Increased IL-6 Release Research Articles

The protein LJM 111 from Lutzomyia longipalpis Salivary Gland Extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model

Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF-α and IFN-γ released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry, TNF-α and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF-α and IFN-γ. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF-α whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils.

Matrix Proteins from Smoke-Exposed Fibroblasts Are Pro-proliferative

Airway remodeling decreases lung function in chronic obstructive pulmonary disease (COPD). Extracellular matrix (ECM) deposition is increased in remodeled airways and drives cellular processes of proliferation, migration, and inflammation. We investigated the role of cigarette smoke in altering the ECM deposited from human lung fibroblasts. Lung fibroblasts isolated from patients with COPD or other lung disease were exposed to cigarette smoke extract (CSE) and 5 ng/ml transforming growth factor-β1 for 72 hours; in some experiments, inhibitors of signaling molecules were added. Deposition of perlecan, fibronectin, and elastin were measured by ELISA, as was release of IL-8 and IL-13. Unstimulated fibroblast cells were reseeded onto deposited matrix and assessed for proliferation and cytokine release. CSE (5%) increased deposition of fibronectin and perlecan from only COPD fibroblasts. Fibronectin and perlecan deposition was attenuated by addition of the NF-κB inhibitor, BMS-345541, and the signal transduction and activator of transcription-1/3 inhibitor, pyridone 6, respectively. CSE (5%) increased IL-8 release from COPD fibroblasts more than non-COPD fibroblasts. This increase was attenuated by BMS-345541. Matrix deposited after 5% CSE stimulation increased proliferation of fibroblasts, but did not alter cytokine release. ECM produced from COPD fibroblasts after CSE exposure has proproliferative effects. Thus, the ECM in patients with COPD may create an environment that promotes airway remodeling.

Graves' Orbitopathy Results in Profound Changes in Tear Composition: A Study of Plasminogen Activator Inhibitor-1 and Seven Cytokines

Secretion of cytokines and expression of cytokine receptors have been reported in the orbital connective tissue in Graves' orbitopathy (GO). Lacrimal glands are putative autoimmune targets, and changes in tear film and ocular surface have also been described. Our aim was to characterize the cytokine profile of tears in patients with Graves' disease (GD) with and without orbitopathy. Tear samples were collected from 54 eyes of GO patients (age 43.4±15.2 years), 18 eyes of GD patients (age 46.8±11.7 years), and 24 control eyes (age 38.6±13.8 years). Patients underwent ophthalmological examination including Clinical Activity Score (CAS). The level of interleukin (IL)-1β, IL-6, IL-13, IL-17A, IL-18, tumor necrosis factor (TNF)-α, and RANTES (regulated upon activation, normal T-cell expressed, and secreted) as well as plasminogen activator inhibitor-1 (PAI-1) were measured by multiplex bead array and release values were calculated. The release of IL-1β, IL-6, IL-13, IL-17A, IL-18, TNF-α, and RANTES were significantly higher in GO patients compared to controls (p<0.05). There was a 2.5-fold increase of IL-6 release. No significant differences were found in cytokine release between the GO and GD groups. In the GO group, significant positive correlation was found between CAS and the release of IL-6 and PAI-1 into tears (r=0.27, p<0.05 and r=0.24, p<0.05, respectively). PAI-1 release was significantly higher in GO than in GD patients and was increased in both the GD and GO groups compared to controls. Impaired cytokine balance has been observed in tears of GO patients. Secretion of IL-6 into tears might be a useful indicator of disease activity in GO.

Candidaalbicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models

Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1β and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.

SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target.

NTPDase1 Controls IL-8 Production by Human Neutrophils

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam(3)CSK(4) (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y(2) receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam(3)CSK(4)-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y(2) activation.

