase (LpL) and lipid peroxidation in rat (wistar) liver and heart homogenates. The patterns of H2O2 induced reactive oxygen species (ROS) generation were observed by confocal microscopy. The activities of antioxidant enzymes and reducing power of the cells were measured in a microplate reader. Gene transcripts of proinflammatory markers in THP-1 and HAECs were assayed by real time PCR and levels of inflammatory protein markers by ELISA or flow cytometry. Phytochemical analyses of TAAE and TAWE were done using liquid chromatography, coupled to mass spectrometry (LC-MS). ResultsTAAE and TAWE inhibited the lipid peroxidation and HMG-CoA reductase but had no effect on LpL. Both the extracts attenuated H2O2 mediated ROS generation in THP-1 cells by promoting catalase (CAT), glutathione peroxidase (GPx) activities, and by sustaining cellular reducing power. TAAE was highly effective in attenuating proinflammatory gene transcripts in THP-1 cells and HAECs, whereas the response to TAWE depended on the type of transcript and cell type. Both extracts decreased the levels of typical inflammatory marker proteins, viz. LPS induced tumor necrosis factor (TNF)-α secreted by THP-1 cells and TNF-α induced cell surface adhesion molecules on HAECs, namely vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Phytochemical analyses indicated the richness in phenolic compounds and terpenes of TAAE and TAWE, while revealing variability in their metabolite profile. ConclusionOur study scientifically validates the antioxidative and antiinflammatory properties of Terminalia arjuna stem bark. The marked effects on cultured human monocytic and aortic endothelial cells (HAEC) provide the biochemical and molecular basis for therapeutic potential of TA-stem bark against cardiovascular diseases (CVD).

643 Elevated Acetoacetate, Oxidative Stress and Mcp-1 Levels in Cord Blood of Infants of Diabetic Mothers

BackgroundInfants of diabetic mothers (IDM) are at increased risk for metabolic complications. Type 1 and some type 2 diabetic patients have elevated levels of ketone bodies acetoacetate (AA) and β-hydroxybutyrate...

Elevated Acetoacetate and Monocyte Chemotactic Protein-1 Levels in Cord Blood of Infants of Diabetic Mothers

Background: Infants of diabetic mothers (IDMs) are at increased risk for metabolic complications. Type 1 and some type 2 diabetic patients have elevated levels of the ketone bodies acetoacetate (AA) and β-hydroxybutyrate (BHB). Objective: The aim of this study was to examine how hyperketonemia in diabetic mothers affects markers of inflammation and oxidative stress in their offspring. Methods: Blood was obtained from 23 diabetic mothers and 13 healthy mothers and their infants’ umbilical cords at delivery. Interleukin-8, monocyte chemotactic protein-1 (MCP-1) and protein carbonyl (protein oxidation) levels were determined by ELISA. U937 human monocyte cell culture was used to examine the effect of AA and BHB on secretion of MCP-1. Results: There was a significant increase in the levels of AA in cord blood of IDMs compared with cord blood of infants of healthy mothers. A significant increase in the levels of protein oxidation (p < 0.05) and MCP-1 levels (p < 0.05) was observed in the cord blood of IDMs. The level of MCP-1 correlated significantly (r = 0.51, p = 0.01) with the concentration of AA in the IDMs. In further experiments with cultured monocytes treated with exogenous AA (0–4 mM), a significant increase in MCP-1 secretion was observed in AA- but not BHB-treated monocytes. Conclusion: Blood levels of AA and MCP-1 are elevated in IDMs, which may contribute to the development of the metabolic complications seen in IDMs.

