The inflammatory balance in human sepsis

Sepsis and its potential degradation into multiple organ failure, reflects a complex immunological process occurring system wide, fueled by local and systemic proinflammatory stimuli, the activity of which is only minimally reflected in the levels of pro- and anti-inflammatory mediators found in the blood. On a cellular level, sepsis encompasses the up-regulation of both pro- and anti-inflammatory pathways. Inflammation is augmented through activation of the intracellular promoter protein nuclear factor κB (NF-κB), which induces synthesis of mRNA coding for many of the pro-inflammatory mediators, including TNF-α, IL-8, and inducible nitric oxide synthase. Similarly, the same stimuli also produce parallel activation of the heat shock protein (hsp) pathways that inhibit NF-κB activation and minimize the inflammatory response. It is the balance between these two pathways that appears to determine the immune responsiveness of the cells both locally and system wide. By characterizing this immune responsiveness, one may understand better the determinants of the ultimate inflammatory response and potential immunotherapy.     

Altered cytokine profiles of mononuclear cells after stimulation in a patient with Blau syndrome

Autoinflammatory Blau syndrome (BS) is associated with NOD2 gene mutations that lead to constitutive NFκB activation. NOD2 functions as an intracellular receptor for the muramyl dipeptide (MDP) component of peptidoglycan (PGN). The objectives of this study are to analyse whether NFκB activation in BS affects immune cell functions, and whether NOD2 and toll-like receptor (TLR) pathways interact. Peripheral blood mononuclear cells (MNCs) from a BS patient and three normal donors were analyzed for their ability to produce pro- and anti-inflammatory cytokines in the presence and absence of MDP, PGN, and lipopolysaccharide (LPS). The results obtained showed that the basal TNF-α and IL-10 production by MNCs over 24 h of incubation was very low for both the patient and the normal donors. However, upon stimulation with MDP, LPS, and PGN, the cells from the BS patient produced much lower levels of TNF-α, IL-10, G-CSF, and IFN-γ than the normal donor cells. We conclude that the pathogenic mechanism responsible for the chronic inflammation that characterizes BS may relate to the impaired production of both pro- and anti-inflammatory cytokines to stimuli. The NOD2 pathway possibly interacts with either the TLR2 or TLR4 pathways.     

Nicotine inhibits tumor necrosis factor-α induced IL-6 and IL-8 secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis

It was recently demonstrated that the cholinergic anti-inflammatory pathway can modulate host inflammatory responses via cholinergic mediators or via electrical stimulation of the vagus nerve. Here, we investigated whether nicotine, a selective cholinergic agonist, plays any anti-inflammatory role in rheumatoid arthritis fibroblast-like synoviocytes (FLS). We observed that low concentrations (0.1–100 μM) of nicotine did not affect FLS viability in lactate dehydrogenase release test or the MTT assay. Nicotine at concentrations of 0.1–10 μM dose reduced the protein and mRNA expression of IL-6 and IL-8 induced by tumor necrosis factor-α (TNFα). Nicotine also inhibited nuclear factor (NF)-κB (p65) translocation from the cytoplasm to the nucleus, based on Western blotting and immunocytochemical analysis. In conclusion, nicotine can inhibit the TNFα dependant inflammatory pathway in synoviocytes by suppressing the activation of the NF-κB pathway.     

HMG-CoA reductase inhibitor simvastatin suppresses Toll-like receptor 2 ligand-induced activation of nuclear factor kappa B by preventing RhoA activation in monocytes from rheumatoid arthritis patients

To investigate whether anti-inflammatory effects of HMG-CoA reductase inhibitor simvastatin (SMV) in rheumatoid arthritis (RA) is mediated by Toll-like receptor-2 (TLR-2) signal via inhibiting activation of RhoA, a small Rho GTPase that plays an important role in inflammatory responses. Peripheral blood monocytes from active RA patients were treated with Staphylococcus aureus peptidoglycan (PG), a ligand of TLR-2, in the presence or absence of SMV. RhoA activity was assessed by a pull-down assay. DNA-binding activity was measured by a sensitive multi-well colorimetric assay. Cytokine secretion was measured by ELISA. PG stimulation increased the level of active GTP-bound RhoA compared with unstimulated monocytes, and the effect of PG on RhoA activity was suppressed with anti-TLR-2 monoclonal antibody. RhoA inhibition either with a specific inhibitor or by siRNA transfection inhibited activation of NF-κB and secretion of TNFα and IL-1β in PG-induced RA monocytes. SMV mitigated PG-induced increase in RhoA activity and NF-κB activation as well as secretion of TNFα and IL-1β. The inhibitory effects of SMV were completely reversed by mevalonate and geranylgeranyl pyrophosphate. Our results indicate the modulation of RhoA on TLR-2-mediated inflammatory signaling in RA and provide a novel evidence for anti-inflammatory effects of statins through influencing TLR-2 signaling via RhoA in RA.     

