Interleukin-8 Protein Release Research Articles

Scoparone inhibits PMA-induced IL-8 and MCP-1 production through suppression of NF-κB activation in U937 cells

Scoparone is a major component of the shoot of Artemisia capillaris (Compositae), which has been used for the treatment of hepatitis and biliary tract infection in oriental countries. In this study, the effects of scoparone on the expression of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) and activation of nuclear factor-kappaB (NF-κB) were examined in U937 human monocytes activated with phorbol 12-myristate 13-acetate (PMA). Scoparone (5–100 μM) had no cytotoxic effect in unstimulated cells and concentration-dependently reversed PMA-induced toxicity in the cells stimulated with PMA. Scoparone concentration-dependently reduced the release of IL-8 and MCP-1 protein and expression of IL-8 and MCP-1 mRNA levels induced by PMA. Moreover, scoparone inhibited the levels of NF-κB-DNA complex and NF-κB activity in the cells stimulated with PMA in a concentration-dependent manner. Scoparone dose-dependently inhibited the phosphorylation of IκBα and nuclear translocation of NF-κB 1 p50, RelA p65, and c-Rel p75. These data suggest that scoparone may inhibit the expression of chemokines (IL-8 and MCP-1) in PMA-stimulated U937 cells and a potential mechanism of scoparone may be inhibition of NF-κB activation, which is linked to inhibition of NF-κB subunits (NF-κB 1 p50, RelA p65, and c-Rel p75) translocation via suppression of IκBα phosphorylation.

Increased expression of interleukin-6 by vasoactive intestinal peptide is associated with regulation of CREB, AP-1 and C/EBP, but not NF-κB, in mouse calvarial osteoblasts

Interleukin-6 (IL-6), and the related cytokines IL-11, leukemia inhibitory factor (LIF) and oncostatin M (OSM), are potent stimulators of osteoclastic bone resorption. In the present study, we have addressed the possibility that the neuropeptide vasoactive intestinal peptide (VIP) may regulate the production of and/or sensitivity to the IL-6 family of cytokines in mouse calvarial osteoblasts. VIP stimulated IL-6 mRNA expression and protein release in a time- and concentration-dependent manner, whereas mRNA expression of the IL-6 receptor, as well as mRNA expressions of IL-11, LIF, OSM and their cognate receptors, were unaffected by VIP. In cells transfected with the IL-6 promoter coupled to luciferase, VIP increased transcriptional activity. The effects of VIP were shared by the related neuropeptide PACAP-38, belonging to the same superfamily of neuropeptides, whereas secretin did not have any effect, indicating that the effects were mediated by VPAC 2 receptors. The effects of VIP were potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram and mimicked by forskolin, indicating the involvement of the cyclic AMP/protein kinase A pathway. This was further demonstrated by the facts that the stimulatory effect of VIP on luciferase activity could be reversed by the PKA inhibitors H-89 and KT5720 and was mimicked by cyclic AMP analogues selective for PKA, but not by those selective for Epac. In addition, VIP enhanced the phosphorylation of CREB, as assessed by both immunocytochemical analysis and Western blot. The DNA binding activity of nuclear extracts to C/EBP was increased by VIP, whereas binding to AP-1 was decreased. In contrast, DNA binding to NF-κB, as well as nuclear translocation of NF-κB and C/EBP, were unaffected by VIP. The mRNA expressions of C/EBPβ, C/EBPδ, C/EBPγ, c-Jun, JunB, c-Fos, Fra-1 and IκBα and protein level of IκBα were all unaffected by VIP. These observations, together, demonstrate that VIP stimulates IL-6 production in osteoblasts by a mechanism likely to be mediated by VPAC 2 receptors and dependent on cyclic AMP/protein kinase A/CREB activation and also involving the transcription factors C/EBP and AP-1.

