Effect of Mahonia aquifolium active compounds on interleukin-8 production in the human monocytic cell line THP-1

The effect of the crude extract and of two alkaloid fractions prepared from Mahonia aquifolium on interleukin-8 (IL-8) production in the lipopolysaccharide (LPS)-stimulated human monocytic cell line THP-1 was studied. The production of IL-8 by cells stimulated with 20 ng/ml LPS after 48 h treatment with 20 microg/ml crude extract was inhibited by about 30 %. LPS-stimulated cells treated with 0.1 microg/ml bisbenzylisoquinoline alkaloid fraction exhibited a 40% inhibition of IL-8 production in comparison with control cells. The fraction of protoberberine alkaloids had no significant inhibitory activity. Weak or no inhibition of IL-8 production was found after treatment with 0.5 microg/ml of protoberberine alkaloids berberine and jatrorrhizine and with berbamine from the group of BBI alkaloids. In contrast, the production was inhibited after treatment with BBI alkaloids baluchistine (about 20%) and aromoline (up to 30%).     

Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten sensitizers

Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10 μM for 24 and 48 h. The direct-binding haptens dinitrochlorobenzene (DNCB), benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (±S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens.     

Changes of cell-surface thiols and intracellular signaling in human monocytic cell line THP-1 treated with diphenylcyclopropenone

Changes of cell-surface thiols induced by chemical treatment may affect the conformations of membrane proteins and intracellular signaling mechanisms. In our previous study, we found that a non-toxic dose of diphenylcyclopropene (DPCP), which is a potent skin sensitizer, induced an increase of cell-surface thiols in cells of a human monocytic cell line, THP-1. Here, we examined the influence of DPCP on intracellular signaling. First, we confirmed that DPCP induced an increase of cell-surface thiols not only in THP-1 cells, but also in primary monocytes. The intracellular reduced-form glutathione/oxidized-form glutathione ratio (GSH/GSSG ratio) was not affected by DPCP treatment. By means of labeling with a membrane-impermeable thiol-reactive compound, Alexa Fluor 488 C5 maleimide (AFM), followed by two-dimensional gel electrophoresis and analysis by liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), we identified several proteins whose thiol contents were modified in response to DPCP. These proteins included cell membrane components, such as actin and β-tubulin, molecular chaperones, such as heat shock protein 27A and 70, and endoplasmic reticulum (ER) stress-inducible proteins. Next, we confirmed the expression in DPCP-treated cells of spliced XBP1, a known marker of ER stress. We also detected the phosphorylation of SAPK/JNK and p38 MAPK, which are downstream signaling molecules in the IRE1α-ASK1 pathway, which is activated by ER stress. These data suggested that increase of cell-surface thiols might be associated with activation of ER stress-mediated signaling.     

Inhibition of L-leucine methyl ester mediated killing of THP-1, a human monocytic cell line, by a new anti-inflammatory drug, T614

T614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) is a member of the family of methanesulfonanilide non-steroidal anti-inflammatory drugs (mNSAIDs), most of which act as cyclooxygenase (COX)-2 inhibitors. L-leucine methyl ester (Leu-OME) is a reagent which has been shown to kill phagocytes following interaction with intracellular proteases. There are two pathways whereby Leu-OME becomes cytotoxic to phagocytes. Within lysosomes, Leu-OME is converted into free Leu, which causes disruption of the lysosomes and subsequent cell necrosis. The other is the conversion of Leu-OME into (Leu-Leu)(n)-OME, which is associated with the induction of apoptosis. In the present study, we examined the action of T614 on Leu-OME mediated killing of THP-1, a human monocytic cell line. We revealed that T614 and phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, inhibited Leu-OME mediated killing of THP-1 cells. All the other mNSAIDs, including nimesulide (NIM-03), fluosulide (CGP28238), FK3311 and NS398, also rescued THP-1 from Leu-OME mediated killing, although to a lesser degree. Of the classical NSAIDs tested, a protective effect was observed with diclofenac at high concentration, but not with naproxen or indomethacin. Unlike conventional lysosomal inhibitors, such as chloroquine and ammonium chloride (NH(4)Cl), T614 and PMSF did not raise lysosomal pH, as measured by flow cytometry using fluorescein isothiocyanate dextran (FITC-dextran). Therefore, the mechanism whereby T614 and PMSF inhibit Leu-OME killing is distinct from that of chloroquine or NH(4)Cl. Based on the similarity of T614 and PMSF, we suggest that, besides their roles as COX-2 inhibitors, T614 and other mNSAIDs may act as lysosomal protease inhibitors.     