Hyperlipidemia stimulates the extracellular release of the nuclear high mobility group box 1 protein

Our aim was to evaluate the effect of hyperlipidemia on the activation of endogenous alarmin, the high mobility group box 1 (HMGB1) protein, related to systemic inflammation associated with the progression of experimental atherosclerosis and to establish whether statin treatment regulates the HMGB1 signaling pathway. Hyperlipidemia was induced in vivo in golden Syrian hamsters and in monocyte cell culture (U937) by feeding the animals with a high-fat Western diet and by exposing the cells to hyperlipidemic serum. Blood samples, heart, lung and cells were harvested for biochemical, morphological, Western blot, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses. The data revealed that, in the atherosclerotic animal model, the protein HMGB1 and its gene expression were increased and that fluvastatin treatment significantly reduced the release of HMGB1 into the extracellular space. The cell culture experiments demonstrated the relocation of HMGB1 protein from the nucleus to cytoplasm under hyperlipidemic stress. The high level of detected HMGB1 correlated positively with the up-regulation of the advanced glycation end product receptors (RAGE) in the lung tissue from hyperlipidemic animals. During hyperlipidemic stress, the AKT signaling pathway could be activated by HMGB1-RAGE interaction. These results support the existence of a direct correlation between experimentally induced hyperlipidemia and the extracellular release of HMGB1 protein; this might be controlled by statin treatment. Moreover, the data suggest new potentials for statin therapy, with improved effects on patients with systemic inflammation induced by hyperlipidemia.

Angiotensin II induces NF-κB, JNK and p38 MAPK activation in monocytic cells and increases matrix metalloproteinase-9 expression in a PKC- andRho kinase-dependent manner

Angiotensin II (ANG II), the main effector of the renin-angiotensin system, is implicated in endothelial permeability, recruitment and activation of the immune cells, and also vascular remodeling through induction of inflammatory genes. Matrix metalloproteinases (MMPs) are considered to be important inflammatory factors. Elucidation of ANG II signaling pathways and of possible cross-talks between their components is essential for the development of efficient inhibitory medications. The current study investigates the inflammatory signaling pathways activated by ANG II in cultures of human monocytic U-937 cells, and the effects of specific pharmacological inhibitors of signaling intermediates on MMP-9 gene (MMP-9) expression and activity. MMP-9 expression was determined by real-time PCR and supernatants were analyzed for MMP-9 activity by ELISA and zymography methods. A multi-target ELISA kit was employed to evaluate IκB, NF-κB, JNK, p38, and STAT3 activation following treatments. Stimulation with ANG II (100 nM) significantly increased MMP-9 expression and activity, and also activated NF-κB, JNK, and p38 by 3.8-, 2.8- and 2.2-fold, respectively (P < 0.01). ANG II-induced MMP-9 expression was significantly reduced by 75 and 67%, respectively, by co-incubation of the cells with a selective inhibitor of protein kinase C (GF109203X, 5 µM) or of rho kinase (Y-27632, 15 µM), but not with inhibitors of phosphoinositide 3-kinase (wortmannin, 200 nM), tyrosine kinases (genistein, 100 µM) or of reactive oxygen species (α-tocopherol, 100 µM). Thus, protein kinase C and Rho kinase are important components of the inflammatory signaling pathways activated by ANG II to increase MMP-9 expression in monocytic cells. Both signaling molecules may constitute potential targets for effective management of inflammation.

Evaluation of inflammatory effects of airborne endotoxin emitted from composting sources

Because of the lack of effective methodology, the biological effects of environmental endotoxin have not been assessed. Here we have collected and measured airborne endotoxin at different locations around composting sites. Increased endotoxin concentrations were observed close to composting activities and also at nearby boundary areas. Analysis of proinflammatory effects of the environmental endotoxin on interleukin (IL)-8 and IL-6 release from human D562 pharyngeal epithelial and MM6 monocytic cell cultures showed an association between endotoxin level and cytokine induction. The cytokine-inducing effect of bioaerosol extracts was inhibited by polymyxin B, indicating that endotoxin was the cause of cytokine responses we found. The environmental endotoxin was also more active for stimulating cytokines in airway epithelial cells than commercially purified Escherichia coli endotoxin. Our results suggest that these in vitro inflammatory cell models may contribute to the assessment of health impacts of environmental endotoxin.

