Journal of Inflammation - Latest articles

Augmentation of Pulmonary Epithelial Cell IL-8 Expression and Permeability by Pre-B-cell Colony Enhancing Factor

2008-09-22

Background: Previous studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI). The molecular mechanism of PBEF involvement in the pathogenesis of ALI is still incompletely understood. This study examined the role of PBEF in regulating pulmonary alveolar epithelial cell IL-8 expression and permeability. Methods: Human pulmonary alveolar epithelial cells (cell line and primary cells) were transfected with human PBEF cDNA or PBEF siRNA and then cultured in the presence or absence of TNFα. PBEF and IL-8 expression were analyzed by RT-PCR and Western blotting. In addition, changes in pulmonary alveolar epithelial and artery endothelial cell barrier regulation with altered PBEF expression was evaluated by an in vitro cell permeability assay. Results: Our results demonstrated that, in human pulmonary alveolar epithelial cells, the overexpression of PBEF significantly augmented basal and TNFα-stimulated IL-8 secretion by more than 5 to 10-fold and increased cell permeability by >30%; the knockdown of PBEF expression with siRNA significantly inhibited basal and TNFα-stimulated IL-8 secretion by 70% and IL-8 mRNA levels by 74%. Further, the knockdown of PBEF expression also significantly attenuated TNFα-induced cell permeability by 43%. Similar result was observed in human pulmonary artery endothelial cells. Conclusion: These results suggest that PBEF may play a vital role in basal and TNFα-mediated pulmonary inflammation and pulmonary epithelial barrier dysfunction via its regulation of other inflammatory cytokines such as IL-8, which could in part explain the role of PBEF in the susceptibility and pathogenesis of ALI. These results lend further support to the potential of PBEF to serve as a diagnostic and therapeutic target to ALI.

15-deoxy-delta12,14-prostaglandin J2 attenuates endothelial-monocyte interaction: implication for inflammatory diseases

Ratna Prasad, Shailendra Giri, Avtar K Singh and Inderjit Singh — 2008-08-08

Background: The Infiltration of leukocytes across the brain endothelium is a hallmark of various neuroinflammatory disorders. Under inflammatory conditions, there is increased expression of specific cell adhesion molecules (CAMs) on activated vascular endothelial cells which increases the adhesion and infiltration of leukocytes. TNFα is one of the major proinflammatory cytokines that causes endothelial dysfunction by various mechanisms including activation of transcription factor NF-κB, a key transcription factor that regulates expression of CAMs. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone superfamily of ligand-activated transcriptional factors. 15-deoxy-δ 12, 14-prostaglandin J2 (15d-PGJ2) is a well recognized natural ligand of PPARγ and possesses anti-inflammatory properties both in vitro and in vivo. This study aims to elucidate the mechanism of 15-PGJ2 on the adhesion of mononuclear cells to activated endothelial cells. Methods: To delineate the signaling pathway of 15d-PGJ2 mediated effects, we employed an in vitro adhesion assay model of endothelial-monocyte interaction. Expression of CAMs was examined using flow cytometry and real time PCR techniques. To define the mechanism of 15d-PGJ2, we explored the role of NF-κB by EMSA (Electrophoretic Mobility Shift Assay) gels, NF-κB reporter and p65-transcriptional activities by transient transfection in the brain-derived endothelial cell line (bEND.3). Results: Using an in vitro adhesion assay model, we demonstrate that 15d-PGJ2 inhibits TNFα induced monocyte adhesion to endothelial cells, which is mediated by downregulation of endothelial cell adhesion molecules in a PPARγ independent manner. 15d-PGJ2 modulated the adhesion process by inhibiting the TNFα induced IKK-NF-κB pathway as evident from EMSA, NF-κB reporter and p65 mediated transcriptional activity results in bEND.3 cells. Conclusion: These findings suggest that 15d-PGJ2 inhibits inflammation at multiple steps and thus is a potential therapeutic target for various inflammatory diseases.

