interleukin-8 and lipoteichoic-acid

Despite similar clinical relevance of Gram-positive and Gram-negative infections, immune activation by Gram-positive bacteria is by far less well understood than immune activation by Gram-negative bacteria. Our group has made available highly purified lipoteichoic acids (LTA) as a key Gram-positive immunostimulatory component. We have characterized the reasons for lower potency of LTA compared to Gram-negative lipopolysaccharide (LPS), identifying lack of IL-12/IFNgamma induction as a general characteristic of TLR2 agonists, and need for presentation of LTA on surfaces for enhanced immunostimulatory potency, as major aspects. Aspects of chemokine induction, where LTA is more potent than LPS, have been addressed. Furthermore, novel complement and plant defence activation, as well as CD36 as a new LTA receptor, were identified. The bacterial costimuli and modulators of LTA inducible responses are being investigated: LTA isolated from so far 16 bacterial species, although different in structure, behave remarkably similar while whole live and killed bacteria differ with regard to the pattern of induced responses. The purification and characterization of the respective components of the bacterial cell wall has begun.

Topics: Animals; Bacteria; CD36 Antigens; Cell Wall; Cytokines; Gram-Positive Bacteria; Humans; Immunity, Innate; Interferon-gamma; Interleukin-12; Interleukin-8; Lipopolysaccharides; Mice; Models, Biological; Monocytes; Teichoic Acids

Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), leading to chronic inflammation, while bacterial components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are often present in airways of COPD patients, especially during exacerbations.We hypothesised that extracellular heat shock protein 70 (eHsp70), a damage-associated molecular pattern elevated in serum of COPD patients, induces inflammation and alters cigarette smoke and LPS/LTA-induced inflammatory effects in the airway epithelium.We used 16HBE cells exposed to recombinant human (rh)Hsp70 and its combinations with cigarette smoke extract (CSE), LPS or LTA to investigate those assumptions, and we determined pro-inflammatory cytokines' secretion as well as TLR2 and TLR4 gene expression.rhHsp70 and CSE alone stimulated IL-6, IL-8 and TNF-α secretion. CSE and rhHsp70 had antagonistic effect on IL-6 secretion, while combinations of LPS or LTA with rhHsp70 showed antagonistic effect on TNF-α release. By using specific inhibitors, we demonstrated that effects of rhHsp70 on cytokines' secretion were mediated via NF-κB and/or MAPK signalling pathways. rhHsp70 increased, and CSE decreased TLR2 gene expression compared to untreated cells, but their combinations increased it compared to CSE alone. LPS and rhHsp70 combinations decreased TLR2 gene expression compared to untreated cells. TLR4 expression was not induced by any of the treatments.In conclusion, we demonstrated that extracellular Hsp70 modulates pro-inflammatory responses of human airway epithelial cells to cigarette smoke and bacterial components LPS and LTA. Simultaneous presence of those compounds and their interactions might lead to inappropriate immune responses and adverse consequences in COPD.

Topics: Cigarette Smoking; HSP70 Heat-Shock Proteins; Humans; Inflammation; Interleukin-6; Interleukin-8; Lipopolysaccharides; NF-kappa B; Nicotiana; Pulmonary Disease, Chronic Obstructive; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Probiotics in livestock feed supplements are considered to be an alternative to antibiotics. However, effector molecules responsible for the beneficial roles of probiotics in pigs are in general not well known. Thus, this study demonstrated that a well-known virulence factor, flagellin of Salmonella typhimurium, significantly induced IL-8 production in porcine peripheral blood mononuclear cells, whereas lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria Lactobacillus plantarum, L. casei, and L. rhamnosus GG, effectively inhibited flagellin-induced IL-8 production at mRNA and protein levels. However, the lipoproteins of L. plantarum, L. casei, and L. rhamnosus GG did not suppress flagellin-induced IL-8 production. While D-alanine-deficient L. plantarum LTA inhibited flagellin-induced IL-8 production, L. plantarum LTA deficient in both D-alanine and acyl chains failed to inhibit it; this suggests that the acyl moieties of L. plantarum LTA are essential for inhibiting flagellin-induced IL-8 production. Taken together, L. plantarum LTA plays an important role in improving anti-inflammatory responses of porcine peripheral blood mononuclear cells.

Topics: Animals; Flagellin; Inflammation; Interleukin-8; Lactobacillus plantarum; Leukocytes, Mononuclear; Lipopolysaccharides; Salmonella typhimurium; Swine; Teichoic Acids

The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte-non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from

Topics: Animals; Cell Survival; Cells, Cultured; Chickens; Coculture Techniques; Enterotoxins; Flagellin; Immunity, Innate; Inflammation Mediators; Interleukin-6; Interleukin-8; Lipopolysaccharides; Liver; Male; Pathogen-Associated Molecular Pattern Molecules; Poly I-C; Spheroids, Cellular; Teichoic Acids; Tetradecanoylphorbol Acetate

Obesity is an important risk factor for severe asthma exacerbations, which are mainly caused by respiratory infections. Dietary fatty acids, which are increased systemically in obese patients and are further increased after high-fat meals, affect the innate immune system and may contribute to dysfunctional immune responses to respiratory infection. In this study we investigated the effects of dietary fatty acids on immune responses to respiratory infection in pulmonary fibroblasts and a bronchial epithelial cell line (BEAS-2B). Cells were challenged with BSA-conjugated fatty acids (ω-6 polyunsaturated fatty acids [PUFAs], ω-3 PUFAs, or saturated fatty acids [SFAs]) +/- the viral mimic polyinosinic:polycytidylic acid (poly[I:C]) or bacterial compound lipoteichoic acid (LTA), and release of proinflammatory cytokines was measured. In both cell types, challenge with arachidonic acid (AA) (ω-6 PUFA) and poly(I:C) or LTA led to substantially greater IL-6 and CXCL8 release than either challenge alone, demonstrating synergy. In epithelial cells, palmitic acid (SFA) combined with poly(I:C) also led to greater IL-6 release. The underlying signaling pathways of AA and poly(I:C)- or LTA-induced cytokine release were examined using specific signaling inhibitors and IB. Cytokine production in pulmonary fibroblasts was prostaglandin dependent, and synergistic upregulation occurred via p38 mitogen-activated protein kinase signaling, whereas cytokine production in bronchial epithelial cell lines was mainly mediated through JNK and p38 mitogen-activated protein kinase signaling. We confirmed these findings using rhinovirus infection, demonstrating that AA enhances rhinovirus-induced cytokine release. This study suggests that during respiratory infection, increased levels of dietary ω-6 PUFAs and SFAs may lead to more severe airway inflammation and may contribute to and/or increase the severity of asthma exacerbations.