Irradiation of mechanically-injured human arterial endothelial cells leads to increased gene expression and secretion of inflammatory and growth promoting cytokines

Radiation therapy is applied to inhibit neointima formation after percutaneous transluminal coronary angioplasty (PTCA). In this study, we evaluated the effect of irradiation on re-endothelialisation of circular denuded tracks made in post-confluent cultures of arterial endothelial cells (ECs) and on cellular factors involved in this process. Image analysis and time-lapse microcinematography revealed cell migration into denuded areas starting 4 h after injury. Fifty percent coverage was achieved at 14.8±2.0 h. Using competitive PCR and flow cytometry techniques, no significant changes in mRNA expression of interleukin-1β (IL-1β), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B) and tissue factor (TF), and surface molecule expression of anti-intercellular adhesion molecule-1 (ICAM-1), anti-vascular cell adhesion molecule-1 (VCAM-1), anti-platelet/endothelial cell adhesion molecule-1 (PECAM-1), MHC-1, TF and Fas were observed. However, injury did significantly ( P<0.05) elevate the release of IL-8 and FGF-2 protein in the cell culture supernatant, as assessed by ELISA. Radiation (15 Gy) given immediately after injury did not affect the kinetics of re-endothelialisation up to 48 h, in spite of the fact that no cell divisions were observed. Thereafter cell density decreased and cultures deteriorated. Compared to cultures exposed to injury alone, radiation induced significant ( P<0.05) increases in mRNA levels of IL-8 (1.35±0.10-fold increase at 4 h), FGF-2 (1.62±0.10-fold at 4 h; 1.76±0.33-fold at 24 h) and IL-1β (2.76±0.40-fold at 24 h), whereas mRNA levels of TGF-β1, PDGF-A and PDGF-B increased about 1.2-fold. IL-8 and FGF-2 protein concentrations in the media were higher than those observed in non-irradiated injured cell cultures; however, this difference was not significant. Radiation induced a 2.3±0.3-fold increase ( P<0.05) in Fas surface expression only. In conclusion, irradiation of mechanically-injured human EC leads to increased gene expression and protein secretion of inflammatory and growth promoting cytokines.

Irradiation of mechanically-injured human arterial endothelial cells leads to increased gene expression and secretion of inflammatory and growth promoting cytokines

<h2>Abstract</h2> Radiation therapy is applied to inhibit neointima formation after percutaneous transluminal coronary angioplasty (PTCA). In this study, we evaluated the effect of irradiation on re-endothelialisation of circular denuded tracks made in post-confluent cultures of arterial endothelial cells (ECs) and on cellular factors involved in this process. Image analysis and time-lapse microcinematography revealed cell migration into denuded areas starting 4h after injury. Fifty percent coverage was achieved at 14.8±2.0h. Using competitive PCR and flow cytometry techniques, no significant changes in mRNA expression of interleukin-1β (IL-1β), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B) and tissue factor (TF), and surface molecule expression of anti-intercellular adhesion molecule-1 (ICAM-1), anti-vascular cell adhesion molecule-1 (VCAM-1), anti-platelet/endothelial cell adhesion molecule-1 (PECAM-1), MHC-1, TF and Fas were observed. However, injury did significantly (<i>P</i><0.05) elevate the release of IL-8 and FGF-2 protein in the cell culture supernatant, as assessed by ELISA. Radiation (15Gy) given immediately after injury did not affect the kinetics of re-endothelialisation up to 48h, in spite of the fact that no cell divisions were observed. Thereafter cell density decreased and cultures deteriorated. Compared to cultures exposed to injury alone, radiation induced significant (<i>P</i><0.05) increases in mRNA levels of IL-8 (1.35±0.10-fold increase at 4h), FGF-2 (1.62±0.10-fold at 4h; 1.76±0.33-fold at 24h) and IL-1β (2.76±0.40-fold at 24h), whereas mRNA levels of TGF-β1, PDGF-A and PDGF-B increased about 1.2-fold. IL-8 and FGF-2 protein concentrations in the media were higher than those observed in non-irradiated injured cell cultures; however, this difference was not significant. Radiation induced a 2.3±0.3-fold increase (<i>P</i><0.05) in Fas surface expression only. In conclusion, irradiation of mechanically-injured human EC leads to increased gene expression and protein secretion of inflammatory and growth promoting cytokines.