Cocaine, not morphine, causes the generation of reactive oxygen species and activation of NF-κB in transiently cotransfected heart cells

This study was designed to determine levels of NF-κB reporter gene activity and free radical generation in cultured striated myocytes (H9C2 cells) exposed to cocaine or morphine in the presence of free radical scavengers. Cells were transiently transfected with a NF-κB reporter gene and changes in luciferase activity were detected, by bioluminescence. Using confocal microscopy and 2′,7′-dichlorofluorescin diacetate, cocaine-induced or morphine-induced free radicals were quantified in H9C2 cells. Cocaine and morphine (0–1×10⁻² M) were tested separately. Cocaine but not morphine significantly activated Nf-κB reporter gene, activity in H9C2 cells. Overexpression of IκB inhibited NF-κB reporter activity at low (1×10⁻⁴ M) but not high (1×10⁻² M) cocaine concentrations. Free radicals were generated in H9C2 cells stimulated with cocaine but not with morphine. The production of free radicals and NF-κB reporter gene activity could be blocked with N-acetylcysteine, glutathione, and to a lesser extent, lipoic acid. The results suggest that cocaine induces free radical production, which leads to the activation of NF-κB signal transduction and possible inflammatory responses.     

Steroid resistance in asthma: Mechanisms and treatment options

Glucocorticoid insensitivity presents a profound management problem in patients with asthma because conventional therapies are not effective. Glucocorticoids, acting through the glucocorticoid receptor (GR), are able to selectively repress inflammatory gene expression by utilizing several distinct mechanisms targeting nuclear factor-κB and activator protein-1 activation complexes and by effects on mitogen-activated protein kinases. Different model systems often activate distinct sets of signaling molecules and different glucocorticoid responsiveness may result from differences in concentrations and timing of steroid treatment of cells, GR expression levels, and the precise inflammatory stimulus used. Thus, abnormal activation of many signaling pathways may affect corticosteroid responsiveness in patients with corticosteroid-resistant asthma. Understanding the molecular mechanisms of GR action and inaction may lead to the development of new anti-inflammatory drugs or enable clinicians to reverse the relative steroid-insensitivity that is characteristic of some patients with severe asthma.     

Activation of NF-κB in intestinal epithelial cells by<Emphasis Type="Italic"> E. coli</Emphasis> strains isolated from the colonic mucosa of IBD patients

Background and aims The involvement of bacteria in the pathogenesis of inflammatory bowel disease has been discussed for several years. In this study we evaluated the ability of E. coli isolates from inflamed and noninflamed colonic mucosa to activate NF-B.Materials and methods Fifteen bacterial strains from inflamed and six from noninflamed colonic tissues from IBD patients. Their ability to induce NF-B activation was examined in vitro by gel-shift assays. The activation of the TNF- promoter was determined by reporter gene assays. Bacterial isolates were characterized by invasion assays, electron microscopy, and PCR.Results Four of 15 E. coli bacterial isolates from inflamed IBD tissues induced NF-B activity in intestinal epithelial cells as determined by gel-shift assays. NF-B activation was only seen with living bacteria but not with heat-inactivated cells. Isolates from noninflamed tissues and a wild-type E. coli control strain induced a weaker or no activation. Reporter gene assays with a construct comprising a luciferase gene driven by the TNF- promoter revealed that isolates from Crohns disease patients induced a stronger activation of the TNF- gene than isolates from ulcerative colitis patients. The isolated bacteria invaded HT-29 cells, although typical virulence genes for enteropathogenic, enterhemorrhagic, or enteroinvasive E. coli, i.e., eae, tir, EspA, Per (A-C), ipaC, were not detected in these cells. Bacterial invasion was additionally confirmed by electron microscopy examination.Conclusion Our results indicate that E. coli strains can be found in the mucosa of some IBD patients which are able to activate NF-B similar to known pathogenic strains. The absence of several virulence genes in these cells suggests that they are members of the luminal flora which acquire as yet unidentified virulence determinants and are therefore involved in the pathophysiology of IBD.     

Lancemaside A ameliorates colitis by inhibiting NF-κB activation in TNBS-induced colitis mice

Purpose  

In a preliminary study, we found that lancemaside A, which is a main constituent of Codonopsis lanceolata used as an herbal medicine for inflammatory diseases, potently inhibits lipopolysaccharide (LPS)-stimulated, TLR-4-linked NF-κB activation of NF-κB luciferase reporter gene-transfected 293-hTLR4-hemagglutinin (HA) cells. Therefore, we investigated its inhibitory effect in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice.     

Kidney-targeting Smad7 gene transfer inhibits renal TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways, and improves diabetic nephropathy in mice

Aims/hypothesis  

The TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes.     