Topics: Adult; Aged; alpha-Linolenic Acid; Arachidonic Acid; Cell Line; Docosahexaenoic Acids; Eicosapentaenoic Acid; Epithelial Cells; Female; Fibroblasts; Gene Expression Regulation; HeLa Cells; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Lung; Male; MAP Kinase Kinase 4; Middle Aged; p38 Mitogen-Activated Protein Kinases; Palmitic Acid; Poly I-C; Primary Cell Culture; Rhinovirus; Signal Transduction; Teichoic Acids

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.

Topics: Cell Death; Cell Survival; HSP70 Heat-Shock Proteins; Humans; Inflammation; Interleukin-1alpha; Interleukin-8; Lipopolysaccharides; Teichoic Acids; THP-1 Cells; Time Factors; Tumor Necrosis Factor-alpha

Staphylococcus epidermidis colonises human skin without apparent inflammation, but a dominance of S. epidermidis and S. aureus is characteristic of cutaneous microbial dysbiosis in atopic dermatitis (AD). While S. aureus can trigger AD, the role of S. epidermidis is less understood. We characterised consequences of innate immune sensing of lipoteichoic acid (LTA) preparations derived from S. epidermidis (epi-LTA) or S. aureus (aureus-LTA). Therefore, dendritic cell (DC) activation and consecutive priming of antigen-specific T cells following exposure of DC to epi-LTA or aureus-LTA were investigated. Mimicking acute AD, exposure of DC to IL-4 and LTAs was analysed. Exposure to epi-LTA or aureus-LTA activated human immune cells and murine dendritic cells (DCs) via TLR2/MyD88, however, resulting in divergent immune profiles. Differences between LTAs were significant for IL-6, IL-12p40 and IL-12p70 but not for IL-10, which was best reflected by the IL-12p70-to-IL-10 ratio being IL-10-balanced for epi-LTA but pro-inflammatory for aureus-LTA. LTA-exposed DCs activated CD4+ T cells; however, while T-cell-derived IL-10 was equivalent between LTAs, IFN-γ and IL-17 were significantly higher for aureus-LTA. Mimicking acute AD by exposing DCs to IL-4 and LTAs revealed that IL-4 significantly and uniformly suppressed epi-LTA-induced cytokine production, keeping the IL-12p70-to-IL-10 ratio balanced. In contrast, exposure of DCs to aureus-LTA and IL-4 enhanced IL-12p70 but suppressed IL-10 levels, further unbalancing the IL-12p70-to-IL-10 ratio. These data demonstrate opposing immune consequences following exposure to staphylococcal LTAs. Epi-LTA induced IL-10-balanced, aureus-LTA pro-inflammatory immune profiles.

Topics: Adaptive Immunity; Animals; Coculture Techniques; Dendritic Cells; Humans; Immunity, Innate; Interleukin-10; Interleukin-12; Interleukin-4; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mice; Myeloid Differentiation Factor 88; Staphylococcus aureus; Staphylococcus epidermidis; STAT6 Transcription Factor; T-Lymphocytes, Helper-Inducer; Teichoic Acids; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue-associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.

Topics: Adenocarcinoma; Animals; Antibodies, Bacterial; Bacterial Infections; Disease Models, Animal; Enterococcus; Female; Gene Expression Regulation, Neoplastic; Hot Temperature; Humans; Interleukin-8; Lipopolysaccharides; Male; Middle Aged; Pancreatic Juice; Pancreatic Neoplasms; Pancreatitis, Chronic; RNA, Messenger; RNA, Ribosomal, 16S; Teichoic Acids; Vascular Endothelial Growth Factor A

Pulmonary infections are frequent complications in lung cancer and may worsen its outcome and survival. Inflammatory mediators are suspected to promote tumor growth in non-small-cell lung cancer (NSCLC). Hence, bacterial pathogens may affect lung cancer growth by activation of inflammatory signalling. Against this background, we investigated the effect of purified lipoteichoic acids (LTA) of Staphylococcus aureus (S. aureus) on cellular proliferation and liberation of interleukin (IL)-8 in the NSCLC cell lines A549 and H226. A549 as well as H226 cells constitutively expressed TLR-2 mRNA. Even in low concentrations, LTA induced a prominent increase in cellular proliferation of A549 cells as quantified by automatic cell counting. In parallel, metabolic activity of A549 cells was enhanced. The increase in proliferation was accompanied by an increase in IL-8 mRNA expression and a dose- and time-dependent release of IL-8. Cellular proliferation as well as the release of IL-8 was dependent on specific ligation of TLR-2. Interestingly, targeting IL-8 by neutralizing antibodies completely abolished the LTA-induced proliferation of A549 cells. The pro-proliferative effect of LTA could also be reproduced in the squamous NSCLC cell line H226. In summary, LTA of S. aureus induced proliferation of NSCLC cell lines of adeno- and squamous cell carcinoma origin. Ligation of TLR-2 followed by auto- or paracrine signalling by endogenously synthesized IL-8 is centrally involved in LTA-induced tumor cell proliferation. Therefore, pulmonary infections may exert a direct pro-proliferative effect on lung cancer growth.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Proliferation; Humans; In Vitro Techniques; Interleukin-8; Lipopolysaccharides; Lung Neoplasms; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptor 2; Tumor Cells, Cultured

Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif-containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this motif was also found to interact with MyD88 in vitro. SARM expression in HEK293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells.