Does carbon monoxide treatment alter cytokine levels after endotoxin infusion in pigs? A randomized controlled study

2008-08-07

Background: Carbon monoxide (CO) has recently been suggested to have anti-inflammatory properties, but data seem to be contradictory and species-specific. Thus, in studies on macrophages and mice, pretreatment with CO attenuated the inflammatory response after endotoxin exposure. On the other hand, human studies showed no effect of CO on the inflammatory response. Anti-inflammatory efficacy of CO has been shown at concentrations above 10% carboxyhaemoglobin. This study was undertaken to elucidate the possible anti-inflammatory effects of CO at lower CO concentrations. Methods: Effects of CO administration on cytokine (TNF-alpha, IL-6, IL-1beta and IL-10) release were investigated in a porcine model in which a systemic inflammatory response syndrome was induced by endotoxin infusion. Endotoxin was infused in 20 anaesthetized and normoventilated pigs. Ten animals were targeted with inhaled CO to maintain 5% COHb, and 10 animals were controls. Results: In the control group, mean pulmonary artery pressure increased from a baseline value of 17 mmHg (mean, n = 10) to 42 mmHg (mean, n = 10) following 1 hour of endotoxin infusion. Similar mean pulmonary artery pressure values were found in animals exposed to carbon monoxide. Plasma levels of all of the measured cytokines increased in response to the endotoxin infusion. The largest increase was observed in TNF-alpha, which peaked after 1.5 hours at 9398 pg/ml in the control group and at 13395 pg/ml in the carbon monoxide-exposed group. A similar peak was found for IL-10 while the IL-6 concentration was maximal after 2.5 hours. IL-1beta concentrations increased continuously during the experiment. There were no significant differences between carbon monoxide-exposed animals and controls in any of the measured cytokines. Conclusion: Our conclusion is that 5% COHb does not modify the cytokine response following endotoxin infusion in pigs.

A novel hybrid aspirin-NO-releasing compound inhibits TNFalpha release from LPS-activated human monocytes and macrophages

2008-07-31

Background: The cytoprotective nature of nitric oxide (NO) led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNFα release from lipopolysaccharide (LPS)-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-κB) activation. Methods: Peripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 μM), their respective furazan NO-free counterparts (B16, B15; 10 μM), aspirin (10 μM), existing nitroaspirin (NCX4016; 10 μM), an NO donor (DEA/NO; 10 μM) or dexamethasone (1 μM), in the presence and absence of LPS (10 ng/ml; 4 h). Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNFα release and by lactate dehydrogenase (LDH) assay for cell necrosis. To assess NF-κB activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-κB, IκBα (assessed by western blotting) and nuclear localisation (assessed by immunofluorescence) of the p65 subunit of NF-κB were determined. Results: B8 significantly reduced TNFα release from LPS-treated macrophages to 36 ± 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNFα release to 28 ± 5, and 49 ± 9% of control, respectively. The B8 effect was equivalent in magnitude to that of dexamethasone, but was not shared by 10 μM DEA/NO, B7, the furazans, aspirin or NCX4016. LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic IκBα and nuclear translocation of the p65 NF-κB subunit was inhibited by the active NO-furoxans. Conclusion: Here we show that furoxan-aspirin, B8, significantly reduces TNFα release from both monocytes and macrophages and suggest that inhibition of NF-κB activation is a likely mechanism for the effect. This anti-inflammatory action highlights a further therapeutic potential of drugs of this class.