A key role for Pre–B cell colony–enhancing factor in experimental hepatitis

Pre-B cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase or visfatin, plays an important role in metabolic, inflammatory, and malignant diseases. Recent evidence suggests that blocking its enzymatic activity using a specific small-molecule inhibitor (FK866) might be beneficial in acute experimental inflammation. We investigated the role of PBEF in human liver disease and experimental hepatitis. PBEF serum levels and hepatic expression were determined in patients with chronic liver diseases. These studies were followed by in vivo experiments using concanavalin A (ConA) and D-galactosamine/lipopolysaccharide (LPS) models of experimental hepatitis. PBEF was either overexpressed by hydrodynamic perfusion or inhibited by FK866. In vivo findings were corroborated studying inflammatory responses of lentivirally PBEF-silenced or control FL83B mouse hepatocytes. Here, we demonstrate that PBEF serum levels were increased in patients with chronic liver diseases irrespective of disease stage and etiology. In particular, we observed enhanced PBEF expression in hepatocytes. Liver-targeted overexpression of PBEF rendered mice more susceptible to ConA- and D-galactosamine/LPS-induced hepatitis compared with control animals. In contrast, inhibition of PBEF using FK866 protected mice from ConA-induced liver damage and apoptosis. Administration of FK866 resulted in depletion of liver nicotinamide adenine dinucleotide+ levels and reduced proinflammatory cytokine expression. Additionally, FK866 protected mice in the D-galactosamine/LPS model of acute hepatitis. In vitro, PBEF-silenced mouse hepatocytes showed decreased responses after stimulation with LPS, lipoteichoic acid, and tumor necrosis factor α. In primary murine Kupffer cells, FK866 suppressed LPS-induced interleukin (IL)-6 production, whereas incubation with recombinant PBEF resulted in increased IL-6 release. Our data suggest that PBEF is of key importance in experimental hepatitis. Its specific inhibition might be considered a novel treatment option for inflammatory liver diseases.

Application of an optimized system for the well-defined exposure of human lung cells to trichloramine and indoor pool air

In this study an in vitro exposure test to investigate toxicological effects of the volatile disinfection by-product trichloramine and of real indoor pool air was established. For this purpose a set-up to generate a well-defined, clean gas stream of trichloramine was combined with biotests. Human alveolar epithelial lung cells of the cell line A-549 were exposed in a CULTEX(®) device with trichloramine concentrations between 0.1 and 40 mg/m(3) for 1 h. As toxicological endpoints the cell viability and the inflammatory response by the cytokines IL-6 and IL-8 were investigated. A decreasing cell viability could be observed with increasing trichloramine concentration. An increase of IL-8 release could be determined at trichloramine concentrations higher than 10 mg/m(3) and an increase of IL-6 release at concentrations of 20 mg/m(3). Investigations of indoor swimming pool air showed similar inflammatory effects to the lung cells although the air concentrations of trichloramine of 0.17 and 0.19 mg/m(3) were much lower compared with the laboratory experiments with trichloramine as the only contaminant. Therefore it is assumed that a mixture of trichloramine and other disinfection by-products in the air of indoor pool settings contribute to that effect.

Peritoneal Dialysis Fluid Induces P38-Dependent Inflammation in Human Mesothelial Cells

Noninfectious upregulation of proinflammatory pathways in mesothelial cells may represent an integral part of their stress response upon exposure to peritoneal dialysis fluids (PDF). The aim of this study was to evaluate the role of the stress-inducible mitogen-activated protein kinase (MAPK) p38 in regulation of inflammatory and stress responses in mesothelial cells following in vitro exposure to PDF. Human peritoneal mesothelial cells were exposed to Dianeal PD4 or Physioneal (Baxter AG, Vienna, Austria) containing 1.36% glucose and then allowed to recover. Phosphorylation of p38, induction of heat shock protein-70 (HSP70), release of lactate dehydrogenase (LDH), secretion of interleukin (IL)-8, gene transcription, and mRNA stability were assessed with and without the MAPK p38 inhibitor SB203580. Exposure to Dianeal resulted in phosphorylation of p38 within 30 minutes (309% of control, p < 0.05) and increased IL-8 release (370% of control, p < 0.05), HSP70 expression (151% of control, p < 0.05), and LDH release (180% of control, p < 0.05). Exposure to Physioneal resulted in attenuated changes in IL-8, HSP70, and LDH. Addition of the p38 inhibitor SB203580 to Dianeal resulted in dampened IL-8 release (-55%; p < 0.05) and basal HSP70 expression, and unchanged LDH release. Effects of p38 on IL-8 were at transcriptional, posttranscriptional, and translational levels. These data confirm concordant p38-dependent upregulation of IL-8 and HSP70 following exposure to bioincompatible PDF. The MAPK p38 pathway therefore links proinflammatory processes and the cellular stress response in human peritoneal mesothelial cells.

