Stimulated THP-1 Cells Research Articles

Transglutaminase 2 silencing reduced the beta-amyloid-effects on the activation of human THP-1 cells

The aberrant expression and activation of transglutaminase 2 (TG2), the ubiquitous enzyme which catalyzes calcium-dependent protein cross-linking reactions, has been reported in many inflammatory diseases. Chronic inflammation, mediated by prolonged activation of brain-resident immunocompetent cells, appears to be involved in the pathogenesis of several age-related diseases, such as Alzheimer's disease. Given that increased TG2 expression has been observed in AD brains, this study was aimed to characterize the role of TG2 in THP-1 monocytes stimulated with amyloid-beta (Aβ). Aβ1-42 treatment dose-dependently increased TG2 expression in THP-1 cells. In particular, a fivefold up-regulation of TG2, compared with control cells, was observed in the presence of 0.5 μM Aβ1-42. At the same concentration, Aβ1-42 was able to promote monocyte maturation as suggested by increased expression of the cell surface antigen CD14 as well as the adhesion-promoting factor fibronectin. The stimulation of THP-1 cells with Aβ1-42 also led to a significant up-regulation of tumor necrosis factor α (TNF-α) and matrix metalloproteinase 9 (MMP-9). Interestingly, THP-1 cell transfection with small interfering RNA directed against TG2 was able to reduce Aβ1-42 increased levels of all the examined markers of monocyte maturation (CD14, fibronectin), and activation (TNF-α, MMP-9). These results indicate that TG2 up-regulation is required for the functional THP-1 monocyte activation induced by Aβ1-42. This work suggests that TG2 inhibition may represent a therapeutic target to ameliorate the inflammation and progression in Alzheimer's disease.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D–E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC50<3nM and inhibit the release of TNFα (IC50<0.3μM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC50=0.05–0.23μM), less potent in cells (IC50<1.1μM), but show good oral absorption. Compound 13E (100mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.

Pro-inflammatory responses in human monocytes are β1-adrenergic receptor subtype dependent

Stress induced circulating catecholamines are hypothesized to selectively activate adrenergic receptors (ARs) on immunocompetent cells modulating their inflammatory response to trauma or environmental toxins. We characterized changes in expression of a pro-inflammatory cytokine modulated by β-AR activation in human primary and immortalized monocytes that had been simultaneously stimulated with lipopolysaccharide (LPS). Results from cytokine antibody arrays demonstrated that half-maximal effective concentrations of the selective β-AR agonist isoproterenol (Iso) qualitatively increased LPS-mediated expression of the soluble cytokine, interleukin-1β (IL-1β). Semi-quantitative immunoblot techniques confirmed a synergistic increase of IL-1β production in both LPS stimulated THP-1 cells and primary human monocytes co-incubated with Iso. Immunoblot techniques as well as radioligand binding studies were also used to characterize the heterogeneous expression of β1- and β2-AR subtypes on THP-1 cells. β-AR activation is classically associated with generation of cAMP in many tissues and cell types. Therefore, using the method of Schild, we generated Iso concentration–response curves in the presence of fixed subtype-selective β-AR antagonist concentrations to demonstrate that β1-AR activation was exclusively linked with the generation of cAMP in THP-1 cells. Furthermore, use of a selective kinase inhibitor demonstrated that Iso potentiated the expression of soluble IL-1β through activation of cAMP-dependent protein kinase A. Finally, discriminating concentrations of subtype-selective β-AR antagonists revealed that β1-AR stimulation alone accounted for the synergistic production of IL-1β in LPS stimulated monocytes co-incubated with Iso. These results demonstrate a unique synergistic pro-inflammatory response mediated through a β1-AR cAMP-dependent mechanism in LPS-challenged monocytic cells.