Topics: Adaptor Proteins, Vesicular Transport; Amino Acid Sequence; Armadillo Domain Proteins; Binding Sites; Blotting, Western; Cytoskeletal Proteins; HEK293 Cells; Humans; Interleukin-8; Lipopolysaccharides; Microscopy, Confocal; Mutation; Myeloid Differentiation Factor 88; Protein Binding; Receptors, Interleukin-1; Signal Transduction; Teichoic Acids; Toll-Like Receptors; Tumor Necrosis Factor-alpha; Up-Regulation

Lipoteichoic acid (LTA) is one of microbe-associated molecular pattern (MAMP) molecules of gram-positive bacteria. In this study, we demonstrated that Clostridium butyricum LTA (bLTA) significantly inhibited the inflammatory response and apoptosis induced by Staphylococcus aureus LTA (aLTA) in HT-29 cells. aLTA stimulated the inflammatory responses by activating a strong signal transduction cascade through NF-κB and ERK, but bLTA did not activate the signaling pathway. bLTA pretreatment inhibited the activation of the NF-κB and ERK signaling pathway induced by aLTA. The expression and release of cytokines such as IL-8 and TNF-α were also suppressed by bLTA pretreatment. aLTA treatment induced apoptosis in HT-29 cells, but bLTA did not affect the viability of the cells. Further study indicated that bLTA inhibited apoptosis in HT-29 cells induced by aLTA. These results suggest that bLTA may act as an aLTA antagonist and that an antagonistic bLTA may be a useful agent for suppressing the pro-inflammatory activities of gram-positive pathogenic bacteria.

Topics: Apoptosis; Cell Survival; Clostridium butyricum; Dose-Response Relationship, Drug; Gene Expression Regulation; HT29 Cells; Humans; Inflammation; Interleukin-8; Lipopolysaccharides; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Signal Transduction; Species Specificity; Staphylococcus aureus; Teichoic Acids; Tumor Necrosis Factor-alpha

Houttuynia cordata (HC) has been commonly used as many traditional remedies in local areas of Japan. Although many pharmacological activities of HC have been reported, the mechanism underlying the effect of HC remains unknown. We conducted the interview survey in Japan to verify how HC was actually used. The interview survey revealed that HC poultice (HCP) prepared from smothering fresh leaves of HC was most frequently used for the treatment of purulent skin diseases including furuncle and carbuncle with high effectiveness. Ethanol extract of HCP (eHCP) showed anti-bacterial effects against methicillin-resistant Staphylococcus aureus (MRSA), and showed an anti-biofilm activity against MRSA. eHCP showed dose-dependent inhibition of S. aureus lipoteichoic acid (LTA)-induced interleukin-8 and CCL20 production in human keratinocyte without any cytotoxicity. These results suggest that HCP is effective for skin abscess and its underlying mechanism might be the complicated multiple activities for both bacteria and host cells.

Topics: Aged; Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Biofilms; Carbuncle; Cell Line, Transformed; Chemokine CCL20; Dose-Response Relationship, Drug; Ethanol; Female; Furunculosis; Houttuynia; Humans; Interleukin-8; Japan; Keratinocytes; Lipopolysaccharides; Male; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Middle Aged; Phytotherapy; Plant Extracts; Plant Leaves; Staphylococcal Infections; Surveys and Questionnaires; Teichoic Acids

Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

Topics: Animals; Cattle; Chemokine CCL2; Chemokine CXCL6; Chemokines; Epithelial Cells; Female; Gene Expression; Interleukin-8; Lipopolysaccharides; Mammary Glands, Animal; Staphylococcus aureus; Teichoic Acids

Nasal secretions include cytokines and inflammatory mediators that are involved in the pathogenesis of upper airway inflammation.. We tried to find unknown biomolecules that are involved in the pathogenesis of chronic rhinosinusitis (CRS).. We collected nasal mucosal secretions from patients who were diagnosed as having CRS and who underwent endoscopic sinus surgery. A total of 63 patients who underwent nasal secretion collection were reviewed. Enzyme-linked immunosorbent assay was performed by using nasal lavage samples to evaluate which biomolecules were associated with the severity of inflammation based on the Lund-Mackay score. By using human nasal epithelial cells, we performed Western blot, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay to evaluate the secretory mechanism of heat shock protein (HSP) 70.. We found that the level of interleukin 8 and HSP70 were significantly associated with the Lund-Mackay score and interleukin 17C, C-X-C motif chemokine 10, and HSP27 were not significantly associated. HSP70 was also significantly associated with the surgical outcome of the enrolled patients. Furthermore, we found that exposure to hypoxia and treatment of lipoteichoic acid induced the secretion of HSP70 but that lipopolysaccharide did not induce the secretion of HSP70 in human nasal epithelial cells.. Our findings indicated that HSP70 might play a role in the pathogenesis of CRS and the possibility of HSP70 as a biomolecule that represents the severity of CRS.

Topics: Adolescent; Adult; Aged; Cells, Cultured; Chronic Disease; Female; HSP70 Heat-Shock Proteins; Humans; Interleukin-8; Lipopolysaccharides; Male; Middle Aged; Rhinitis; Severity of Illness Index; Sinusitis; Teichoic Acids

In spite of advances in neonatal care and the new generation of antibiotics, neonatal sepsis is still a major cause of morbidity and mortality. Early diagnosis of neonatal sepsis is difficult because clinical signs are non-specific. Thus, new biomarkers are still needed for diagnosis. Gelsolin is an actin-binding plasma protein. Furthermore, extracellular gelsolin binds lipopolysaccharide and lipoteichoic acid, which are major virulence factors of Gram-negative and Gram-positive bacteria. The result of this binding is the inhibition of gelsolin's F-actin depolymerizing activity. Thus, gelsolin inhibits the release of IL-8 from human neutrophils subjected to lipoteichoic acid, lipopolysaccharide and heat-inactivated bacteria treatment. Our hypothesis is that pGSN levels decrease in neonatal infants with sepsis and this decrease might be used as a reliable biological marker. Forty patients who were diagnosed with severe sepsis at a neonatal intensive care unit were enrolled in the sepsis group. Twenty patients who were followed for prematurity were enrolled in the control group. The pGSN level at the time of diagnosis in the sepsis group was 33.98±11.44μg/ml, which was significantly lower than that of control group (60.05±11.3μg/ml, P<0.001) and after treatment (53.38±31.26μg/ml, P=0.003). Area under ROC curve was 0.96 (p: 0.0001, 95% CI; 0.90-0.99). Sensitivity was 90.32 (95% CI; 74.2-97.8), specificity was 95 (95% CI; 75.1-99.2). Plasma gelsolin significantly decreased in septic patient and recovery of decreased gelsolin levels correlated with clinical improvement. Thus, plasma gelsolin may be a usable marker for severe sepsis.