Anti-inflammatory effects of ciprofloxacin in S. aureus Newman induced nasal inflammation in vitro

F Sachse, C von Eiff, K Becker and C Rudack — 2008-07-29

ObjectivesChronic rhinosinusitis (CRS) is a chronic inflammatory disease of the nasal mucosa. Recent studies suggest that S. aureus enterotoxins may play an etiologic role in the development of CRS. Apart from surgery and repeated courses of steroids, macrolide antibiotics have been reported to exert anti-inflammatory effects in CRS. Similar effects have been reported for fluoroquinolones on various cell types. Since these effects have poorly been characterized in CRS, we examined anti-inflammatory effects of ciprofloxacin on human nasal epithelial cells (HNECs). Methods: Inflammation was induced in HNECs cultured from nasal turbinate mucosa with supernatants of S. aureus Newman for 12 hours. Subsequently, HNECs were coincubated with S. aureus Newman and ciprofloxacin (1.5 × 10-5 M), clarithromycin (10-6 M) or prednisolone (10-5 M) for another 12 hours. IL-8 synthesis was quantified after 12 and 24 hours by ELISA. Results: Stimulation with S. aureus Newman supernatants was associated with an increase of IL-8 synthesis after 12 hours in all experiments. During the second 12 hours, IL-8 synthesis decreased and this effect was independent from any stimulus or inhibitor. However, coincubation of HNECs with ciprofloxacin was associated with a more extensive decrease of IL-8 synthesis. Similarly, addition of clarithromycin was associated with a reduction of IL-8 synthesis although this effect was not significant. Coincubation with prednisolone resulted in a significant reduction of IL-8 levels. Conclusion: Ciprofloxacin exerts anti-inflammatory effects in S. aureus Newman driven nasal inflammation. Inhibitory effects were comparable to those of prednisolone and clarithromycin.

Serum tumor necrosis factor-alpha concentrations are negatively correlated with serum 25(OH)D concentrations in healthy women

Catherine A Peterson and Mary E Heffernan — 2008-07-24

Background: Circulating 25 hydroxyvitamin D (25 (OH)D), an accurate measure of vitamin D status, is markedly greater in individuals with increased exposure to ultraviolet B (UVB) light via sunlight or the use of artificial UV light. Aside from the known relationship between vitamin D and bone, vitamin D has also been implicated in immune function and inflammation. Furthermore, a mass of evidence is accumulating that vitamin D deficiency could lead to immune malfunction. Our overall objective was to study the relationship between vitamin D status (as determined by serum 25(OH) D concentrations) and inflammatory markers in healthy women. Methods: This observational study included 69 healthy women, age 25–82 years. Women with high UVB exposure and women with minimal UVB exposure were specifically recruited to obtain a wide-range of serum 25(OH)D concentrations. Health, sun exposure and habitual dietary intake information were obtained from all subjects. Body composition was determined by dual-energy-x-ray absorptiometry. A fasting blood sample was collected in the morning and analyzed for serum 25(OH)D, parathyroid hormone (iPTH), estradiol (E2), cortisol, and inflammatory markers [tumor necrosis factor -alpha (TNF-α), interleukin-6 and -10 (IL-6, IL-10), and C-reactive protein (CRP)]. Results: Women with regular UVB exposure (Hi-D) had serum 25(OH)D concentrations that were significantly higher (p < 0.0001) and iPTH concentrations that were significantly lower (p < 0.0001) than women without regular UVB exposure (Lo-D). Although IL-6, IL-10, and CRP did not have a statistically significant relationship with 25(OH)D concentrations, linear regression models revealed a significant inverse relationship between serum 25(OH)D and TNF-α concentrations. This relationship remained significant after controlling for potential covariates such as body fat mass, menopausal status, age, or hormonal contraceptive use. Conclusion: Serum 25(OH)D status is inversely related to TNF-α concentrations in healthy women, which may in part explain this vitamin's role in the prevention and treatment of inflammatory diseases. Results gleaned from this investigation also support the need to re-examine the biological basis for determining optimal vitamin D status.