Indirect Induction of Endothelial Cell Injury by PU- or PTFE-Mediated Activation of Monocytes

Polyurethanes (PUs) and polytetrafluoroethylene (PTFE) are widely used for making cardiovascular devices, but thrombus formation on the surfaces of these devices is inevitable. Since endothelial injury can lead to thrombosis, most of the studies on PUs or PTFE focused on their damage to endothelial cells. However, few studies have attempted to clarify whether the use of foreign objects as biomaterials can cause endothelial injury by activating the innate immune system. In this study, we aimed to investigate the roles of PU- or PTFE-stimulated immune cells in endothelial-cell injury. First, monocytes (THP-1 cells) were stimulated with PU or PTFE for 24 h and, subsequently, human umbilical vein endothelial cells (HUVECs) were treated with the supernatants of the stimulated cells for 24 h. We measured the generation of intracellular reactive oxygen species (ROS) from THP-1 cells treated with PU and PTFE for 24 h, meanwhile hydrogen dioxide (H2O2), tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the supernatants were also detected. Then, we assessed the apoptosis rate of the HUVECs and determined the expression of NO, inducible nitric oxide synthase (iNOS), and apoptosis-related proteins (p53, Bax, Bcl-2) in the HUVECs. The results showed that large amounts of ROS and low levels of pro-inflammatory cytokines (TNF-α and IL-1β) were produced by the stimulated THP-1 cells. After culturing with the supernatants of the PU- or PTFE-stimulated THP-1 cells, the apoptosis rate, NO production and expression of iNOS, p53 and Bax in the HUVECs were up-regulated, while Bcl-2 expression was down-regulated. In conclusion, the release of ROS by PU- or PTFE-treated THP-1 cells may induce iNOS expression and cause apoptosis in HUVECs via the p53, Bax and Bcl-2 proteins. These data provide the interesting finding that endothelial injury in the process of biomaterial-induced thrombosis can be initiated through the release of soluble mediators by monocytes.

Assay conditions can influence the outcome of cytotoxicity tests of nanomaterials: Better assay characterization is needed to compare studies

Background Reliable in vitro studies that generate consistent toxicity data on nanomaterials on a high-throughput scale will be of invaluable significance in the next few years. Aim In this study, we checked the influence of several steps of the experimental design on the outcome: we investigated the role of cell density, viability assay and particle dispersion method, including the influence of serum and effect of a surfactant (Tween 80). Methods The dose–response curve was assessed for ground multiwalled carbon nanotubes (CNT) and the silica benchmark Min-U-Sil, on lung epithelial cells (A549 cells) and macrophages (stimulated THP-1 cells). Results The cell density used in the cytotoxicity study has an impact on the outcome: for the same concentration of Min-U-Sil, the viability of A549 cells varied from 10% to 55% with increasing cell density. Whereas foetal calf serum attenuated the cytotoxicity of Min-U-Sil, this effect was not seen for CNT. The results show how cell culture conditions can modify the outcome of a toxicological experiment, as shown in this study for Tween 80 to disperse the test agent. Conclusions These experiments illustrates that results reported in literature can only be compared when, in addition to the use of a benchmark particle, a detailed method description is available. Therefore, more emphasis is needed on a standardized design for cytotoxicity studies.

Azithromycin Reduces Tumor Necrosis Factor-Alpha Production in Lipopolysaccharide-Stimulated THP-1 Monocytic Cells by Modification of Stress Response and p38 MAPK Pathway

Macrolide antibiotics are known to have a variety of immunomodulatory effects in addition to antimicrobial activity, but the mechanisms of immunomodulation are still unclear. We investigated in vitro the effect of azithromycin on tumor necrosis factor alpha (TNF-α) production in lipopolysaccharide (LPS)-stimulated THP-1 cells, a human monocytic cell line, and compared the results with those for other macrolides, minocycline and ofloxacin. In the presence of LPS, treatment with azithromycin (AZM) resulted in a significant decrease in LPS-induced TNF-α production compared to that with other antimicrobial agents. The results of phosphorylation of three MAPKs, ERK, JNK and p38, indicated that the phospho-p38 level was reduced by AZM. IκB-α, an inhibitor of NFκB, was not disrupted by the antibiotics. LPS-induced TNF-α release from THP-1 cells was inhibited in the presence of KNK437, a potent 70-kDa heat shock protein (HSp-70) inhibitor. Interestingly, the induction of HSp-70 by LPS was attenuated with the concurrent addition of AZM in the cells. AZM was found to restrain TNF-α production by monocytes at least in part by modifying the HSP-70 and p38 related signaling pathways to LPS stimulation.