Hyperketonemia decreases mitochondrial membrane potential and its normalization with chromium (III) supplementation in monocytes

Altered cellular mitochondrial membrane potential (MMP) has been implicated in the increased insulin resistance and the risk for diabetes. Hyperketonemia is increasingly being identified in type 2 diabetic patients in addition to those with type 1 diabetes. No previous study has examined the effect of hyperketonemia and trivalent chromium on cellular mitochondrial membrane potential (MMP) in any cell type. Using a U937 monocyte cell culture model, this study examined the hypothesis that hyperketonemia decreases and trivalent chromium normalizes the cellular MMP level. Cells were cultured with control and ketone bodies [acetoacetate (AA), β-hydroxybutyrate (BHB)] in the absence or the presence (0.5-100 μM) of Cr(3+) at 37°C for 24 h. The MMP was determined using DiOC6 and flow cytometry. The results show a significant decrease in MMP in cells treated with AA, but not in the cells treated with BHB. The effect of AA on cellular MMP was prevented in chromium (III)-pretreated cells. Thus, hyperketonemia decreases the MMP, and supplementation with chromium (III) normalizes altered MMP, which may play a role in the improvement in glucose metabolism seen after chromium (III) supplementation in some studies with diabetic animals and patients.

Corrosion of phosphate‐enriched titanium oxide surface dental implants (TiUnite®) under in vitro inflammatory and hyperglycemic conditions

Endosseous dental implants use is increasing in patients with systemic conditions that compromise wound healing. Manufacturers recently have redesigned implants to ensure more reliable and faster osseointegration. One design strategy has been to create a porous phosphate-enriched titanium oxide (TiUnite) surface to increase surface area and enhance interactions with bone. In the current study, the corrosion properties of TiUnite implants were studied in cultures of monocytic cells and solutions simulating inflammatory and hyperglycemic conditions. Furthermore, to investigate whether placement into bone causes enough mechanical damage to alter implant corrosion properties, the enhanced surface implants as well as machined titanium implants were placed into human cadaver mandibular bone, the bone removed, and the corrosion properties measured. Implant corrosion behavior was characterized by open circuit potentials, linear polarization resistance, and electrical impedance spectroscopy. In selected samples, THP1 cells were activated with lipopolysaccharide prior to implant exposure to simulate an inflammatory environment. No significant differences in corrosion potentials were measured between the TiUnite implants and the machined titanium implants in previous studies. TiUnite implants exhibited lower corrosion rates in all simulated conditions than observed in PBS, and EIS measurements revealed two time constants which shifted with protein-containing electrolytes. In addition, the TiUnite implants displayed a significantly lower corrosion rate than the machined titanium implants after placement into bone. The current study suggests that the corrosion risk of the enhanced oxide implant is lower than its machined surface titanium implant counterpart under simulated conditions of inflammation, elevated dextrose concentrations, and after implantation into bone.

The generation of nanocolloidal wear from stainless steel and titanium alloy and its toxic effects in a monocyte cell culture model

AbstractThis study addresses the aspect of submicron‐sized / nanocolloidal wear comparing stainless steel (316L) and titanium alloy (TiAl6V4) for their toxic and inflammatory potentials. Wear was generated in a tribometer using the disc‐on‐pin‐method with pin and disc submerged in a sterile cell culture medium setting. The wear medium was separated according to Stokes' law into a fraction smaller 200 nm (nanocolloids) and a fraction greater 200 nm (particles). Vitality and inflammatory potential was measured in a cell culture model employing murine macrophages (J774). Cells were incubated with increasing concentrations (12.5, 25 and 50vol%) of either wear medium (particles and nanocolloids) from 316L and TiAl6V4. Vitality was measured by MTT assay and inflammatory reactions were quantified by TNF‐α ELISA.Nanocolloids from stainless steel and titanium induced strong, dose dependant toxic effects in the MTT assay while particles did not affect vitality in a dose dependant manner. The inflammatory response remained unaltered in all four groups. We conclude that interactions between soluble metallic wear and proteins forming nanocolloidal wear should be considered when conduction experiments addressing the aspect of biocompatibility in metallic implant materials.