Topics: Actins; Area Under Curve; Biomarkers; Female; Gelsolin; Humans; Infant, Newborn; Interleukin-8; Lipopolysaccharides; Male; Morbidity; Neonatal Sepsis; Neutrophils; Prospective Studies; ROC Curve; Sensitivity and Specificity; Sepsis; Teichoic Acids

Lactobacilli are probiotic bacteria that are considered to be beneficial in the gastrointestinal tract of humans. Although lactobacilli are well known to alleviate intestinal inflammation, the molecular basis of this phenomenon is poorly understood. In this study, we investigated the effect of Lactobacillus plantarum lipoteichoic acid (Lp.LTA), which is a major cell wall component of this species, on the production of interleukin (IL)-8 in human intestinal epithelial Caco-2 cells. Treatment with Pam2CSK4, a synthetic lipopeptide that is known to mimic Gram-positive bacterial lipoproteins as an important virulence factor, significantly induced IL-8 expression in Caco-2 cells. However, neither heat-inactivated L. plantarum nor L. plantarum peptidoglycan inhibited Pam2CSK4-induced IL-8 mRNA expression. In addition, both a deacylated form and a dealanylated form of Lp.LTA failed to inhibit Pam2CSK4-induced IL-8 expression, indicating that the lipid and D-alanine moieties are critical for Lp.LTA-mediated inhibition. Moreover, Lp.LTA inhibited Pam2CSK4-induced activation of p38 kinase, JNK, and NF-κB transcription factor by suppressing toll-like receptor 2 activation. Collectively, these results suggest that Lp.LTA exerts anti-inflammatory effects on human intestinal epithelial cells by blocking IL-8 production.

Topics: Alanine; Caco-2 Cells; Enterocytes; Humans; Interleukin-8; Lactobacillus plantarum; Lipopeptides; Lipopolysaccharides; Mitogen-Activated Protein Kinases; Peptidoglycan; Phosphorylation; Teichoic Acids; Toll-Like Receptor 2

Binding of allergen to IgE on basophils positively affects allergic inflammation by releasing inflammatory mediators. Recently, basophils were shown to express pattern-recognition receptors, such as toll-like receptors (TLRs), for recognizing microbe-associated molecular patterns (MAMPs) that are independent of allergen-IgE binding. In this study, we investigated whether MAMP alone can induce IL-6 production in a human basophil cell line, KU812. Stimulation with flagellin in the absence of allergen-IgE association induced IL-6 expression in KU812 cells, while stimulation with lipoteichoic acid, peptidoglycan, or poly I:C did not under the same condition. Flagellin-induced IL-6 expression was also observed in human primary basophils. Flow cytometric analysis showed that KU812 cells expressed flagellin-recognizing TLR5 both on the cell surface and in the cytoplasm while TLR2 and TLR3 were observed only in the cytoplasm. We further demonstrated that although flagellin augmented the phosphorylation of mitogen-activated protein kinases including p38 kinase, ERK, and JNK, flagellin-induced IL-6 production was attenuated by inhibitors for p38 kinase and ERK, but not by JNK inhibitors. In addition, flagellin enhanced phosphorylation of signaling molecules including CREB, PKCδ, and AKT. The inhibitors for PKA and PKC also showed inhibitory effects. Interestingly, flagellin-induced IL-6 production was further enhanced by pretreatment with inhibitors for PI3K, implying that PI3K negatively affects the flagellin-induced IL-6 production. Furthermore, DNA binding activities of NF-κB, AP-1, and CREB, which play pivotal roles in the induction of IL-6 gene expression, were increased by flagellin. These results suggest that flagellin alone is sufficient to induce IL-6 gene expression via TLR5 signaling pathways in human basophils.

Topics: Allergens; Basophils; Cell Line; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Extracellular Signal-Regulated MAP Kinases; Flagellin; Humans; Hypersensitivity; Immunoglobulin E; Inflammation; Interleukin-6; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Peptidoglycan; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Poly I-C; Protein Kinase C-delta; Proto-Oncogene Proteins c-akt; Receptors, Pattern Recognition; RNA, Messenger; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 3; Toll-Like Receptor 5; Transcription Factor AP-1

Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.

Topics: Adult; Aggregatibacter actinomycetemcomitans; CCAAT-Enhancer-Binding Proteins; Enterococcus faecalis; Female; Fibroblasts; Gene Expression Regulation; Humans; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Lipopolysaccharides; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Periodontal Ligament; Phosphorylation; Primary Cell Culture; RNA, Small Interfering; Signal Transduction; Teichoic Acids; Toll-Like Receptor 4; Transcription Factor AP-1

Staphylococcus aureus can cause the intestinal inflammatory diseases. However, little is known about the molecular mechanism of S. aureus infection in the intestine. In the present study, we investigated whether S. aureus could stimulate human intestinal epithelial cells triggering inflammation. When the human intestinal epithelial cell-line, Caco-2, and the primary colon cells were stimulated with ethanol-inactivated S. aureus, IL-8 expression was induced in a dose-dependent manner. The inactivated S. aureus preferentially stimulated Toll-like receptor (TLR) 2 rather than TLR4. Lipoproteins, lipoteichoic acid (LTA), and peptidoglycan (PGN) are considered as potential TLR2 ligands of S. aureus. Interestingly, S aureus lipoproteins and Pam2CSK4 mimicking Gram-positive bacterial lipoproteins, but not LTA and PGN of S. aureus, significantly induced IL-8 expression in Caco-2 cells. Furthermore, lipoprotein-deficient S. aureus mutant strain failed to induce IL-8 production. Collectively, these results suggest that S. aureus stimulates the human intestinal epithelial cells to induce the chemokine IL-8 production through its lipoproteins, potentially contributing the development of intestinal inflammation.