Responses to Amyloids of Microbial and Host Origin Are Mediated through Toll-like Receptor 2

Curli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like beta-amyloid 1-42, which is associated with brain inflammation and Alzheimer's disease, curli fibrils have been implicated in the induction of host inflammatory responses. However, the underlying mechanisms of amyloid-induced inflammation are not fully understood. In a mouse sepsis model, we show that curli fibrils contributed to Nos2 expression, a hallmark of inflammation, by stimulating Toll-like receptor (TLR) 2. The TLR2 agonist activity was reduced by an amyloidogenicity-lowering amino acid substitution (N122A) in CsgA. Amyloid-forming synthetic peptides corresponding to beta-amyloid 1-42 or CsgA 111-151 stimulated Nos2 production in macrophages and microglia cells through a TLR2-dependent mechanism. This activity was abrogated when an N122A substitution was introduced into the synthetic CsgA peptide. The induction of TLR2-mediated responses by bacterial and eukaryotic amyloids may explain the inflammation associated with amyloids and the resulting pathologies.

Cathepsin X prevents an effective immune response against Helicobacter pylori infection

Cathepsin X, a cysteine protease, has been shown to regulate an immune response by activating β-2 integrin receptors. In this study we demonstrate its role in regulating the immune response to infection with H. pylori. The level of cathepsin X was determined in THP-1 monocyte cells primed with H. pylori antigens isolated from subjects suffering from gastritis, who had either eradicated or not the disease after the antibiotic therapy. We show that the specific clinical outcome of H. pylori eradication therapy correlates strongly with the membrane expression of cathepsin X in stimulated THP-1 cells, being significantly higher after stimulation with H. pylori strains from those subjects who did not respond to antibiotic therapy. The same antigens elicit a more vigorous immune response, increased expression of MHC II, however trigger inadequate cytokine profile (IFN-γ and IL-4) to eradicate the pathogen. We propose that cathepsin X mediated activation of β-2 integrin receptor Mac-1 suppresses the stimulatory signal in the form of cytokines. Cathepsin X co-localizes on the membrane of THP-1 cells with Mac-1 integrin receptor and its inhibition increases homotypic aggregation and mononuclear cell proliferation, events that are associated with low Mac-1 activity. Our study highlights the diversity of the innate immune response to H. pylori antigens leading to either successful eradication of the infection or maintenance of chronic inflammation, revealing cathepsin X location and activity as a regulator of the effectiveness of H. pylori eradication.

Therapeutic effects of rectal administration of muscovite on experimental colitis in rats

The aim of this study was to investigate whether rectal administration of muscovite can ameliorate colonic inflammation in a rat model of experimental colitis, and its possible mechanism. Female Sprague-Dawley (SD) rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis were treated with rectal administration of muscovite or 5-aminosalicylic acid (5-ASA) daily for 14 days. The changes in body weight, macroscopic damage and histologic scores were subsequently evaluated. Gene expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), mucin2 (MUC2) and trefoil factor 3 (TFF3) in the colonic tissues was assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) while protein levels of TNF-alpha and IL-1beta were detected by ELISA. Mucin2 expression in colonic mucosa was detected by immunohistochemistry. The capacity of muscovite to adsorb cytokines in vitro was determined by the changes in the amount of TNF-alpha, IL-1beta secreted by lipopolysaccharide (LPS)-stimulated THP-1 cells and IL-8 secreted by LPS-stimulated HT-29 cells. Rectal administration of muscovite improved the loss of body weight, macroscopic and histologic scores of TNBS-induced colitis in a dose-dependent manner. Trinitrobenzene sulfonic acid-induced expression of TNF-alpha and IL-1beta was reduced by muscovite and 5-ASA treatment. Reduction of MUC2 expression in colitis rats was reversed by muscovite and 5-ASA treatment. However, the expression of TFF3 mRNA in colonic mucosa was not affected. In addition, we found muscovite inhibited the expression of TNF-alpha, IL-1beta secreted by THP-1 and IL-8 secreted by HT-29 cells in a dose-dependent manner. Our study demonstrated for the first time that rectal administration of muscovite can ameliorate colonic inflammation of TNBS-induced colitis. Further confirmatory studies are needed to prove that muscovite might be a potential therapeutic agent for the treatment of ulcerative colitis.