Effects of mycophenolic acid on IL-6 expression of human renal proximal and distal tubular cells in vitro.

Interleukin-6 (IL-6) is a multifunctional cytokine which regulates immune responses and host defence mechanisms. IL-6 has been found to be increased in certain inflammatory conditions of the kidney, in which tubular epithelial cells play a pivotal role. Human renal tubular cells express IL-6. Until now no data about the effect of the immunosuppressant drug mycophenolic acid (MPA) on IL-6 expression were available. Proximal and distal tubular epithelial cells (PTC/DTC) have been isolated immunomagnetically. Confluent monolayers were stimulated with interleukin-1beta (IL-1beta; 25 U/ml), IL-1beta+ MPA (0.25-50 micro M) or MPA alone for 48 h. Release of IL-6 protein into the supernatant was evaluated with an enzyme immunoassay, IL-6 mRNA expression was evaluated using the Quantikine mRNA kit. After IL-1beta stimulation, a highly significant 2.6- (PTC) and 3.8-fold (DTC) upregulation of IL-6 expression was detectable. IL-6 mRNA was upregulated by IL-1beta [1.57- (PTC) and 2.03-fold (DTC)]. MPA inhibited this cytokine-induced IL-6 expression in a dose-dependent manner. Incubation with the lowest MPA concentration had no effect on the stimulated upregulation, whereas all higher doses significantly decreased IL-6 expression. Dexamethasone significantly inhibited the cytokine-induced IL-6 protein release in PTC, but not in DTC. In this study we demonstrated for the first time an inhibitory effect of MPA on the stimulated IL-6 expression of renal tubular epithelial cells. In contrast to older data, which showed a synergistic upregulation of the expression of a CC-chemokine by a combination of cytokines and MPA, in the present study we could demonstrate an immunosuppressive effect of MPA on the expression of an important cytokine.

PM 10 -mediated IL-8 Release from Epithelial Cells Involves Histone Acetylation

Increases in the levels of environmental particulate matter (PM 10 ) in the air are associated with a variety of adverse health effects, particularly in susceptible patients with chronic lung and cardiovascular diseases. The adverse effects in the lungs are probably caused by oxidative stress leading to lung inflammation. The expression of many genes, including those encoding proinflammatory mediators, involves the remodelling of the chromatin structure provided by histone proteins. Chromatin is tightly coiled around histone proteins and therefore the access of transcription factors to the transcriptional machinery is inhibited. Histone acetylation causes the unwinding of the chromatin structure, thereby allowing transcription factor access to promoter sites, whereas histone deacetylation has the opposite effect of winding chromatin and inhibiting transcription. Nuclear histone acetylation is reversible and is regulated by a group of histone acetyltransferases (HATs), which promote acetylation, and histone deacetylases (HDACs), which promote deacetylation. There are several co-activators, transcription factors and nuclear proteins that have HAT activity. The aim of this study was to determine whether the PM 10 -mediated mRNA expression and release of interleukin-8 (IL-8) from alveolar airspace epithelial cells is associated with histone acetylation and oxidative stress. We studied the effects of PM 10 and an HDAC inhibitor (trichostatin-A, TSA) on the release of IL-8 by enzyme-linked immunosorbent assay, and the acetylation of histone 4 (H4) was assessed by immunocytochemistry in human alveolar type II cells. PM10 and H2O2 increased IL-8 protein release from A549 cells after 20 h treatment, and this was enhanced by HDAC inhibition (TSA co-treatment). PM 10 and H 2 O 2 treatment also increased the HAT activity. PM 10 enhanced H4 acetylation, which was mediated by oxidative stress, as shown by thiol antioxidant inhibition. The PM10and TSA-mediated increases in IL-8 and histone acetylation were associated with increases in nuclear factor-κB activation. These data suggest that the remodelling of chromatin by histone acetylation plays a role in the PM10-mediated pro-inflammatory responses in the lungs. This PM 10 response is mediated by oxidative stress.