Topics: Anti-Bacterial Agents; Caco-2 Cells; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Flow Cytometry; Gene Expression Regulation; Humans; Inflammation; Interleukin-8; Intestines; Lipopolysaccharides; Lipoproteins; Peptidoglycan; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptor 2

The cutaneous inflammation associated with acne vulgaris is caused by the anaerobic bacterium Propionibacterium acnes through activation of the innate immune system in the skin. Current standard treatments for acne have limitations that include adverse effects and poor efficacy in many patients, making development of a more effective therapy highly desirable. In the present study, we demonstrate the protective effects of a novel customized α-helical cationic peptide, P5, against P. acnes-induced inflammatory responses in vitro and in vivo. Application of P5 significantly reduced expression of two inflammatory cytokines IL-8 and TNF-α in P. acnes-treated primary human keratinocytes, where P5 appeared to act in part by binding to bacterial lipoteichoic acid, thereby suppressing TLR2-to-NF-κB signaling. In addition, in a mouse model of acne vulgaris, P5 exerted both anti-inflammatory and antimicrobial effects against P. acnes, but exerted no cytotoxic effects against skin cells. These results demonstrate that P5, and perhaps other cationic antimicrobial peptides, offer the unique ability to reduce numbers P. acnes cells in the skin and to inhibit the inflammation they trigger. This suggests these peptides could potentially be used to effectively treat acne without adversely affecting the skin.

Topics: Acne Vulgaris; Animals; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; Gram-Positive Bacterial Infections; Humans; Interleukin-8; Keratinocytes; Lipopolysaccharides; Mice; Propionibacterium acnes; Signal Transduction; Teichoic Acids; Tumor Necrosis Factor-alpha

Alpha-melanocyte stimulating hormone (α-MSH) is a highly conserved 13-aa neuropeptide derived from pro-opiomelanocortin by post-translational processing, which has been reported to exhibit potent anti-inflammatory activity and a wide range of immunosuppressive activities in the skin. However, the regulatory effect of α-MSH is not completely clear in cutaneous innate immunity. In this study, we investigate the functional regulation of α-MSH in TLR2-mediated inflammatory responses in normal human keratinocytes (HKs). α-MSH pretreatment down-regulated the Staphylococcus aureus LTA-induced expression of both TLR2 and IL-8 as well as NF-κB nuclear translocation in HK cells. The inhibitory effect of α-MSH was blocked by agouti signaling protein (ASP), an α-MSH receptor-1 antagonist. To investigate the mechanism of this response in more detail, siRNA of IRAK-M, a negative regulator of TLR signaling, was utilized in these studies. The α-MSH suppressive effect on IL-8 production and NF-κB transactivation was inhibited by IRAK-M siRNA transfection in HK cells. These results indicate that α-MSH is capable of suppressing keratinocyte TLR2-mediated inflammatory responses induced by S. aureus-LTA, thus demonstrating another novel immunomodulatory activity of α-MSH in normal human keratinocytes.

Topics: Active Transport, Cell Nucleus; alpha-MSH; Cell Nucleus; Gene Expression Regulation, Enzymologic; Gene Silencing; Humans; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Keratinocytes; Lipopolysaccharides; NF-kappa B; RNA, Small Interfering; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptor 2

During bacterial infections, pathogen-associated molecular patterns (PAMPs) induce cytokine/chemokine release in immunoactive cells. This increases corticosteroid-resistant airway inflammation in chronic obstructive pulmonary disease (COPD) and leads to exacerbations. Anti-inflammatory therapies other than corticosteroids are required and resveratrol is currently under discussion. Resveratrol is an activator of sirtuins, which are class III histone deacetylases (HDACs). We suggested that human airway smooth muscle cells (HASMCs) release COPD-associated cytokines/chemokines in response to lipoteichoic acid (LTA), a major PAMP of gram-positive bacteria and that resveratrol is superior to the corticosteroid dexamethasone in suppressing these cytokines/chemokines. Cultivated HASMCs of patients with COPD were pre-incubated with resveratrol or dexamethasone before stimulation with LTA. CCL2, GM-CSF, IL-6 and IL-8 were analysed in culture supernatants by enzyme-linked immunosorbent assay. Drug effects were investigated in the absence and presence of trichostatin A (TSA), an inhibitor of class I/II HDACs, and EX527, an inhibitor of the sirtuin SIRT1. LTA induced robust cytokine/chemokine release. Resveratrol was superior to dexamethasone in reducing CCL-2, IL-6 and IL-8 in LTA-exposed HASMCs of patients with COPD. Both drugs were equally effective in reducing GM-CSF. Resveratrol effects were partially reversed by EX527 but not by TSA. Dexamethasone effects were partially reversed by TSA but not by EX527. We conclude that HASMCs contribute to the increase in airway inflammation in COPD exacerbations caused by gram-positive bacterial infections. Our data suggest resveratrol as an alternative anti-inflammatory therapy in infection-induced COPD exacerbations. Resveratrol and corticosteroids suppress cytokine/chemokine expression through activation of SIRT1 or interaction with class I/II HDACs, respectively, in HASMCs.

Topics: Adrenal Cortex Hormones; Aged; Anti-Inflammatory Agents; Carbazoles; Chemokine CCL2; Cytokines; Dexamethasone; Female; Gram-Positive Bacteria; Granulocyte-Macrophage Colony-Stimulating Factor; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Interleukin-6; Interleukin-8; Lipopolysaccharides; Male; Middle Aged; Myocytes, Smooth Muscle; Pulmonary Disease, Chronic Obstructive; Respiratory System; Resveratrol; Sirtuin 1; Stilbenes; Teichoic Acids

Large amounts of anti-inflammatory mediators, such as interleukin (IL)-10, are produced and found early in the course of sepsis. We explore the role of IL-10 on neutrophil (PMN) activation/function using an in vitro model. Isolated human PMN were pre-incubated with lipopolysaccharide (LPS) and/or IL-10 for 18h. Subsequently, a second LPS exposure was performed and CD11b and CD66b up-regulation, and the reactive oxygen species (ROS) generation were measured 2h later. We found that IL-10 prevented PMN activation and the secretion of TNF-α and IL-8 induced by the first LPS contact. In the absence of IL-10, a second LPS exposure induced additive effects that were prevented by IL-10. Only ROS generation was highly affected by the blockade of PMN-secreted TNF-α or IL-8. Additionally, IL-10 prevented other possible mechanisms of LPS priming. Therefore, IL-10 modulates PMN activation preventing autocrine activating loops and priming mechanisms, rendering PMN less responsive to a second LPS exposure.