Macrophage inflammatory protein-1β induced cell adhesion with increased intracellular reactive oxygen species

To investigate the role of macrophage inflammatory protein-1 β (MIP-1β) in the development of atherosclerosis, we designed an in vitro study to elucidate the mechanisms of monocyte-endothelium adhesion via intracellular reactive oxygen species (ROS). Angiotensin II (AngII) was used as a positive control. Furthermore, we examined the efficacy of MIP-1β as a predictor of stroke and cardiovascular events in hypertensive patients. MIP-1β or AngII stimulation significantly increased ROS production and adhesion of THP-1 cells to inflamed human umbilical vein endothelial cells. Cell adhesion and ROS production were inhibited in stimulated THP-1 cells by: inhibition of ROS signaling with N-acetylcysteine, diphenyleneiodonium, or PEG-Catalase; inhibition of PI3Kγ with siRNA or LY294002; and by Rac1 siRNA. The MIP-1 β or AngII stimulation did not increase surface expression of integrins, very late antigen 4 (VLA-4) and lymphocyte function-associated antigen 1 (LFA-1), but cell adhesion was reduced by using an antiVLA-4 or an antiLFA-1 antibody. Moreover, cell adhesion and ROS production stimulated with MIP-1β or AngII were completely inhibited by fluvastatin. In our clinical study, patients with the highest quartile of MIP-1β showed a higher risk of stroke and cardiovascular events by a Cox proportional-hazards model. In conclusion, MIP-1β directly induced cell adhesion to endothelial cells through oxidative stress via PI3k–Rac1 cascades. Serum MIP-1β level might be a useful predictor for cerebro-cardiovascular events in hypertensive patients. Condensed abstractWe designed an in vitro investigation to examine the role of MIP-1β on the development of atherosclerosis, including cell adhesion involving CAMs and ROS production, compared with angiotensin II. Furthermore, we investigated the prognostic impact of MIP-1β on stroke and cardiovascular events in hypertensive patients in a small cohort study.

Proton pump inhibitors exert anti-inflammatory effects and decrease human microglial and monocytic THP-1 cell neurotoxicity

To explore whether proton pump inhibitors (PPIs) possess anti-inflammatory effects on microglia, we investigated the effect of lansoprazole (LPZ) and omeprazole (OPZ) on the toxic action towards SH-SY5Y neuroblastoma cells of supernatants from human microglia and THP-1 cells stimulated by lipopolysaccharide combined with interferon-γ. In addition, we studied the effect of LPZ and OPZ on the THP-1 cell production of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 using enzyme-linked immunosorbent assays. We found that both PPIs had a protective effect on the toxicity of supernatants and that there was a synergism of this effect with S-ibuprofen (IBP), a typical non-steroidal anti-inflammatory drug (NSAID). A similar protective effect of LPZ was observed with supernatants from stimulated human microglia. We also found that both PPIs significantly reduced the TNF-α secretion from stimulated THP-1 cells in a concentration dependent manner and that there was a trend towards such reduction of IL-6. These results indicate that PPIs possess anti-inflammatory effects and can decrease human microglial and monocytic neurotoxicity. They suggest that PPIs combined with NSAIDs may be effective in the treatment of a broad spectrum of neurodegenerative diseases associated with activated microglia.

Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia Stimulate Peripheral Blood Mononuclear Cells to Secrete TNF-α and IFN-γ

Moesin is an intracellular protein that links the cell membrane and cytoskeleton, while also mediating the formation of microtubules and cell adhesion sites as well as ruffling of the cell membrane. To determine the roles of anti-moesin Abs derived from the serum of patients with aplastic anemia (AA) in the pathophysiology of bone marrow failure, we studied the expression of moesin on various blood cells and the effects of anti-moesin Abs on the moesin-expressing cells. The proteins recognized by anti-moesin mAbs were detectable on the surface of T cells, NK cells, and monocytes from healthy individuals as well as on THP-1 cells. The peptide mass fingerprinting of the THP-1 cell surface protein and the knock-down experiments using short hairpin RNA proved that the protein is moesin itself. Both the anti-moesin mAbs and the anti-moesin polyclonal Abs purified from the AA patients' sera stimulated THP-1 cells and the PBMCs of healthy individuals and AA patients to secrete 60-80% as much TNF-alpha as did LPS 100 ng/ml. Although the polyclonal Abs induced IFN-gamma secretion from the PBMCs of healthy individuals only when the PBMCs were prestimulated by anti-CD3 mAbs, the anti-moesin Abs were capable of inducing IFN-gamma secretion from the PBMCs of AA patients by themselves. Anti-moesin Abs may therefore indirectly contribute to the suppression of hematopoiesis in AA patients by inducing myelosuppressive cytokines from immunocompetent cells.