Evaluation of Anti-inflammatory and Atrophogenic Effects of Glucocorticoids on Reconstructed Human Skin

Topical glucocorticoids (GCs) are extensively used in the treatment of inflammatory skin diseases. However, their long-term use is often accompanied by severe and eventually irreversible adverse effects, with atrophy being the most important limitation. Currently, most non-clinical studies involve animal testing, so the results are not always representative of the situation in humans. The aim of this project was to establish an in vitro test protocol for the evaluation of the anti-inflammatory and atrophic potential of topically applied GCs in reconstructed human skin. Initial studies with fibroblasts and keratinocytes confirmed the anti-inflammatory and atrophogenic effects of GCs, as evidenced by decreased cytokine production and collagen mRNA expression. In non-pretreated reconstructed human skin (EpiDermFT™), the topical application of GCs for seven days strongly reduced the secretion of interleukin (IL)-6. GC-induced skin atrophy, known to appear only after prolonged treatment, was not detected by the analysis of epidermal thickness and collagen mRNA expression. However, reproducible epidermal inflammation was established for the first time in reconstructed human skin. Topical treatment with tumour necrosis factor (TNF) increased IL-6 release and strongly reduced epidermal thickness accompanied by severe parakeratosis. GC treatment of reconstructed human skin reduced IL-6 levels and completely resolved parakeratosis, leading to the normalisation of epidermal thickness. These induced inflammatory conditions mimic more closely the clinical situations in which GCs are used, and therefore appear to be more suitable for future investigations for the establishment of a human-based in vitro test protocol for evaluating wanted and unwanted GC effects.

C1q/TNF-related protein-3 (CTRP-3) is secreted by visceral adipose tissue and exerts antiinflammatory and antifibrotic effects in primary human colonic fibroblasts

The adipokine CTRP-3 (C1q/TNF-related protein-3) belongs to the C1q/TNF-related protein family which antagonizes the effects of lipopolysaccharide (LPS). The aim was to investigate the antiinflammatory and antifibrotic role of CTRP-3 in Crohn's disease (CD). Mesenteric adipose tissue (MAT) of patients with CD or colonic cancer (CC) was resected. Human primary colonic lamina propria fibroblasts (CLPF) were isolated from controls and CD patients. Concentrations of chemokines and cytokines in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Expression of connective tissue growth factor (CTGF), collagen I, and collagen III was analyzed by real-time polymerase chain reaction (PCR). Recombinant CTRP-3 expressed in insect cells was used for stimulation experiments. CTRP-3 is synthesized and secreted by MAT resected from patients with CD, ulcerative colitis (UC), CC, and sigma diverticulitis as well as by murine and human mature adipocytes. CTRP-3 had no effect on the basal secretion of MCSF, MIF, or RANTES in MAT of CD and control patients. LPS-stimulation (10 ng/mL) significantly increased IL-8 release in CLPF of CD patients and, to a lesser extent, in cells of controls and of fibrotic CD tissue. CTRP-3 significantly and dose-dependently reduced LPS-induced IL-8 secretion in CLPF within 8 hours after LPS exposure, whereas LPS-induced IL-6 and TNF release was not affected. CTRP-3 inhibited TGF-β production and the expression of CTGF and collagen I in CLPF, whereas collagen III expression remained unchanged. CTRP-3 exerts potent antiinflammatory and antifibrotic effects in CLPF by antagonizing the LPS pathway and by targeting the TGF-β-CTGF-collagen I pathway.

Hydrogen Sulfide Inhibits Proliferation and Release of IL-8 from Human Airway Smooth Muscle Cells

Hydrogen sulfide (H(2)S) is synthesized intracellularly by the enzymes cystathionine-γ-lyase and cystathionine-β-synthase (CBS), and is proposed to be a gasotransmitter with effects in modulating inflammation and cellular proliferation. We determined a role of H(2)S in airway smooth muscle (ASM) function. ASM were removed from resection or transplant donor lungs and were placed in culture. Proliferation of ASM was induced by FCS and the proinflammatory cytokine, IL-1β. Proliferation of ASM and IL-8 release were measured by bromodeoxyuridine incorporation and ELISA, respectively. Exposure of ASM to H(2)S "donors" inhibited this proliferation and IL-8 release. Methemoglobin, a scavenger of endogenous H(2)S, increased DNA synthesis induced by FCS and IL-1β. In addition, methemoglobin increased IL-8 release induced by FCS, but not by IL-1β, indicating a role for endogenous H(2)S in these systems. Inhibition of CBS, but not cystathionine-γ-lyase, reversed the inhibitory effect of H(2)S on proliferation and IL-8 release, indicating that this is dependent on CBS. CBS mRNA and protein expression were inhibited by H(2)S donors, and were increased by methemoglobin, indicating that CBS is the main enzyme responsible for endogenous H(2)S production. Finally, we found that exogenous H(2)S inhibited the phosphorylation of extracellular signal-regulated kinase-1/2 and p38, which could represent a mechanism by which H(2)S inhibited cellular proliferation and IL-8 release. In summary, H(2)S production provides a novel mechanism for regulation of ASM proliferation and IL-8 release. Therefore, regulation of H(2)S may represent a novel approach to controlling ASM proliferation and cytokine release that is found in patients with asthma.