Topics: Autocrine Communication; Cell Degranulation; Cell Separation; Humans; Interleukin-10; Interleukin-8; Lipopolysaccharides; Neutrophil Activation; Neutrophils; Reactive Oxygen Species; Teichoic Acids

Previous studies have suggested that odontoblasts sense gram-positive bacteria components through Toll-like receptor 2 (TLR2) and trigger dental pulp immunity by producing proinflammatory cytokines. Currently, the factors that modulate odontoblast TLR2 activation are unknown. Our aim was to investigate lipopolysaccharide-binding protein (LBP) effects on the TLR2-mediated odontoblast response.. Human odontoblast-like cells were stimulated with lipoteichoic acid (LTA) (a TLR2 ligand), LBP, CD14 (a TLR2 cofactor), or various combinations of LTA/LBP, LTA/CD14, or LTA/CD14/LBP. CXCL8, IL6, and TLR2 gene expression was assessed by real-time polymerase chain reaction. CXCL8 and interleukin (IL)-6 production was determined by enzyme-linked immunosorbent assay in culture supernatants of cells stimulated with LTA, LTA/CD14, or LTA/CD14/LBP. LBP effects on nuclear factor kappa B (NF-κB), p38, JNK, ERK, STAT3, and p70S6 signaling pathways were determined in LTA-stimulated odontoblast-like cells with a multiplex biometric immunoassay. LBP effects were compared with specific inhibitors of these signaling pathways. LBP transcript and protein were investigated in vivo in healthy and inflamed dental pulps by real-time polymerase chain reaction and immunohistochemistry.. Activation of CXCL8, IL6, and TLR2 gene expression and CXCL8 and IL-6 secretion in LTA- and LTA/CD14-stimulated odontoblast-like cells was significantly decreased by LBP. LBP inhibited NF-κB and p38 signaling pathways in LTA-stimulated cells in a similar way to NF-κB and p38 inhibitors. LBP transcript and protein were detected in vivo in inflamed dental pulps but not in healthy ones.. These results demonstrate that LBP reduces TLR2-dependent production of inflammatory cytokines by odontoblast-like cells. We suggest that in this way it could modulate host defense in human dental pulp.

Topics: Acute-Phase Proteins; Carrier Proteins; Cell Culture Techniques; Extracellular Signal-Regulated MAP Kinases; Gram-Positive Bacteria; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Glycoproteins; NF-kappa B; Odontoblasts; p38 Mitogen-Activated Protein Kinases; Pulpitis; Ribosomal Protein S6 Kinases, 70-kDa; STAT3 Transcription Factor; Teichoic Acids; Toll-Like Receptor 2

To investigate the suitability of blood granulocyte and monocyte sensitivity, as measured by the quantity of different agonists required to induce CD62L shedding, for assessment of perioperative immune changes in patients undergoing cardiac surgery with cardiopulmonary bypass.. Patients scheduled for aortocoronary bypass grafting or for valve surgery were included in this prospective observational study. Blood samples were drawn before anesthesia induction, directly after surgery and 48 hours after anesthesia induction. We determined the concentration of two different inflammatory stimuli--lipoteichoic acid (LTA) and tumor necrosis factor alpha (TNF)--required to induce shedding of 50% of surface CD62L from blood granulocytes and monocytes. In parallel monocyte surface human leukocyte antigen (HLA)-DR, and plasma interleukin (IL)-8, soluble (s)CD62L, soluble (s)Toll-like receptor (TLR)-2 and ADAM17 quantification were used to illustrate perioperative immunomodulation.. 25 patients were enrolled. Blood granulocytes and monocytes showed decreased sensitivity to the TLR 2/6 agonist Staphylococcus aureus LTA immediately after surgery (p = 0.001 and p = 0.004 respectively). In contrast, granulocytes (p = 0.01), but not monocytes (p = 0.057) displayed a decreased postoperative sensitivity to TNF. We confirmed the presence of a systemic inflammatory response and a decreased immune sensitivity in the post-surgical period by measuring significant increases in the perioperative plasma concentration of IL-8 (p ≤ 0.001) and sTLR (p = 0.004), and decreases in monocyte HLA-DR (p<0.001), plasma sCD62L (p ≤ 0.001). In contrast, ADAM17 plasma levels did not show significant differences over the observation period (p = 0.401).. Monitoring granulocyte and monocyte sensitivity using the "CD62L shedding assay" in the perioperative period in cardiac surgical patients treated with the use of cardiopulmonary bypass reveals common changes in sensitivity to TLR2/6 ligands and to TNF stimulus. Further long-term follow-up studies will address the predictive value of these observations for clinical purposes.

Topics: ADAM Proteins; ADAM17 Protein; Aged; Cardiopulmonary Bypass; Enzyme-Linked Immunosorbent Assay; Female; Heart Diseases; HLA-DR Antigens; Humans; Inflammation; Interleukin-8; L-Selectin; Ligands; Lipopolysaccharides; Male; Middle Aged; Predictive Value of Tests; Prospective Studies; Staphylococcus aureus; Teichoic Acids; Tumor Necrosis Factor-alpha

Enterococcus faecalis is frequently found in the root canal of teeth, is a major microorganism of endodontic therapy failure, and is associated with chronic apical periodontitis. Human β-defensins (HBDs) are known to play critical roles in defending the host against infectious microbes and producing dental pulp in healthy and patients. The purpose of the present study was to investigate the bactericidal and neutralizing effects of HBDs on E. faecalis and E. faecalis lipoteichoic acid (Ef LTA) as a major virulence factor of E. faecalis.. HBD-1, -2, -3, and -4 were synthesized and investigated the susceptibility against E. faecalis. Also, the neutralizing effects of HBDs on cytokine and intercellular adhesion molecule 1 (ICAM-1) expression by activity of E. faecalis and Ef LTA were analyzed using enzyme-linked immunosorbent assay and flow cytometry.. HBD-1 and -2 were weakly susceptible, and HBD-3 and HBD-4 were strongly susceptible to E. faecalis. All of the HBDs exhibited neutralizing effects on the activity of Ef LTA, and HBD-3 strongly neutralized the activity of E. faecalis in tumor necrosis factor-α, interleukin-8, and ICAM-1 expression. The neutralizing effects of HBDs were to inhibit E. faecalis or Ef LTA binding to the host cells.. These results suggest that the induction of HBDs might have great potential as endodontic therapeutic agents.