Lithium Induces NF-κB Activation and Interleukin-8 Production in Human Intestinal Epithelial Cells

Lithium has been documented to regulate apoptosis and apoptotic gene expression via NF-kappa B and mitogen-activated protein (MAP) kinase-dependent mechanisms. Since both NF-kappa B and MAP kinases are also important mediators of inflammatory gene expression, we investigated the effect of lithium on NF-kappa B- and MAP kinase-mediated inflammatory gene expression. Incubation of human intestinal epithelial cells with lithium induced both enhanced NF-kappa B DNA binding and NF-kappa B-dependent transcriptional activity. In addition, lithium stimulated activation of both the p38 and p42/44 MAP kinases. This lithium-induced up-regulation of NF-kappa B and MAP kinase activation was associated with an enhancement of interleukin-8 mRNA accumulation as well as an increase in interleukin-8 protein release. These proinflammatory effects of lithium were, in large part, mediated by activation of Na(+)/H(+) exchangers, because selective blockade of Na(+)/H(+) exchangers prevented the lithium-induced intestinal cell inflammatory response. These results demonstrate that lithium stimulates inflammatory gene expression via NF-kappa B and MAP kinase activation.

Gastrin induces CXC chemokine expression in gastric epithelial cells through activation of NF-kappaB.

Although hypergastrinemia is frequently observed in individuals with a chronic Helicobacter pylori infection, its pathophysiological significance in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of CXC chemokines in gastric epithelial cells. Human and rat gastric epithelial cells, transfected with gastrin receptor, were stimulated with gastrin. The expression of mRNAs for human interleukin-8 (IL-8) and rat cytokine-induced neutrophil chemoattractant-1 and release of human IL-8 protein were then determined by Northern blot analysis and ELISA, respectively. Gastrin not only induced the expression of mRNAs for these chemokines but also stimulated IL-8 protein release. A luciferase assay using IL-8 promoter genes showed that nuclear factor (NF)-kappaB is absolutely required and activator protein-1 (AP-1) is partly required for the maximum induction of IL-8 by gastrin. An electrophoretic mobility shift assay revealed that gastrin is capable of activating both NF-kappaB and AP-1. In addition, the inhibition of NF-kappaB abrogated gastrin-induced chemokine expression. These results suggest that gastrin is capable of upregulating CXC chemokines in gastric epithelial cells and therefore may contribute to the progression of the inflammatory process in the stomach.

Homocysteine induces expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human aortic endothelial cells: implications for vascular disease.

Proinflammatory cytokines play key roles in atherogenesis and disease progression. Because hyperhomocysteinemia is an independent risk factor for cardiovascular disease, we hypothesized that homocysteine could be atherogenic by altering the expression of specific cytokines in vascular endothelial cells. Northern blot and RNase protection assays showed that DL-homocysteine induced mRNA expression of the proinflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in cultured human aortic endothelial cells (HAECs). Homocysteine had no effect on expression of other cytokines, namely tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-1beta, and transforming growth factor-beta. MCP-1 mRNA expression increased 1 hour after homocysteine treatment, reached a maximum within 2 to 4 hours, and declined to basal levels over the next 24 hours. Induction of mRNA expression for both chemokines was observed with as little as 10 micromol/L DL-homocysteine, and maximal expression was achieved with 50 micromol/L DL-homocysteine. Homocysteine also triggered the release of MCP-1 and IL-8 protein from HAECs into the culture medium. The induction was specific for homocysteine, because equimolar concentrations of L-homocystine, L-cysteine, and L-methionine had no effect on mRNA levels and protein release. Furthermore, L-homocysteine induced chemokine expression, but D-homocysteine did not, thus demonstrating enantiomeric specificity. The culture medium from homocysteine-treated HAECs promoted chemotaxis in human peripheral blood monocytes and U937 cells. Anti-human recombinant MCP-1 antibody blocked the migration. Pathophysiological levels of L-homocysteine alter endothelial cell function by upregulating MCP-1 and IL-8 expression and secretion. This suggests that L-homocysteine may contribute to the initiation and progression of vascular disease by promoting leukocyte recruitment.