Differential monocyte responses to TLR ligands in children with autism spectrum disorders

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1β, IL-6, IL-8, TNFα, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1β, IL-6, and TNFα responses following TLR 2, and IL-1β response following TLR 4 stimulation in monocyte cultures from children with ASD ( p < 0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1β, IL-6, GM-CSF, and TNFα responses in monocyte cell cultures from children with ASD compared with controls ( p < 0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.

An Atypical NF-κB-Regulated Pathway Mediates Phorbol Ester-Dependent Heme Oxygenase-1 Gene Activation in Monocytes

Heme oxygenase (HO)-1 catalyzes the rate-limiting step of heme degradation and plays an important anti-inflammatory role via its enzymatic products carbon monoxide and biliverdin. In this study it is reported that the HO-1 gene is transcriptionally induced by the phorbol ester PMA in cell cultures of monocytic cells with a regulatory pattern that is different from that of LPS-dependent HO-1 induction in these cells. Activation of HO-1 by PMA was mediated via a newly identified kappaB element of the proximal rat HO-1 gene promoter region (-284 to -275). This HO-kappaB element was a nuclear target for the NF-kappaB subunit p65/RelA as determined by nuclear binding assays and transfection experiments with luciferase reporter gene constructs in RAW264.7 monocytes. Moreover, PMA-dependent induction of endogenous HO-1 gene expression and promoter activity was abrogated in embryonic fibroblasts from p65(-/-) mice. PMA-dependent HO-1 gene activation was reduced by an overexpressed dominant negative mutant of IkappaBalpha, but not by dominant negative IkappaB kinase-2, suggesting that the classical NF-kappaB pathway was not involved in this regulation. The antioxidant N-acetylcysteine and inhibitors of p38 MAPK or serine/threonine kinase CK2 blocked PMA-dependent HO-1 gene activation. Finally, it is demonstrated by luciferase assays with a Gal4-CHOP fusion protein that the activation of p38 MAPK by PMA was independent of CK2. Taken together, induction of HO-1 gene expression by PMA is regulated via an IkappaB kinase-independent, atypical NF-kappaB pathway that is mediated via the activation of p38 MAPK and CK2.

Corrosion of machined titanium dental implants under inflammatory conditions

The effects of hyperglycemia, altered cell function, or inflammatory mediators on implant corrosion are not well studied; yet, these effects are critical to implant biocompatibility and osseointegration. Because implant placement is burgeoning, patients with medically compromising systemic conditions such as diabetes are increasingly receiving implants, and the role of other inflammatory diseases on implant corrosion also needs investigation. In the current study, the corrosion properties of commercially available, machined titanium implants were studied in blood, cultures of monocytic cells, and solutions containing elevated dextrose concentrations. Implant corrosion was estimated by open circuit potentials, linear polarization resistance, and electrical impedance spectroscopy (EIS) for 26 h. In selected samples, THP1 monocytic cells were activated for 2 h with Lipopolysaccharide prior to implant exposure, and IL-1beta secretion was measured to assess the affect of the implants on monocyte activation. Implants under conditions of inflammatory stress exhibited more negative E(corr) values, suggesting an increased potential for corrosion. Linear polarization measurements detected increased corrosion rates in the presence of elevated dextrose conditions over PBS conditions. EIS measurements suggested that implants underwent surface passivation reactions that may have limited corrosion over the short term of this test. This result was supported by cyclic polarization tests. IL-1beta secretion was not altered under conditions of corrosion or implant exposure. The results suggest that inflammatory stress and hyperglycemia may increase the corrosion of dental endosseous titanium-based implants, but that longer, more aggressive electrochemical conditions may be necessary to fully assess these effects.