Mechanical stress induces tumor necrosis factor-α production through Ca2+ release-dependent TLR2 signaling

We studied centrifugation-mediated mechanical stress-induced tumor necrosis factor-alpha (TNF-alpha) production in the monocyte-like cell line THP-1. The induction of TNF-alpha by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF-alpha after 1 h of stimulation. TNF-alpha production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like receptor-2 (TLR2) mRNA in proportion to the expression of TNF-alpha. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF-alpha expression response to mechanical stress. After transient overexpression of TLR2 in HEK-293 cells, mechanical stress induced TNF-alpha mRNA production. Interestingly, mechanical stress activated the c-Src-dependent TLR2 phosphorylation, which is necessary to induce Ca(2+) fluxes. When THP-1 cells were pretreated with BAPTA-AM, thapsigargin, and NiCl(2).6H(2)O, followed by mechanical stimulation, both TLR2 and TNF-alpha production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF-alpha production through Ca(2+) releases from intracellular Ca(2+) stores following TLR2 phosphorylation. In addition, TNF-alpha treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the preincubation of anti-TNF-alpha antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF-alpha produced by mechanically stimulated THP-1 cells affected TLR2 production. We concluded that TNF-alpha production induced by centrifugation-mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with Ca(2+) release and that TNF-alpha production induced by mechanical stress affects TLR2 production.

Expression and function of ephrin-B1 and its cognate receptor EphB2 in human atherosclerosis: from an aspect of chemotaxis

Although several cytokines and chemokines have been demonstrated to play pivotal roles in the pathophysiological conditions of atherosclerosis, few findings exist regarding the expression and function of cytokine-modulating molecules such as ephrin-Bs and their cognate receptors, EphBs, in human atherosclerosis. Therefore, in the present study, we screened novel genes modulating atherogenesis by cDNA array and quantitatively determined them by real-time RT (reverse transcription)-PCR in human carotid atherosclerotic plaques. Ephrin-B1 and EphB2, key regulators of embryogenesis, were significantly up-regulated in plaques compared with those in adjacent control tissues [ephrin-B1, 0.638+/-0.106 compared with 0.831+/-0.152, or 130% (P<0.05); EphB2, 1.296+/-0.281 compared with 2.233+/-0.506, or 172% (P<0.05)]. Immunohistological analysis demonstrated that both ephrin-B1 and EphB2 were expressed in macrophages and T-lymphocytes in plaques as well as in monocytes, T-lymphocytes and arterial endothelial cells isolated from healthy adults. Interestingly, the extracellular domains of ephrin-B1 and EphB2, the expression of which were both enhanced in stimulated THP-1 cells, significantly inhibited spontaneous (22.5 and 27.6% respectively; P<0.01) and MCP-1 (monocyte chemoattractant protein-1)-dependent (29.7 and 22.6% respectively; P<0.01) migration of monocytes. In conclusion, these results demonstrate that ephrin-B1 and EphB2 are overexpressed in atherosclerotic tissue and might locally regulate cell migration, possibly through modulating cytokine-related chemotaxic activity; however, the functional role of these molecules in atherogenesis should be investigated further.

QS382. The Effect of Phosphodiesterase Inhibition on NF-κB Activation in LPS Stimulated THP1 Cells

Background: The activation of the transcriptional factor NF-κB has been shown to play a critical role in the host response to sepsis by increasing cellular adhesion molecules and proinflammatory cytokine synthesis. NF-κB resides in its mature form in the cytoplasm, complexed to the Inhibitor kappaB (IκB) proteins. Cell activation by various stimuli, including LPS, causes phosphorylation and degradation of IκB, which is catalyzed by the phosphorylation of IKKβ. Following degradation of IκB, NF-κB translocates to the nucleus and activates the transcription of its target genes.

Mechanistic analysis of macrophage response to IRAK-1 gene knockdown by a smart polymer-antisense oligonucleotide therapeutic

An excessive inflammatory response is a clinical problem following major infections and severe injury that may lead to Sepsis Syndrome and Multiple Organ Failure (MOF), including the Acute Respiratory Distress Syndrome (ARDS). Management of excessive inflammation may be possible through control of key inflammatory pathways such as those mediated by the important interleukin-1 receptor associated kinase-1 (IRAK-1). In the current study, we report the impact on gene expression induced by lipopolysaccharide (LPS) stimulation of THP-1 cells treated with an antisense oligonucleotide (ASODN) against the IRAK-1 gene using cDNA microarrays and quantitative RT-PCR. The therapeutic ASODN was delivered using a pH-sensitive, membrane-interactive polymer that destabilizes the endosomal membrane to enhance access cytoplasmic delivery in targeted cells. Following LPS stimulation, the anti-inflammatory activity of ASODN against the IRAK-1 gene expression is evidenced by the lower expression of inflammatory chemokines, cytokines and acute-phase proteins compared to control cells. These results provide a larger mechanistic picture of IRAK-1 knockdown by this polymer therapeutic in macrophage-like cells.