Cytokine induction as a measure of cutaneous toxicity in primary and immortalized porcine keratinocytes exposed to jet fuels, and their relationship to normal human epidermal keratinocytes

The purpose of this study was to identify biomarkers of toxicity in primary porcine keratinocytes (PKC) and an immortalized porcine keratinocyte cell line (MSK3877) exposed to jet fuels Jet A, JP-8, and JP-8+100. Cells were exposed to 0.1% jet fuels and assayed for interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) mRNA using the TaqMan real time quantitative reverse transcriptase PCR assay. IL-8 and TNF-α protein release was measured using an ELISA. PKC exposed to jet fuels caused a slight upregulation of TNF-α mRNA at early time points, but no significant differences in TNF-α protein production were detected. IL-8 mRNA was increased at 4 h following exposure, and IL-8 protein was increased at 8 h. In MSK 3877 cells, jet fuels were shown to increase the production and expression of TNF-α mRNA and protein at 30 min and 1 h following exposure, respectively. IL-8 mRNA was only slightly induced compared to control. IL-8 protein release was suppressed by jet fuel exposure. These results were compared with those of a previous study in our laboratory to evaluate the utility of using porcine cells in lieu of normal human epidermal keratinocytes (NHEK). Similarities exist between PKC and NHEK with respect to both TNF-α and IL-8 production. The expression profile of TNF-α in MSK3877 cells mimics that of NHEK. In contrast, the profile of IL-8 expression opposes that of PKC and NHEK. These results suggest that porcine keratinocytes are susceptible to jet fuel toxicity. However, the responses of immortalized cells may vary from those of PKC and NHEK necessitating cautious interpretation of such data.

Shiga Toxin-1 Regulation of Cytokine Production by Human Glomerular Epithelial Cells

Background/Aims: Inflammatory cytokines may enhance renal injury in post-diarrheal hemolytic uremic syndrome (Stx HUS) by enhancing the cytotoxic effect of Shiga toxins (Stx). The sources of inflammatory cytokines in Stx HUS are unclear. Since Stx-1 potently inhibits protein synthesis by glomerular epithelial cells (GEC) and increases cytokine release by renal epithelial cells, we examined Stx-1 regulation of cytokine production by human GEC. Methods: Stx-1 (and cycloheximide (CHX), another protein synthesis inhibitor) cytotoxicity, protein synthesis inhibition, and effect on interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) release and mRNA levels were determined. Results: Stx-1 alone had a modest stimulatory effect on inflammatory cytokine production by GEC that occurred at toxin concentrations ranging from minimal to 50% inhibition of protein synthesis. CHX, at concentrations that produced similar inhibition of protein synthesis, increased IL-1, IL-6, and TNF protein release and mRNA accumulation, but in a different time- and dose-dependent pattern than Stx. Lipopolysaccharide (LPS) did not change IL-1, but stimulated IL-6 and TNF production. LPS and Stx-1 combined stimulated production of all three cytokines to a greater extent than either toxin alone. Conclusion: These data indicate that: (1) Stx-1 alone modestly stimulates GEC inflammatory cytokine production; (2) LPS and Stx-1 combined can potently enhance GEC cytokine release, and (3) this action of Stx-1 may relate in part to inhibition of protein synthesis but cannot be fully attributed to this effect.

Identification of a possible association between carbon tetrachloride-induced hepatotoxicity and interleukin-8 expression.