Topics: Anti-Bacterial Agents; Bacterial Adhesion; Bacteriological Techniques; beta-Defensins; Cell Line; Chromatography, High Pressure Liquid; Enterococcus faecalis; Flow Cytometry; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Lipopolysaccharides; Monocytes; Tandem Mass Spectrometry; Teichoic Acids; Tumor Necrosis Factor-alpha; Virulence Factors

We recently observed that lipoteichoic acid (LTA) isolated from Lactobacillus plantarum inhibited endotoxin-mediated inflammation of the immune cells and septic shock in a mouse model. Here, we examined the inhibitory role of L. plantarum LTA (pLTA) on the inflammatory responses of intestinal epithelial cells (IEC). The human colon cell line, HT-29, increased interleukin (IL)-8 expression in response to recombinant human tumor necrosis factor (TNF)-alpha, but not in response to bacterial ligands and interferon (IFN)-gamma. TNF-α also increased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and intercellular adhesion molecule 1 (ICAM-1) through activation of p38 mitogen-activated protein kinase (MAPK) from HT-29 cells. However, the inflammatory response of HT-29 on TNF-α stimulation was significantly inhibited by pLTA treatment. This pLTA-mediated inhibition accompanied the inhibition of nuclear factor (NF)-kappa B and MAPKs. Our data suggest that pLTA regulates cytokine-mediated immune responses and may be a good candidate for maintaining intestinal homeostasis against excessive inflammation.

Topics: Cell Adhesion; Cell Line, Tumor; Epithelial Cells; HT29 Cells; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-8; Intestinal Mucosa; Intestines; Lactobacillus plantarum; Lipopolysaccharides; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Teichoic Acids; Tumor Necrosis Factor-alpha

Pattern-recognition receptors such as Toll-like receptors (TLRs) are essential sensors implicated in the early and efficient innate immune response against pathogens. We have previously demonstrated that leukotriene B(4)(LTB(4)) has the capacity to enhance leukocyte responses to TLR9 ligands and to control viral infection. In this report, we provide evidence that LTB(4) treatment of human neutrophils leads to a potentiation in proinflammatory cytokine secretion induced by various myeloid differentiation factor 88-dependent TLR agonists. LTB(4) failed to enhance TLR mRNA levels as well as expression of TLR2 and TLR4 receptors, suggesting that LTB(4) acts through intracellular mechanism(s) to potentiate neutrophil responses to TLR ligands. We found that while IRAK can be activated by LTB(4), this process is dispensable to LTB(4) to potentiate neutrophil responses to TLR ligands since pretreatment of neutrophils with IRAK1/4 inhibitor did not affect its potentiating effects. However, our data clearly show that LTB(4) treatment of neutrophils led to the phosphorylation of downstream signaling molecules, TAK1 and p38, a process found essential to observe an increased secretion of cytokines by neutrophils activated with TLR ligands. Pretreatment of neutrophils with TAK1 or p38 kinase inhibitors strongly repressed the effect of LTB(4) on cytokine synthesis by neutrophils stimulated with LTA, LPS or CpG. The same pattern was observed in agonist-treated human embryonic kidney 293 cells transfected with TAK1-targeting siRNA where secretion of IL-8 was significantly reduced to basal levels. These results indicate that TAK1 and p38 kinases appear to be central in the 'priming effect' of LTB(4) on neutrophils to enhance response to TLR ligands.

Topics: Blotting, Western; Cells, Cultured; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; HEK293 Cells; Humans; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Leukotriene B4; Ligands; Lipopolysaccharides; MAP Kinase Kinase Kinases; Neutrophils; Oligodeoxyribonucleotides; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 9; Tumor Necrosis Factor-alpha

In the intestine, bacterial components activate innate responses that protect the host. We hypothesize that bacterial components reduce Interleukin-8 (IL-8) production in intestinal epithelial cells stimulated by flagellin via the Toll-like receptor (TLR) signaling pathway. Caco-2 cells were pretreated with various doses of lipopolysaccharide (LPS), lipoteichoic acid (LTA), or low-dose flagellin (LDFL) for 24h. Cells were then treated with flagellin (FL) 500 ng/ml (HDFL) for another 48 h. IL-8 production was measured in the cell culture medium by ELISA. Eighty-four genes in the TLR pathway were evaluated by RT Profiler PCR Array. Pathway Studio 8.0 software was used for altered pathway analysis. HDFL induced IL-8 production by 19-fold (p<0.01). Pretreatment with LDFL at 20, 10 or 1 ng/ml reduced HDFL-induced IL-8 production by 61%, 52% and 40%, respectively (p<0.05). LPS at 50 μg/ml decreased HDFL-induced IL-8 production by 38% (p<0.05). HDFL up-regulated CXCL10, IL1B, IL-8, IRAK2, NF-κB1 and I-κB (all p<0.05). Pathway Studio analysis showed that HDFL induced cell processes including inflammation, cell death and apoptosis. Pretreatment with LDFL at 10 ng/ml down-regulated FADD, FOS, MAP4K4, MyD88, TLR2, TLR3 and TNFERSF1A compared to HDFL (all p<0.05). These down-regulated genes are integral for numerous cell functions including inflammatory response, cell death, apoptosis and infection. These results demonstrate that LPS and LDFL provoke tolerance to HDFL-induced IL-8 production. This tolerance effect was accompanied by a complex interaction of multiple genes related to inflammatory as well as other responses in the TLR pathway rather than a single gene alteration.