Mycoplasma genitalium Lipoproteins Induce Human Monocytic Cell Expression of Proinflammatory Cytokines and Apoptosis by Activating Nuclear Factor κB

This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-κB (NF-κB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-κB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-κB. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-κB.

Regulation of constitutive and microbial pathogen‐induced human macrophage migration inhibitory factor(MIF) gene expression

The cytokine macrophage migration inhibitory factor (MIF) is an important regulator of innate immunity, inflammation and oncogenesis. However, four decades after its identification, the molecular mechanism(s) regulating the expression of the MIF gene remain largely unknown. Analyses of human monocytic (THP-1), epithelial (HeLa and A549) and keratinocytic (HaCat) cells transfected with wild-type, truncated and mutated MIF promoter reporter constructs, and electrophoretic mobility shift assay, chromatin immunoprecipitation, and siRNA inhibition indicated that the transcription factors specificity protein (Sp)1 and cAMP response element-binding protein (CREB) are critical positive regulators of constitutive human MIF gene expression. Albeit located in a cytosine guanine dinucleotide island, the MIF gene was found to be hypomethylated, an observation consistent with high baseline transcriptional activity. Moreover, stimulation of THP-1 cells and of peripheral blood mononuclear cells with microbial products up-regulated phosphorylated Sp1 nuclear content, Sp1 DNA-binding activity, MIF promoter activity and MIF mRNA levels in a MEK1/2-, Sp1-dependent manner. Taken together with previous observations of an important role for MIF in pro-inflammatory macrophage responses, these present findings suggest a key role for Sp1 and CREB in transcriptional regulation of MIF gene expression and MIF-dependent host antimicrobial innate immune defense.

Synthesis of 1-aryl-3-(3,4-dihydro-2 H-chromen-5-yl) ureas as TNF-α inhibitors

A new series of compounds, 1-aryl-3-(3,4-dihydro-2 H-chromen-5-yl) ureas, have been synthesized and their structures were confirmed by FAB-MS and 1H NMR. The preliminary pharmacological screening showed that these compounds inhibited TNF-α production in lipopolysaccharide (LPS)-stimulated THP-1 cells.

A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation

In humans, the relaxin hormone family includes H1, H2 and H3 isoforms and insulin-like peptides 3 to 6. The ever-increasing interest in relaxin as potential new drug requires reliable methods to compare bioactivity of different relaxins. The existing bioassays include in vivo or ex vivo methods evaluating the organ-specific responses to relaxin and in vitro methods based on measurement of cAMP increase in relaxin receptor-bearing cells. We previously demonstrated that relaxin dose-dependently inhibits platelet aggregation. On this basis, we have developed a simple, reliable bioassay for relaxin used to compare purified porcine relaxin, assumed as reference standard, with two recombinant human H2 relaxins, H3 relaxin, insulin-like peptides 3 and 5. Pre-incubation of platelets with relaxins (3, 10, 30,100, 300 ng/ml; 10 min.) caused the inhibition of ADP-induced platelet aggregation. Within the 10–100 ng/ml range, porcine relaxin showed the highest effects and a nearly linear dose–response correlation. Lower peptide concentrations were ineffective, as were insulin-like peptides 3 and 5 at any concentration assayed. Platelet inhibition was mediated by specific RXFP1 relaxin receptor and cGMP, whose intracellular levels dose-dependently increased upon relaxin. For comparison, we stimulated THP-1 cells, a relaxin receptor-bearing cell line, with porcine relaxin, human H2 and H3 relaxins at the above concentrations (15 min.). We observed a dose-related increase of intracellular cAMP similar to the trend of platelet inhibition. Insulin like peptide 5 was ineffective. In conclusion, this study shows that inhibition of platelet aggregation may be used to assess bioactivity of relaxin preparations for experimental and clinical purposes.