Hepatotoxicants can elicit liver damage by various mechanisms that can result in cell necrosis and death. The changes induced by these compounds can vary from gross alterations in DNA repair mechanisms, protein synthesis, and apoptosis, to more discrete changes in oxidative damage and lipid peroxidation. However, little is known of the changes in gene expression that are fundamental to the mechanisms of hepatotoxicity. We have used DNA microarray technology to identify gene transcription associated with the toxicity caused by the hepatotoxicant carbon tetrachloride. Labeled poly A+ RNA from cultured human hepatoma cells (HepG2) exposed to carbon tetrachloride for 8 hours was hybridized to a human microarray filter. We found that 47 different genes were either upregulated or downregulated more than 2-fold by the hepatotoxicant compared with dimethyl formamide, a chemical that does not cause liver cell damage. The proinflammatory cytokine interleukin-8 (IL-8) was upregulated over 7-fold compared with control on the array, and this was subsequently confirmed at 1 hour and 8 hours by Northern blot analyses. We also found that carbon tetrachloride caused a time-dependent increase in interleukin-8 protein release in HepG2 cells, which was paralleled by a decrease in cell viability. These data demonstrate that carbon tetrachloride causes a rapid increase in IL-8 mRNA expression in HepG2 cells and that this increase correlates with a later and significant increase in the levels of interleukin-8 protein. These results illustrate the potential of microarray technology in the identification of novel gene changes associated with toxic processes.

Erythromycin Suppresses Nuclear Factor-κB and Activator Protein-1 Activation in Human Bronchial Epithelial Cells

Erythromycin (EM), and related 14-member macrolide antibiotics, has attracted attention for its effectiveness in airway diseases including diffuse panbronchiolitis and sinobronchial syndrome. However, its molecular mechanisms remain unknown. We evaluated the effects of EM on activation of several transcription factors, including nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) in human bronchial epithelial cell line BET-1A, which are known to regulate the expression of many proinflammatory cytokines and chemokines such as interleukin-8 (IL-8). BET-1A cells were cultured with hormonally defined Ham's F12 medium, and were stimulated by phorbol myristate acetate (PMA). EM suppressed mRNA expression as well as the release of IL-8 at therapeutic and noncytotoxic concentrations (% inhibition of IL-8 protein release: 42.2 ± 5.5%, at 10−6 M). Furthermore, electrophoretic mobility shift assays revealed that EM inhibited the activations of NF-κB and AP-1 induced by PMA in BET-1A cells. These data indicate that EM has inhibitory effects not only on the mRNA expression and release of IL-8, but also on the activation of transcription factors NF-κB and AP-1. Our findings support the concept that the recruitment of neutrophils in airway diseases may be regulated by NF-κB and AP-1.

Modulation of Helicobacter pylori induced interleukin-8 synthesis in gastric epithelial cells mediated by cag PAI encoded VirD4 homologue.

BACKGROUND: Strains of Helicobacter pylori carrying the virulence associated cag pathogenicity island (PAI) induce gastric epithelial synthesis of the chemokine interleukin-8 (IL-8), a neutrophil chemoattractant, and thereby a strong inflammatory...

Shiga toxin-1 regulation of cytokine production by human proximal tubule cells

Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) levels are elevated in kidneys of patients with post-diarrheal hemolytic uremic syndrome (D+HUS) and may contribute to renal dysfunction. The renal cellular sources of these inflammatory cytokines in D+HUS are largely unknown, however, the proximal tubule has emerged as a potentially important candidate. Since Shiga toxin-1 (Stx-1) has been implicated in the genesis of D+HUS, we examined the effect of Stx-1 on cytokine production by human proximal tubule cells. Stx-1 cytotoxicity, protein synthesis inhibition, and effect on IL-1, IL-6, and TNF protein release and mRNA levels were determined. The effect of another protein synthesis inhibitor, cycloheximide (CHX), on these parameters was also evaluated. Stx-1 greatly increased TNF release and mRNA levels while CHX, at concentrations that produced similar inhibition of protein synthesis, had no effect on TNF production. In contrast, Stx-1 and CHX caused comparable elevations in IL-1 release and mRNA accumulation. Stx-1 and CHX also stimulated IL-6 mRNA accumulation, but only at concentrations that either were cytotoxic or substantially blocked protein synthesis. Finally, lipopolysaccharide, which is likely to be elevated in the circulation of patients with D+HUS, had no effect alone, but synergized with Stx-1 to increase IL-1 production. These results indicate that Stx-1 stimulates proximal tubule inflammatory cytokine production and that this effect is due partially to nonspecific induction of mRNA levels as well as activation of Stx-1-specific mechanisms.