Bioavailable constituents/metabolites of pomegranate (Punica granatum L) preferentially inhibit COX2 activity ex vivo and IL-1beta-induced PGE2 production in human chondrocytes in vitro

2008-06-13

Several recent studies have documented that supplementation with pomegranate fruit extract inhibits inflammatory symptoms in vivo. However, the molecular basis of the observed effects has not been fully revealed. Although previous studies have documented the inhibition of nitric oxide and cyclooxygenase (COX) activity in vitro by plant and fruit extracts added directly into the culture medium but whether concentrations of bioactive compounds sufficient enough to exert such inhibitory effects in vivo can be achieved through oral consumption has not been reported. In the present study we determined the effect of rabbit plasma obtained after ingestion of a polyphenol rich extract of pomegranate fruit (PFE) on COX enzyme activity ex vivo and the IL-1β-induced production of NO and PGE2 in chondrocytes in vitro. Plasma samples collected before and 2 hr after supplementation with PFE were tested. Plasma samples collected after oral ingestion of PFE were found to inhibit the IL-1β-induced PGE2 and NO production in chondrocytes. These same plasma samples also inhibited both COX-1 and COX-2 enzyme activity ex vivo but the effect was more pronounced on the enzyme activity of COX-2 enzyme. Taken together these results provide additional evidence of the bioavailability and bioactivity of compounds present in pomegranate fruit after oral ingestion. Furthermore, these studies suggest that PFE-derived bioavailable compounds may exert an anti-inflammatory effect by inhibiting the inflammatory cytokine-induced production of PGE2 and NO in vivo.

IFN-gamma regulation of ICAM-1 receptors in bronchial epithelial cells: soluble ICAM–1 release inhibits human rhinovirus infection

Suzanne C Whiteman and Monica A Spiteri — 2008-06-05

Background: Intercellular adhesion molecule-1 (ICAM-1) is a critical target-docking molecule on epithelial cells for 90% of human rhinovirus (HRV) serotypes. Two forms of ICAM-1 exist, membranous (mICAM-1) and soluble (sICAM-1), both expressed by bronchial epithelial cells. Interferon-gamma (IFN-γ), a crucial Th-1 immuno-regulatory mediator, can modulate mICAM-1 expression; however its simultaneous effects on mICAM-1: sICAM-1 levels and their consequent outcome on cell infectivity have not been previously explored. Methods: Primary normal human bronchial epithelial cells were pre-stimulated with IFN-γ (1 ng/ml for 24 h) and subsequently inoculated with HRV-14 or HRV-1b (TCID50 10 2.5). Epithelial surface ICAM-1 expression and soluble ICAM-1 release were measured at the protein and gene level by immunofluorescence and ELISA respectively; mRNA levels were semi-quantified using RT-PCR. Molecular mechanisms regulating ICAM-1 isoform expression and effects on epithelial cell infectivity were explored. Results: In IFN-γ-biased cells infected with HRV-14, but not HRV-1b, mICAM-1 expression is down-regulated, with simultaneous induction of sICAM-1 release. This differential effect on HRV-14 receptor isoforms appears to be related to a combination of decreased IFN-γ-induced JAK-STAT signalling and proteolytic receptor cleavage of the membranous form in IFN-γ-biased HRV-14 infected cells. The observed changes in relative mICAM-1: sICAM-1 expression levels are associated with reduced HRV-14 viral titres. Conclusion: These findings support the hypothesis that in epithelial cells conditioned to IFN-γ and subsequently exposed to HRV-14 infection, differential modulation in the ratio of ICAM-1 receptors prevails in favour of an anti-viral milieu, appearing to limit further target cell viral attachment and propagation.