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production

Six Thr¹ (O-glyco)-derivatives of the “phagocytosis stimulating peptide” tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(β-d -GlcNAc)-Ser-Thr-OH, which correspond to the “tuftsin-region” at the Fc-domain of immunoglobulin G (amino acid residues 289–299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.     

Apoptosis, necrosis and cell proliferation-inhibition by cyclosporine A in U937 cells (a human monocytic cell line).

The immunosuppressive drug cyclosporine A (CsA) has been used in both organ transplantation and the treatment of autoimmune disorders. However, the drug causes adverse effects in the kidney, liver and nervous system, characterized by cellular loss in the affected area. Apoptosis has been shown to play a role in CsA-induced cytotoxicity. Because permeabilization of the mitochondrial membrane is a common criterion in most apoptotic settings in vertebrate cells, here we evaluated whether CsA causes loss of mitochondrial function in the pathway leading to cellular cytotoxicity. We found that CsA caused a concentration- and time-dependent loss of cell viability in the U937 cell line. Treatment of cells at a dose of 10 microM CsA resulted in G0/G1 arrest with a concurrent decrease in the number of cells in the S and G2/M phases of the cell cycle. In mechanistic studies related to the loss of viability, treating cells with 10 microM CsA for 24 h resulted in both DNA fragmentation and an increase of annexin-V-positive cells. CsA treatment also increased activity of the cysteine protease caspase-3, decreased the mitochondrial membrane potential and induced the release of cytochrome c into the cytosol. Furthermore, CsA treatment increased the number of cells in the sub-G0/G1 peak, indicative of a reduction in DNA, although this increase was not observed when cells were pre-treated with a broad caspase inhibitor. In the study, we also found that a higher dose of CsA induces LDH release when the cells were incubated for a longer period. Taken together, these data suggest that the mode of cell death induced by CsA is dose- and time-dependent. Short-term incubation with lower doses of CsA arrests cell growth; this arrest overlaps with the occurrence of apoptosis and then with necrosis after longer treatment periods with higher doses of CsA.     

MIP-1beta, a novel biomarker for in vitro sensitization test using human monocytic cell line.

In order to seek a novel biomarker for predicting skin sensitization, changes in the gene expression profile of THP-1 cells on exposure to 2,4-dinitrochlorobenzene (DNCB), p-phenylenediamine (pPD) and nickel sulfate (Ni) were assessed using oligo-DNA microarrays. While the change in gene expression varied depending on the sensitizers, up-regulation of MIP-1 beta mRNA expression was detected in both DNCB-treated and Ni-treated THP-1 cells. This finding was validated by RT-PCR and confirmed at the protein level by ELISA. Secretion of MIP-1 beta from THP-1 was detected after 24-h treatment with sensitizers such as DNCB, Ni, 2-mercaptobenzothiazole (2-MBT) and cobalt sulfate (Co), while pPD and non-sensitizers such as sodium dodecyl sulfate (SDS) and benzalkonium chloride (BC) had no effect. The use of both MIP-1 beta production and CD86 expression as criteria reduced the number of false-negatives, and the results were in good agreement with those of in vivo assays. MIP-1 beta may be useful as a novel biomarker in in vitro sensitization assay using THP-1 cells, either alone or in combination with known markers.     