Inhibition of TNF-alpha-induced NF-kappaB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132.

The working hypothesis of the studies described herein was that inhibition of proteasome-mediated IkappaB degradation would inhibit TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappaB site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of IkappaB from the cytoplasm of cells, associated with a corresponding increase in NF-kappaB-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 microM) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on IkappaB degradation as determined by Western blot analysis. IkappaB phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappaB binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated IkappaB degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappaB-mediated gene transcription.

Regulation of Interleukin-8 Gene Expression by All-trans Retinoic Acid

We have studied the relationship between interleukin-8 (IL-8) and interleukin-1α (IL-1α) release after stimulation with all-trans retinoic acid (ATRA) and tumor necrosis factor-α (TNF-α) in the human epithelial ovarian cancer cell line HOC-7. Both IL-1α and IL-8 protein release were enhanced by treatment with ATRA and TNF-α after 48 h exposure. Blocking of IL-1α activity in HOC-7 cells with either IL-1 receptor antagonist (IL-1ra) or a neutralizing antibody directed against IL-1α resulted in a dose-dependent decrease of IL-8 release by ATRA, TNF-α and IL-1α treated HOC-7 cells. Expression of IL-8 mRNA was enhanced by the individual stimuli, whereas cotreatment with IL-lra resulted in a loss of IL-8 specific transcripts, except in TNF-α treated cells. Inhibition of de novo protein synthesis by cycloheximide (CHX) and simultaneous blocking of IL-1α activity by IL-1ra for 24 h revealed that ATRA controls IL-8 gene expression transcriptionally and that the extent of IL-8 protein release can be markedly influenced by cellular expressed IL-1α.

Study on IL-8 Expression in Peripheral Blood Monocytes

Background: Peripheral blood monocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities, monocytes/macrophage produce a vast array of regulatory and chemotactic cytokines. Interleukin-8(IL-8), a potent neutrophil-activating and chemotactic peptide, is produced in large quantities by mononuclear phagocytes and may be an important mediator of local and systemic inflammation. Overexpression by IL-8 of such inflammation may be an important step of tissue injury frequently seen in inflammatory reaction. So it could be hypothesized that the agents which block the production of IL-8 can decrease the inflammatory reaction and tissue injury. To evaluate this, we described the effect of Dexamethasone, , Indomethacin and Interferon-(IFN-) on IL-8 mRNA and protein expression from LPS-stimulated human peripheral blood monocytes(PBMC). Method: PBMC was isolated from healthy volunteers. To evaluate the effect of Dexamethasone, & Indomethacin, these drug were treated for 1 hour before and after LPS stimulation and IFN- was only treated I hour before the LPS stimulation. Northern blot analysis for IL-8 mRNA and ELISA for immunoreactive IL-8 protein in culture supernatant were performed. We repeated above experiment three times for Northern blot analysis and two times for ELISA and got the same result. Results: 1) Pre- and post-treatment of Dexamethasone suppressed both the LPS stimulated IL-8 mRNA expression and IL-8 protein release in PBMC. 2) IFN- pre-treatment suppressed the IL-8 mRNA expression and IL-8 protein release in unstimulated cells. 3) In LPS stimulated cells, IFN- suppressed the IL-8 mRNA expression but IL-8 protein release suppression was not observed. 4) and Indomethacin exert no effect on the LPS-stimulated IL-8 mRNA and protein expression in concentration used in this experiment (, Indomethacin; ). Conclusion: One of the mechanism of antiinflammatory action of Dexamethasone can be explained by the suppressing effect of IL-8 production in some extent and by this antiinflammatory effect, dexamethasone can be used to suppress local and systemic inflammation mediated by IL-8.