Cilomilast counteracts the effects of cigarette smoke in airway epithelial cells

Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Cilomilast, a phosphodiesterase-4 inhibitor, inhibits cigarette smoke-induced neutrophilia. This study was aimed to explore whether cilomilast, in a human bronchial epithelial cell line (16-HBE), counteracted CSE effects. In particular, TLR4 expression, IP-10 and IL-8 release, lymphocyte and neutrophil chemotactic activity and ERK and IkBa phosphorylation in CSE and LPS-stimulated 16-HBE were assessed. CSE increased TLR4 expression, reduced IP-10 release and lymphocyte chemotactic activity and increased IL-8 release and neutrophil chemotactic activity. Cilomilast reduced TLR4 expression, IL-8 release and neutrophil chemotactic activity as well as it increased IP-10 release and lymphocyte chemotactic activity. All these cilomilast mediated effects were associated with a reduced ERK1/2 and with an increased IkBa phosphorylation. In conclusion, the present study provides compelling evidences that cilomilast may be considered a possible valid therapeutic option in controlling inflammatory processes present in smokers.

S144 Cigarette smoke dysregulates pro-inflammatory cytokine release from airway epithelial cells and macrophages in response to haemophilus influenzae

BackgroundChronic obstructive pulmonary disease (COPD) is characterised by repeated viral or bacterial exacerbations which increase morbidity, mortality and accelerate lung function decline. Chronic bacterial colonisation, frequently recognised in stable COPD...

Tolerance Induction in Response to Liver Inflammation

Background/Aims: The liver plays an important role in immunological tolerance due to its anatomical location, as it links the gastrointestinal tract and the systemic venous circulation. Therefore, immune reactions against dietary or bacterial antigens from the gut have to be avoided. However, immune responses resulting in elimination of harmful hepatotrophic pathogens have to be induced. We investigated mechanisms of tolerance induction in response to liver inflammation in a murine model of immune-mediated liver injury. Methods: Liver damage was induced by injection of the plant lectin concanavalin A (ConA). Cytokine levels were measured in plasma and liver tissue. The frequencies of intrahepatic and splenic cell subsets were measured by FACS analyses. Results: ConA hepatitis was mediated by activation of CD4+ T cells, NKT cells and Kupffer cells releasing IFN-γ and TNF-α. Tolerance developed towards ConA rechallenge within 8 days, lasted for several weeks and was characterized by significantly reduced plasma transaminase activities, decreased Th1/Th17 responses and an increased IL-10 release, the latter being produced by CD4+CD25+FoxP3+ regulatory T cells and Kupffer cells. Moreover, regulatory T cells from ConA-tolerant mice displayed a higher immunosuppressive potential in vitro and in vivo compared to those from non-tolerant animals. Interestingly, ConA hepatitis was aggravated in CCR5^–/– and CXCR3^–/– mice. Conclusion: These results suggest that ConA tolerance is mediated by induced IL10+ regulatory T cells, probably trafficking into the liver depending on the IFN-γ-inducible chemokine receptors CCR5 and CXCR3.

Mercury induces inflammatory mediator release from human mast cells

BackgroundMercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD) have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl2) on human mast cell activation.MethodsHuman leukemic cultured LAD2 mast cells and normal human umbilical cord blood-derived cultured mast cells (hCBMCs) were stimulated by HgCl2 (0.1-10 μM) for either 10 min for beta-hexosaminidase release or 24 hr for measuring vascular endothelial growth factor (VEGF) and IL-6 release by ELISA.ResultsHgCl2 induced a 2-fold increase in β-hexosaminidase release, and also significant VEGF release at 0.1 and 1 μM (311 ± 32 pg/106 cells and 443 ± 143 pg/106 cells, respectively) from LAD2 mast cells compared to control cells (227 ± 17 pg/106 cells, n = 5, p < 0.05). Addition of HgCl2 (0.1 μM) to the proinflammatory neuropeptide substance P (SP, 0.1 μM) had synergestic action in inducing VEGF from LAD2 mast cells. HgCl2 also stimulated significant VEGF release (360 ± 100 pg/106 cells at 1 μM, n = 5, p < 0.05) from hCBMCs compared to control cells (182 ± 57 pg/106 cells), and IL-6 release (466 ± 57 pg/106 cells at 0.1 μM) compared to untreated cells (13 ± 25 pg/106 cells, n = 5, p < 0.05). Addition of HgCl2 (0.1 μM) to SP (5 μM) further increased IL-6 release.ConclusionsHgCl2 stimulates VEGF and IL-6 release from human mast cells. This phenomenon could disrupt the blood-brain-barrier and permit brain inflammation. As a result, the findings of the present study provide a biological mechanism for how low levels of mercury may contribute to ASD pathogenesis.