一氧化氮和白细胞介素—10对小鼠肺泡巨噬细胞产生肿瘤坏死因？…

目的 观察一氧化氮和ＩＬ－１０对肺泡巨噬细胞炎症反应的调节作用,方法：小鼠肺泡汇噬细胞（ＡＭ）受脂多糖（ＬＰＳ）１０ｍｇ．Ｌ＾－１刺激同时,加入一氧化氮合酶抑制剂Ｓ－硫酸甲基异硫脲（ＳＭＴ）或一氧化氮供体Ｓ－亚硝基乙酰青霉胺（ＳＮＡＰ）,ＥＬＩＳＡ法测定上清液中ＴＮＦα,ＩＬ－１β,ＩＬ－６和ＩＬ－１０浓度,结果：ＡＭ受ＬＰＳ刺激后,ＴＮＦα,ＩＬ－１β和ＩＬ－６释放峰值分别在６,１２和２４小时,     

Sensitization assays: monocyte-derived dendritic cells versus a monocytic cell line (THP-1)

Dendritic cells (DCs) play a critical role in the skin sensitization process of contact allergens. Many efforts have been made to develop in vitro sensitization tests that employ DCs, but more recently protocols were introduced that use cell lines other than DCs. The potential of the cell line THP-1 compared to monocyte-derived DCs (MoDCs) was evaluated using a known potent sensitizer 2,4-dinitrochlorobenzene (DNCB) and the terpenoid ascaridol (1,4-epodioxy-p-menth-2-ene), an ingredient present in oxidized tea tree oil. Activation of these cells was studied by estimation of the CD86 and CD54 cell surface expression. Overall, comparable results were found. The expression of CD86 was augmented by ascaridol in THP-1 and MoDCs, while the expression of CD54 was not reproducibly increased. These results encourage the further development of THP-1 cells as a short-term model for sensitization testing.     

Fraxetin inhibits the induction of anti-Fas IgM, tumor necrosis factor-alpha and interleukin-1beta-mediated apoptosis by Fas pathway inhibition in human osteoblastic cell line MG-63

The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients with inflamed synovium, such as in rheumatoid arthritis (RA). By means of alkaline phosphatase (ALP) activity and osteocalcin ELISA assay, we have shown that fraxetin exhibits a significant induction of differentiation in the human osteoblast-like cell line MG-63. In addition, we also assessed whether fraxetin affects inflammatory cytokine-mediated apoptosis in osteoblast cells. TNF-alpha or IL-1beta enhance apoptotic DNA fragmentation in anti-Fas IgM-treated MG-63 cells by increasing Fas receptor expression. However, TNF-alpha or IL-1beta treatment alone does not induce apoptosis. Treatment of MG-63 cells with fraxetin not only inhibited anti-Fas IgM-induced apoptosis, but also blocked the synergetic effect of anti-Fas IgM with TNF-alpha or IL-1beta on cell death. The apoptotic inhibition of fraxetin is associated with inhibition of TNF-alpha and IL-1beta-mediated Fas expression and enhancement of FLIP expression, resulting in a decrease of caspase-8 and caspase-3 activation. These results indicate a potential use of fraxetin in preventing osteoporosis by inhibiting inflammatory cytokine-mediated apoptosis in osteoblast cells.     

Regulation of glutathione S-transferase P1-1 gene expression by NF-kappaB in tumor necrosis factor alpha-treated K562 leukemia cells

Glutathione S-transferases (GSTs) play an important role in the protection of cells against xenobiotics and lipid hydroperoxides generated by oxidative stress. In human, the GSTP1-1 expression is commonly increased in many tumors and involved in the development of antineoplastic drug resistance. Reactive oxygen species are released at inflammation sites and oxidative stress conditions enhance the expression of genes encoding antioxidant enzymes such as GSTs. Here we investigated the regulation of the GSTP1-1 gene expression in the K562 cell line by nuclear factor kappaB (NF-kappaB) and the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). By studying GSTP1-1 mRNA expression and NF-kappaB/GSTP1-1 promoter interactions, we showed the implication of NF-kappaB in the GSTP1-1 gene expression and we described a new specific TNFalpha-inducible NF-kappaB binding site upstream of the minimal promoter. Moreover, TNFalpha treatment as well as cotransfection of NF-kappaB signaling pathway intermediates induced an activation of the GSTP1-1 gene promoter in K562 cells. Site-directed mutagenesis of the NF-kappaB site strongly inhibited TNFalpha- and NF-kappaBp65-induced promoter activation. Altogether, we showed that a sequence located at -323/-314 within the GSTP1-1 promoter bound NF-kappaB p50/65 and p65/p65 dimers and that this kappaB site was involved in the regulation of the gene by TNFalpha.     