Mycobacterial infection induces the secretion of high-mobility group box 1 protein

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that acts as a pro-inflammatory cytokine and is released by monocytes and macrophages. Necrotic cells also release HMGB1 at the site of tissue damage which induces a variety of cellular responses, including the expression of pro-inflammatory mediators. This study investigated the secretion of HMGB1 in mycobacterial infection by macrophages in vitro and in the lungs of infected guinea pigs. We observed that infection by mycobacterium effectively induced HMGB1 release in both macrophage and monocytic cell cultures. Culture filtrate proteins from Mycobacterium tuberculosis induced maximum release of HMGB1 compared with different subcellular fractions of mycobacterium. We demonstrated that HMGB1 is released in lungs during infection of M. tuberculosis in guinea pigs and increased HMGB1 secretion in lungs of guinea pigs was delayed by prior vaccination with Mycobacterium bovis BCG. The secretion of cytokines like tumour necrosis factor alpha (TNF-alpha) and Interleukin-1beta was significantly increased when M. bovis BCG-infected cultures of J774A.1 cells were incubated with HMGB1. Among different mycobacterial toll-like receptor ligands, heat-shock protein 65 (HSP65) was found to be more potent in inducing HMGB1 secretion in RAW 264.7 cells. Pharmacological suppression of p38 or extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases with specific inhibitors failed to inhibit HSP65-induced HMGB1 release, but inhibition of c-Jun NH(2)-terminal kinase activation attenuated HMGB1 release. Inhibition of the inducible NO synthase and neutralizing antibodies against TNF-alpha also reduced HMGB1 release stimulated by HSP65. We conclude that HMGB1 is secreted by macrophages during tuberculosis and it may act as a signal of tissue or cellular injury and enhances immune response.

Therapeutic dendritic cell vaccine preparation using tumor RNA transfection: a promising approach for the treatment of prostate cancer.

BackgroundEarly prostate adenocarcinoma can be diagnosed through seric prostate-specific antigen (PSA) screenings. However, a fraction of patients progress to an incurable metastatic disease. Therefore, novel therapies for treating these patients are extremely desirable. Therapeutic vaccines based on Dendritic Cells (DCs) carrying tumor antigens have emerged as a promising strategy to initiate an immune response against tumor cells. These vaccines can be prepared using different methodologies, such as the application of tumor mRNA described in this work.MethodsMature and immature DCs were obtained in vitro by adding specific cytokines to monocyte cell cultures. RNA extracted from prostate tumor lineage (LNCAP) was introduced into these cells by electroporation and co-incubation. Transfection success was measured by immunocytochemistry of the PSA expression level in DCs.ResultsCell surface markers, including CD14, CD80, CD86, CCR7, CD11c, and CD1a, confirmed mature and immature DC phenotypes. Both cell maturation stages were successfully used for RNA introduction as shown by PSA characterization.ConclusionOur data support the use of mature and immature DCs for vaccine preparation with either RNA electroporation or RNA co-incubation. The highest efficiency, however, was observed when RNA was delivered by electroporation into mature DCs. Due to in vitro RNA transcription, this method allows small tumors to be used for DC vaccine preparation; it is therefore a promising approach for the treatment of metastatic prostate cancer.