3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells

3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ( 32P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IκB-α (suggesting activation of NF-κB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-κB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

Occludin gene expression as an early in vitro sign for mild eye irritation assessment

Purpose To test a new multiple endpoint analysis (MEA) including occludin gene expression for screening the ocular irritation potential of tear substitutes on human corneal epithelium (HCE), an in vitro model proposed to limit the use of animal testing in pre-clinical studies. Methods Four chemically-preserved and two non chemically-preserved tear substitutes were tested after acute (24 h, 24 h + 24 h post incubation) and repeated applications (for 72 h) and compared to the positive control, benzalkonium chloride (BAK) at 0.1% and 0.01%, by assessing complementary parameters. Cellular viability was evaluated using MTT, histomorphologic analysis was performed on H&E stained vertical sections, IL-8 release was measured by ELISA, and occludin gene expression was quantified using qRT-PCR. Results Cellular viability was moderately reduced by Perborate and Polyquad-preserved tear substitutes and dramatically reduced by BAK and by Thiomersal ® and Oxyd ® preserved tear substitutes. Thiomersal ® also increased IL-8 release. Occludin expression profiles were modified by the four chemically-preserved tear substitutes and by the mechanically-preserved Comod ®, but not by the mechanically-preserved Abak ®. The behavior of BAK and tear substitutes led us to propose a prediction model for the classification of different levels of irritants, mainly based on the occludin transcriptional study. Conclusion The versatility and sensitivity of the HCE model allowed the modeling of cumulative effects that may approach conditions obtained after long term application of tear substitutes. Thus, the modified MEA proposed in this study represents a valuable tool for in vitro eye irritation assessment with the power to detect mild irritants and subclinical eye irritant potential.

Low concentrations of LL-37 alter IL-8 production by keratinocytes and bronchial epithelial cells in response to proinflammatory stimuli

The immunomodulatory cationic host defence peptide LL-37 plays an important role in epithelial innate immunity; at higher concentrations (20-50 microg mL(-1)) associated with inflammation, LL-37 elicits the production of cytokines and chemokines. It was demonstrated here that lower, physiologically relevant LL-37 concentrations (2-3 microg mL(-1)) altered epithelial cell responses to proinflammatory stimuli. In combination with interleukin-1beta (IL-1beta) and the Toll-like receptor-5 (TLR5) agonist flagellin, these low concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes and by bronchial epithelial cells. In combination with the TLR2/1 agonist PAM3CSK4, LL-37 synergistically induced transcription and the release of both IL-8 and IL-6 from primary bronchial epithelial cells; the IL-8 response was demonstrated to be regulated by epidermal growth factor receptor signalling. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist polyI:C resulted in synergistic increases in IL-8 release and cytotoxicity. These data indicate that low concentrations of LL-37 may alter epithelial responses to infecting microorganisms in vivo.

Hypothermia and Surgery

To examine cellular and immunologic mechanisms by which intraoperative hypothermia affects surgical patients. Avoidance of perioperative hypothermia has recently become a focus of attention as an important quality performance measure, aimed at optimizing the care of surgical patients. Anesthetized surgical patients are particularly at risk for hypothermia, which has been directly linked to the development of sequelae, such as coagulopathy, infection, morbid myocardial events, and death after surgery. However, many of the underlying immunologic mechanisms remain unclear. Venous blood samples from healthy volunteers were exposed for up to 4 hours to various temperatures following the addition of a 1 ng/mL lipopolysaccharide challenge. Innate immune function, assessed by the ability of monocytes to present antigen and coordinate cytokine release, was determined by qualitative and quantitative measurements of HLA-DR surface expression 2 hours following incubation, and proinflammatory tumor necrosis factor-alpha (TNF-alpha) and anti-inflammatory (IL-10) cytokine release in the first 4 hours. Monocyte incubation at hypothermic temperatures (34 degrees C) reduced HLA-DR surface expression, delayed TNF-alpha clearance, and increased IL-10 release. Conversely, hyperthermia (40 degrees C) increased monocyte antigen presentation and resulted in rapid decay of TNF-alpha. However, IL-10 release was also increased. Normothermia (37 degrees C) attenuated IL-10 release following the initial proinflammatory surge. Hypothermia exerts multiple effects at the cellular level, which impair innate immune function, and are associated with increased septic complications and mortality. These findings provide a physiological basis for perioperative temperature monitoring, which is a valid surgical performance measure that can be used to reduce surgical complications associated with avoidable hypothermia.