Effect of recombinant human interleukin-1 beta and tumor necrosis factor alpha on liver cytochrome P-450 and serum alpha-1-acid glycoprotein concentrations in the rat

Two hepatocyte-related effects of recombinant human interleukin-1 beta and tumor necrosis factor alpha alone or in association were tested following iv administration to Fischer 344 rats. Within 24 hr, both monokines induced a dose-dependent decrease in cytochrome P-450 levels, whereas serum alpha-1-acid glycoprotein concentrations were strongly increased. The largest variation of both parameters was observed using a combination of the two monokines. Dexamethasone, which possesses anti-interleukin-1 properties and is known to stimulate alpha-1-acid glycoprotein synthesis in rats, also depressed cytochrome P-450 levels, suggesting that alpha-1-acid glycoprotein might mediate the monokine-related inhibition of drug metabolism. Nevertheless, at the lowest doses of monokines tested, only cytochrome P-450 levels were modified. The transfer of a post-dexamethasone serum to normal rats did not change cytochrome P-450 levels.     

An ent-kaurene diterpene enhances apoptosis induced by tumor necrosis factor in human leukemia cells

Some antitumor agents, including tumor necrosis factor-alpha (TNF-alpha) and camptothecin (CPT), often cause resistance of tumor cells to antitumor agents through activation of the nuclear factor-kappa B (NF-kappa B) pathway that leads to up-regulation of anti-apoptotic proteins. Therefore, co-treatment of an inhibitor of the NF-kappa B pathway with antitumor agents is a useful strategy for chemotherapy. Here we report that ent-11 alpha-hydroxy-16-kauren-15-one (KD) selectively inhibits NF-kappa B-dependent gene expression due to treatment with TNF-alpha. KD in combination with TNF-alpha caused a dramatic increase in apoptosis in human leukemia cells accompanied by activation of caspases. A broad-spectrum inhibitor of caspases decreased the apoptosis induced by treatment with KD and TNF-alpha. KD in combination with CPT also caused an increase in apoptosis. These results suggest that the apoptotic potency of co-treatment of KD with TNF-alpha or CPT is elicited through selective inhibition of NF-kappa B-dependent anti-apoptotic proteins and thus may provide a basis for the development of useful approaches to the treatment of leukemia.     

Synergistic effects of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha: central monoamine, corticosterone, and behavioral variations.

The proinflammatory cytokines interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) influence neuroendocrine activity, promote central neurotransmitter alterations, and induce a constellation of symptoms collectively referred to as sickness behaviors. These cytokines may also elicit anxiety and anhedonia, and have been associated with psychological disturbances in humans. In the present investigation, systemic IL-1beta and TNF-alpha dose-dependently and synergistically disrupted consumption of a highly palatable food source (chocolate milk), possibly reflecting anorexia or anhedonia engendered by the treatments. As well, these cytokines synergistically increased plasma corticosterone levels. Although IL-1beta and TNF-alpha provoked variations of amine turnover in the hypothalamus, locus coeruleus, and central amygdala, synergistic effects were not evident in this respect. Nevertheless, in view of the central amine variations induced by the cytokines, it is suggested that immune activation may come to influence complex behavioral processes, as well as affective state.     

The proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha activate serotonin transporters.

Proinflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of major psychiatric disorders. We have demonstrated that activation of p38 mitogen-activated protein kinase (MAPK) induces a catalytic activation of the serotonin transporter (SERT) arising from a reduction in the SERT Km for 5-hydroxytryptamine (5-HT). As inflammatory cytokines can activate p38 MAPK, we hypothesized that they might also activate neuronal SERT. Indeed, Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulated serotonin uptake in both the rat embryonic raphe cell line, RN46A, and in mouse midbrain and striatal synaptosomes. In RN46A cells, IL-1beta stimulated 5-HT uptake in a dose- and time-dependent manner, peaking in 20 min at 100 ng/ml. This was abolished by IL-1ra (20 ng/ml), an antagonist of the IL-1 receptor, and by SB203580 (5 microM), a p38 MAPK inhibitor. TNF-alpha also dose- and time-dependently stimulated 5-HT uptake that was only partially blocked by SB203580. Western blots showed that IL-1beta and TNF-alpha activated p38 MAPK, in an SB203580-sensitive manner. IL-1beta induced an SB203580-sensitive decrease in 5-HT Km with no significant change in Vmax. In contrast, TNF-alpha stimulation decreased 5-HT Km and increased SERT Vmax. SB203580 selectively blocked the TNF-alpha-induced change in SERT Km. In mouse midbrain and striatal synaptosomes, maximal stimulatory effects on 5-HT uptake occurred at lower concentrations (IL-1beta, 10 ng/ml; TNF-alpha, 20 ng/ml), and over shorter incubation times (10 min). As with RN46A cells, the effects of IL-1beta and TNF-alpha were completely (IL-1beta) or partially (TNF-alpha) blocked by SB203580. These results provide the first evidence that proinflammatory cytokines can acutely regulate neuronal SERT activity via p38 MAPK-linked pathways.     