High Glucose and Ketosis (Acetoacetate) Increases, and Chromium Niacinate Decreases, IL-6, IL-8, and MCP-1 Secretion and Oxidative Stress in U937 Monocytes

Elevated blood levels of the proinflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and MCP-1 (monocyte chemoattractant protein-1) increase insulin resistance and the risk of cardiovascular disease (CVD). There is no previous study that has examined the effect of ketosis and trivalent chromium on IL-6, IL-8, or MCP-1 secretion in any cell type or in human or animal model. The authors examined the hypothesis that ketosis increases and trivalent chromium decreases the levels of cytokines and oxidative stress in diabetes using a U937 monocyte cell culture model. Cells were cultured with control, high glucose (HG), and acetoacetate (AA) in the absence or presence (0.5-10 microM) of CrCl(3), chromium picolinate (Cr-P), or chromium niacinate (Cr-N) at 37 degrees C for 24 h. The data show a significant stimulation of IL-6, IL-8, and MCP-1 secretion and an increase in oxidative stress in cells treated with HG or AA. The effect of HG on cytokine secretion was reduced by Cr-N, and to a lesser extent by CrCl(3) and Cr-P. The effect of HG on oxidative stress was reduced by Cr-N and CrCl 3, but not by Cr-P. Similarly, Cr-N decreased the cytokine secretion in HG + AA-treated cells. Cr-N significantly decreased standard oxidant (H(2)O(2)) induced cytokine secretion, which suggests that reduction of cytokine secretion by Cr-N is in part mediated by its antioxidative effect. In a cell culture model, Cr-N appears to be the most effective form of chromium in inhibiting oxidative stress and proinflammatory cytokine secretion by monocytes. This study suggests that chromium niacinate supplementation may be useful in reducing vascular inflammation and the risk of CVD in diabetes.

Binding of Caspase-3 Prodomain to Heat Shock Protein 27 Regulates Monocyte Apoptosis by Inhibiting Caspase-3 Proteolytic Activation

Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.

Mycobacterial Infection induces the secretion of High mobility group box 1 (HMGB1) protein (43.20)

Abstract High mobility group box protein 1 (HMGB1) is a nonhistone nuclear protein that acts as a pro-inflammatory cytokine and is released by monocytes and macrophages. Necrotic cells also secrete HMGB1 at the site of tissue damage that induces a variety of cellularresponses, including expression of proinflammatory mediatorssuch as TNF- , IL-1, and NO. This study investigated the secretion of HMGB1 during mycobacterial infection of macrophages in vitro and in the lungs of infected guinea pigs. We observed that infection with mycobacterium effectively induced HMGB1 release in both macrophage and monocytic cell cultures. Culture filtrate proteins from Mycobacterium tuberculosis (including Ag85A, Ag85B, ESAT-6 and CFP-10) induced maximum release of HMGB1 among different cellular components of mycobacterium. We demonstrated that HMGB1 is released by lung cells during infection of M. tuberculosis in guinea pig and increased HMGB1 secretion in lungs of guinea pigs was delayed by vaccination with BCG. The secretion of cytokines like TNF-α and IL-1β were significantly increased when J774A.1 cell cultures were incubated with HMGB1 during infection with M. bovis BCG. We conclude that HMGB1 is secreted by macrophages during tuberculosis and it may act as a signal of tissue or cellular injury and may act as a precursor to enhance the immune response. Funding for this research was provided by NIH AI40091 (A.A.I.).

Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1

High mobility group box 1 (HMGB1) can be actively secreted by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli (such as bacterial endotoxin, TNF-alpha, IL-1, and IFN-gamma) or passively released by necrotic cells and mediates innate and adaptive inflammatory responses to infection and injury. Here, we demonstrated that a reactive oxygen species, hydrogen peroxide (H(2)O(2)), induces active and passive HMGB1 release from macrophage and monocyte cultures in a time- and dose-dependent manner. At nontoxic doses (e.g., 0.0125-0.125 mM), H(2)O(2) induced HMGB1 cytoplasmic translocation and active release within 3-24 h. At higher concentrations (e.g., 0.25 mM), however, H(2)O(2) exhibited cytotoxicity to macrophage and monocyte cell cultures and consequently, triggered active and passive HMGB1 release. In addition, H(2)O(2) stimulated potential interaction of HMGB1 with a nuclear export factor, chromosome region maintenance (CRM1), in macrophage/monocyte cultures. Inhibitors specific for the JNK (SP600125) and MEK (PD98059), but not p38 MAPK (SB203580), abrogated H(2)O(2)-induced, active HMGB1 release. Together, these data establish an important role for oxidative stress in inducing active HMGB1 release, potentially through a MAPK- and CRM1-dependent mechanism.