JNK pathway is involved in the inhibition of inflammatory target gene expression and NF-kappaB activation by melittin

2008-05-29

Background: Bee venom therapy has been used to treat inflammatory diseases including rheumatoid arthritis in humans and in experimental animals. We previously found that bee venom and melittin (a major component of bee venom) have anti-inflammatory effect by reacting with the sulfhydryl group of p50 of nuclear factor-kappa B (NF-κB) and IκB kinases (IKKs). Since mitogen activated protein (MAP) kinase family is implicated in the NF-κB activation and inflammatory reaction, we further investigated whether activation of MAP kinase may be also involved in the anti-inflammatory effect of melittin and bee venom. Methods: The anti-inflammatory effects of melittin and bee venom were investigated in cultured Raw 264.7 cells, THP-1 human monocytic cells and Synoviocytes. The activation of NF-κB was investigated by electrophoretic mobility shift assay. Nitric oxide (NO) and prostaglandin E2 (PGE2) were determined either by Enzyme Linked Immuno Sorbent Assay or by biochemical assay. Expression of IκB, p50, p65, inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) as well as phosphorylation of MAP kinase family was determined by Western blot. Results: Melittin (0.5–5 μg/ml) and bee venom (5 and 10 μg/ml) inhibited lipopolysaccharide (LPS, 1 μg/ml) and sodium nitroprusside (SNP, 200 μM)-induced activation of c-Jun NH2-terminal kinase (JNK) in RAW 264.7 cells in a dose dependent manner. However, JNK inhibitor, anthra [1,9-cd]pyrazole-6 (2H)-one (SP600215, 10–50 μM) dose dependently suppressed the inhibitory effects of melittin and bee venom on NF-κB dependent luciferase and DNA binding activity via suppression of the inhibitory effect of melittin and bee venom on the LPS and SNP-induced translocation of p65 and p50 into nucleus as well as cytosolic release of IκB. Moreover, JNK inhibitor suppressed the inhibitory effects of melittin and bee venom on iNOS and COX-2 expression, and on NO and PGE2 generation. Conclusion: These data show that melittin and bee venom prevent LPS and SNP-induced NO and PGE2 production via JNK pathway dependent inactivation of NF-κB, and suggest that inactivation of JNK pathways may also contribute to the anti-inflammatory and anti-arthritis effects of melittin and bee venom.

The tripeptide feG inhibits leukocyte adhesion

Ronald D Mathison, Emily Christie and Joseph S Davison — 2008-05-20

Background: The tripeptide feG (D-Phe-D-Glu-Gly) is a potent anti-inflammatory peptide that reduces the severity of type I immediate hypersensitivity reactions, and inhibits neutrophil chemotaxis and adhesion to tissues. feG also reduces the expression of β1-integrin on circulating neutrophils, but the counter ligands involved in the anti-adhesive actions of the peptide are not known. In this study the effects of feG on the adhesion of rat peritoneal leukocytes and extravasated neutrophils to several different integrin selective substrates were evaluated. Results: The adhesion of peritoneal leukocytes and extravasated neutrophils from rats to adhesive proteins coated to 96-well plates was dependent upon magnesium (Mg2+) ion, suggestive of integrin-mediated adhesion. feG inhibited leukocyte adhesion, but only if the cells were stimulated with PAF (10-9M), indicating that feG's actions in vitro require cell activation. In the dose range of 10-10M to 10-12M feG inhibited the adhesion of peritoneal leukocytes to fibrinogen and fibronectin, but not IgG, vitronectin or ICAM-1. feG inhibited the binding of extravasated neutrophils to heparin, IgG, fibronectin and CD16 antibody. Antigen-challenge of sensitized rats reduced the adhesion of peritoneal leukocytes to most substrates and abolished the inhibitory effects of feG. However, pretreating the animals with intraperitoneal feG (100 μg/kg) 18 h before collecting the cells from the antigen-challenged animal restored the inhibition of adhesion by in vitro feG of peritoneal leukocytes and extravasated neutrophils to fibronectin. Conclusion: The modulation of leukocyte adhesion by feG appears to involve actions on αMβ2 integrin, with a possible interaction with the low affinity FcγRIII receptor (CD16). The modulation of cell adhesion by feG is dual in nature. When administered in vivo, feG prevents inflammation-induced reductions in cell adhesion, as well as restoring its inhibitory effect in vitro. The mechanism by which in vivo treatment with feG exerts these effects remains to be elucidated.