Inhibitory effect of sofalcone on tumor necrosis factor-alpha and interleukin-1 beta production in human monocytes stimulated by Helicobacter pylori water extract.

We investigated the effect of sofalcone, a synthetic flavonoid derivative of sophoradin, on tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta production in human monocytes stimulated by Helicobacter pylori water extract. H. pylori water extract significantly stimulated TNF-alpha and IL-1 beta production by monocytes while incubation with sofalcone (10 micrograms/ml and 50 micrograms/ml) significantly inhibited this increase in TNF-alpha and IL-1 beta production. These results suggest that sofalcone could be used to improve H. pylori-associated gastric mucosal inflammation through inhibition of proinflammatory cytokine production.     

Induction by interleukin-1, tumor necrosis factor and lipopolysaccharides of histidine decarboxylase in the stomach and prolonged accumulation of gastric acid

1. Injection of interleukin-1 (IL-1) into pylorus-ligated rats has been shown strongly to inhibit gastric secretion. However, in the present study, we found that an intraperitoneal injection of IL-1 into intact (non-pylorus-ligated) fasted mice rapidly (within 30 min) induced an accumulation of gastric acid (`early response''). When the dose of IL-1 was larger, the accumulation lasted for a longer period.
2. Injection of IL-1 also caused a later elevation of the activity of histidine decarboxylase (HDC), the histamine-forming enzyme, in the stomach (`later response'').
3. Cimetidine, an antagonist of histamine H₂-receptors, suppressed the accumulation of gastric acid in both the early and later periods. An irreversible inhibitor of HDC, α-fluoromethylhistidine, partially inhibited the accumulation in the later period.
4. IL-1, when injected 1 h after feeding in mice fasted overnight, markedly retarded gastric emptying.
5. Tumour necrosis factor (TNF) and lipopolysaccharide (LPS) or endotoxin from E. coli both had IL-1-like effects on the stomach, and their effects are presumably mediated by IL-1.
6. These results support the idea that an inhibition of gastric emptying and an elevation of HDC activity in the stomach may explain the findings that a long-lasting accumulation of gastric acid is induced by IL-1 despite its potent inhibition of gastric acid secretion.
7. On the basis of these results, and in the light of the known actions of histamine, the possible roles of IL-1 in gastric inflammation and ulceration are discussed.

     

Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration "in vivo" by indirect mechanisms

The alpha and beta forms of recombinant interleukin-1 (IL-1 alpha and IL-1 beta) and of recombinant Tumor Necrosis Factor (TNF alpha and TNF beta) induced dose-dependent neutrophil migration into rat peritoneal cavities. Migration induced by both IL-1s showed a bell-shaped dose-response curve and IL-1 beta was 3-fold more potent than IL-1 alpha. Pretreatment of the animals with dexamethasone or depletion of the peritoneal macrophage population, abolished the neutrophil migration induced by the four cytokines. "In vitro" stimulation of macrophage monolayers with IL-1 beta and the TNFs released a factor into the supernatant which, unlike these cytokines, induced neutrophil migration in dexamethasone pretreated animals. These results suggest that the neutrophil migration induced by IL-1 alpha, IL-1 beta, TNF alpha and TNF beta is not due to a direct effect on neutrophils, but occurs via the release of a chemotactic factor(s) from resident macrophages.