Ethyl pyruvate exerts combined anti-inflammatory and anticoagulant effects on human monocytic cells

Sepsis is characterized by a concurrent activation of inflammation and coagulation. Recently, recombinant human activated protein C was shown to decrease mortality in patients with severe sepsis presumably due to a combined anti-inflammatory and anticoagulant effect. These promising findings led to a search for other products that influence both the inflammatory and the procoagulant response to severe infection. Ethyl pyruvate (EP) was recently identified as an experimental anti-inflammatory agent during endotoxemia and sepsis. The aim of the present study was to investigate whether EP influences coagulation besides its anti-inflammatory effects. For this we investigated the effects of EP on the expression and function of tissue factor (TF), the principal initiator of coagulation activation in sepsis, in human monocytic (THP-1) cell cultures. EP dose-dependently inhibited the production of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by lipopolysaccharide (LPS)-stimulated THP-1 cells at mRNA and protein level, thereby confirming its anti-inflammatory properties in this in-vitro system. In addition, EP dose-dependently attenuated the increases in TF mRNA levels, TF-protein-surface expression and cell-surface-associated TF activity in LPS-stimulated THP-1 cells. These results demonstrate for the first time that EP is a compound with combined anti-inflammatory and anticoagulant effects.

Au(III), Pd(II), Ni(II), and Hg(II) alter NFκB signaling in THP1 monocytic cells

The transcription factor NFkappaB plays a key role in the tissue inflammatory response. Metal ions released into tissues from biomaterials (e.g., Au, Pd, Ni, Hg) are known to alter the binding of NFkappaB proteins to DNA, thereby modulating the effect of NFkappaB on gene activation and, ultimately, the tissue response to biomaterials. Little is known about the effect of these metals on key signaling steps prior to NFkappaB-DNA binding such as transcription factor activation or nuclear translocation, yet these steps are equally important to modulation of the pathway. Oxidative stress is known to alter NFkappaB proteins and is suspected to play a role in metal-induced NFkappaB signaling modulation. Our aim in the current study was to assess the effects of sublethal levels of Ni, Hg, Pd, and Au ions on NFkappaB activation and nuclear translocation in the monocyte, which is acknowledged as an important orchestrator of the biological response to materials and the pathogenesis of chronic disease. Sublethal concentrations of Au(III), Ni(II), Hg(II), and Pd(II) were added to cultures of human THP1 monocytic cells for 72 h. In parallel cultures, lipopolysaccharide (LPS) was added for the last 30 min to activate the monocytic cells. Then cellular cytoplasmic and nuclear proteins were isolated, separated by electrophoresis, and probed for IkappaBalpha degradation (activation) and NFkappaB p65 translocation. Protein levels were digitally quantified and statistically compared. The levels of reactive oxygen species (ROS) in the monocytic cells were measured as a possible mechanism of metal-induced NFkappaB modulation. Only Au(III) activated IkappaBalpha degradation by itself. Au(III) and Pd(II) enhanced LPS-induced IkappaBalpha degradation, but Hg(II) and Ni(II) suppressed it. Au(III), Ni(II), and Pd(II) activated p65 nuclear translocation without LPS, and all but Ni(II) enhanced LPS-induced translocation. Collectively, the results suggest that these metal ions alter activation and translocation of NFkappaB, each in a unique way at unique concentrations. Furthermore, even when these metals had no overt effects on signaling by themselves, all altered activation of signaling by LPS, suggesting that the biological effects of these metals on monocytic function may only be manifest upon activation. None of the metal ions elevated levels of ROS at 72 h, indicating that ROS were probably not direct modulators of the NFkappaB activation or translocation at this late time point.