Inhibition of Lipid Peroxidation, NF-κB Activation and IL-8 Production by Rebamipide in Helicobacter pylori-Stimulated Gastric Epithelial Cells

The present study aimed to investigate whether rebamipide, a novel antiulcer agent that has an oxygen radical scavenging activity, would inhibit lipid peroxidation, NF-B activation, and IL-8 production by H. pylori. Human gastric epithelial cells (AGS and KATO III), treated with rebamipide or not were incubated in the absence or the presence of H. pylori. As a result, H. pylori significantly stimulated IL-8 production, which was similar to time course stimulation of lipid peroxidation. Other cytokines (IL-1, IL-1, IL-6, TNF-) were not stimulated by H. pylori. Treatment with H. pylori resulted in the activation of two species of NF-B dimers (a p50/p65 heterodimer and a p50 homodimer). Rebamipide significantly inhibited lipid peroxidation as an indicative of oxidative damage, NF-B complex formation, and IL-8 production by H. pylori. In conclusion, rebamipide may attenuate H. pylori-induced gastric inflammation by inhibiting lipid peroxidation and oxidant-mediated activation of NF-B and thereby decreasing IL-8 production.     

Inhibition of NF-κB Renders Human Juvenile Costal Chondrocyte Cell Lines Sensitive to TNF-α-Mediated Cell Death

Background. Recently, therapeutics employing knowledge on various signaling pathways are being developed, with NF-B being one of the most promising targets. NF-B has been suggested to play a role not only in the induction of inflammatory mediators, but also in the protection from cell death. Objectives. This study pursued the role of the NF-B pathway in the regulation of chondrocyte death induced by tumor necrosis factor alpha (TNF-) and of the pertinent target molecules involved. Methods. The human chondrocyte cell line C28/I2 was used for the experiment. Chondrocytes were transduced with adenovirus-encoding IkappaB (IB) superrepressor which inhibits NF-B activation, and treated with TNF-. The proportion of cell death was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide (MTT) assay. Activation of p38 mitogen activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K) by TNF- was inhibited with SB202190 and Ly 294002 respectively. The expression of apoptosis related protein was analyzed with western blot assay, and the activation of c-Jun N-terminal kinase (JNK) by solid-phase kinase assay. Results. Treatment with TNF- led to cell death in 23% and 50% of ad-IB-SR infected chondrocytes after 24 and 72 h respectively. The expression of Bcl-XL, Bcl-2, and XIAP significantly decreased, and activation of JNK was prolonged for up to 6 h in infected cells treated with TNF-. Preincubation with p38 inhibitor or PI3K inhibitor before TNF- led to a significant increase in cell death in ad-IB-SR transduced chondrocytes, resulting in 53% and 30% cell death after 24 h for p38 inhibitor and PI3K inhibitor respectively. Conclusion. In our experimental system, specific inhibition of NF-B activation rendered chondrocytes susceptible to cell death induced by TNF-. The cell death was enhanced by inhibition of another signaling pathway such as p38 MAP kinase or PI3K. The expression of Bcl-XL, Bcl-2 and XIAP and activation of JNK were affected by ad-IB-SR transduction, implying a role in the NF-B regulated cell survival signaling in human chondrocytes.     

Diacerein has a weak effect on the catabolic pathway of human osteoarthritis synovial fibroblast--comparison to its effects on osteoarthritic chondrocytes

Objectives. Synoviocytes play a crucial role in the inflammatoryresponse leading to structural damage in OA. Our aim was toassess the effects of diacerein and NSAIDs on cellular responsesof synoviocytes associated with inflammation and structuralintegrity of cartilage in OA. Methods. The effects of diacerein, celecoxib, diclofenac, meloxicamand indomethacin on prostaglandin (PG) E2 production, cyclo-oxygenase-2(COX-2) protein expression, nitrite levels, presence of MMP-1and -13, and activation of nuclear factor-B (NF-B) were studiedon stimulated OA synoviocytes and chondrocytes. Results. Diacerein and NSAIDs inhibited IL-1β-stimulatedNF-B activation in synoviocytes and chondrocytes except indomethacinin synoviocytes. Diacerein further increased COX-2 protein expressionand PGE2 synthesis in synoviocytes stimulated with IL-1β,while no effect was observed on stimulated chondrocytes. NSAIDsdiminished until almost basal levels PGE2 release in both cellsand, surprisingly, these drugs also diminished COX-2 proteinexpression both in synoviocytes and chondrocytes. With regardto structural mediators, diacerein decreased MMP-13 levels insynoviocytes but did not modify MMP-1 presence. NSAIDs induceda significant increase in MMP-1 levels in both cell types andin MMP-13 levels in chondrocytes. Conclusions. Diacerein does not seem to reduce but rather increaseinflammatory mediators in synoviocytes, while it does not overallaffect chondrocyte inflammatory profile. KEY WORDS: Osteoarthritis, Cytokines and inflammatory mediators, Extracellular matrix, Synovium, Non-steroidal anti-inflammatory drugs Submitted 2 November 2007; revised version accepted 15 February 2008.