Topics: Apoptosis; Caco-2 Cells; Chemokine CXCL10; Down-Regulation; Epithelial Cells; Escherichia coli; Fas-Associated Death Domain Protein; Flagellin; Gene Expression Profiling; Humans; I-kappa B Proteins; Inflammation; Interleukin-1 Receptor-Associated Kinases; Interleukin-1beta; Interleukin-8; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; Myeloid Differentiation Factor 88; NF-kappa B; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-fos; Signal Transduction; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 3; Toll-Like Receptors; Up-Regulation

Probiotic bacteria are increasingly used as immunomodulatory agents. Yet detailed molecular knowledge on the immunomodulatory molecules of these bacteria is lagging behind. Lipoteichoic acid (LTA) is considered a major microbe-associated molecular pattern (MAMP) of Gram-positive bacteria. However, many details and quantitative data on its immune signalling capacity are still unknown, especially in beneficial bacteria. Recently, we have demonstrated that a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having modified LTA molecules, has an enhanced probiotic efficacy in a DSS-induced colitis model as compared to wild-type.. In this study, the importance of D-alanylated and acylated LTA for the pro-inflammatory activity of LGG was studied in vitro. Purified native LTA of LGG wild-type exhibited a concentration-dependent activation of NF-κB signalling in HEK293T cells after interaction with TLR2/6, but not with TLR2 alone. Chemical deacylation of LTA interfered with the TLR2/6 interaction, while a moderate effect was observed with chemical dealanylation. Similarly, the dltD mutant of LGG exhibited a significantly reduced capacity to activate TLR2/6-dependent NF-κB signalling in a HEK293T reporter cell line compared to wild-type. In addition, the dltD mutant of LGG showed a reduced induction of mRNA of the chemokine IL-8 in the Caco-2 epithelial cell line compared to wild-type. Experiments with highly purified LTA of LGG confirmed that LTA is a crucial factor for IL-8 mRNA induction in Caco-2 epithelial cells. Chemical dealanylation and deacylation reduced IL-8 mRNA expression.. Taken together, our results indicate that LTA of LGG is a crucial MAMP with pro-inflammatory activities such as IL-8 induction in intestinal epithelial cells and NF-κB induction in HEK293T cells via TLR2/6 interaction. The lipid chains of LGG LTA are needed for these activities, while also the D-alanine substituents are important, especially for IL-8 induction in Caco-2 cells.

Topics: Caco-2 Cells; HEK293 Cells; Humans; Interleukin-8; Lacticaseibacillus rhamnosus; Lipopolysaccharides; NF-kappa B; RNA, Messenger; Signal Transduction; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 6

Human odontoblasts trigger immune response s to oral bacteria that invade dental tissues during the caries process. To date, their ability to regulate the expression of the nucleotide-binding domain leucine-rich repeat containing receptor NOD2 when challenged by Gram-positive bacteria is unknown. In this study, we investigated NOD2 expression in healthy and inflamed human dental pulps challenged by bacteria, and in cultured odontoblast-like cells stimulated with lipoteichoic acid (LTA), a Toll-like receptor (TLR) 2 agonist which is specific for Gram-positive bacteria. We found that NOD2 gene expression was significantly up-regulated in pulps with acute inflammation compared to healthy ones. In vitro, LTA augmented NOD2 gene expression and protein level in odontoblast-like cells. The increase was more pronounced in odontoblast-like cells compared to dental pulp fibroblasts. Blocking experiments in odontoblast-like cells with anti-TLR2 antibody strongly reduced the NOD2 gene expression increase, whereas stimulation with the synthetic TLR2 ligand Pam(2)CSK(4) confirmed NOD2 gene up-regulation following TLR2 engagement. These data suggest that NOD2 up-regulation is part of the odontoblast immune response to Gram-positive bacteria and might be important in protecting human dental pulp from the deleterious effects of cariogenic pathogens.

Topics: Antibodies, Monoclonal; Carrier Proteins; Cells, Cultured; Dental Pulp; Fibroblasts; Gene Expression; Humans; Interleukin-8; Lipopolysaccharides; Molar; Nod2 Signaling Adaptor Protein; Odontoblasts; Pulpitis; Teichoic Acids; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

Whole blood culture (C(wb)) is a method to evaluate leukocyte response to stimuli. We used C(wb) to evaluate the inflammatory response to pathogen associated molecular patterns (PAMPs) in cats. Blood was collected from diluted with RPMI and stimulated with various concentrations of lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PG) or control (PBS). Multiple concentrations of LPS, LTA and PG significantly stimulated tumor necrosis factor (TNF), interleukin (IL)-1β and CXC chemokine ligand (CXCL)-8 in feline C(wb). All PAMPs failed to stimulate IL-6 production and PG failed to stimulate CXCL-8 production. Lipopolysaccharide was a more potent inducer of IL-1β and CXCL-8 than LTA or PG and LTA is a more potent inducer of CXCL-8 than PG. Based on these data, PAMPs from gram positive and negative bacteria induce TNF, IL-1β and CXCL-8 production in feline whole blood. Cats appear to be relatively more sensitive to gram negative compared to gram positive bacteria.

Topics: Animals; Cats; Interleukin-1beta; Interleukin-6; Interleukin-8; Leukocytes; Lipopolysaccharides; Peptidoglycan; Teichoic Acids; Tumor Necrosis Factor-alpha

The role of the different major cell wall components of Gram-positive bacteria for immune stimulation is controversial. We thus compared the cytokine inducing capacity of different Staphylococcus aureus (SA) mutants lacking either lipoproteins (SA 113Δlgt), or wall teichoic acids (WTA) (ΔTA), or possessing a reduced d-alanine content in lipoteichoic acid (LTA) (SA 113Δdlt) to its corresponding wildtype (SA 113wt). Inactivated whole bacteria and their purified cell wall components peptidoglycan and LTA, were used to stimulate human whole blood and macrophages from TLR2 knock-out mice. We found that all S. aureus strains induced similar amounts of TNF, IL-8 and IL-10 and none of them was dependent on the presence of TLR2. Surprisingly, in case of SA 113Δlgt a significant attenuated release of only IL-1β protein and mRNA in human whole blood was observed. Highly purified peptidoglycan from all strains in contrast to LTA had a very low activity in stimulating cytokine release. Taken together these results demonstrate that major cell wall alterations like lack of WTA, lipoproteins or alterations in d-alanine content do not affect the overall cytokine inducing potential of whole bacteria and thus cytokine induction is initiated by redundant mechanisms.

Topics: Alanine; Animals; Cell Wall; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-10; Interleukin-1beta; Interleukin-8; Lipopolysaccharides; Lipoproteins; Macrophages; Mice; Mice, Knockout; Peptidoglycan; RNA, Messenger; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptor 2; Tumor Necrosis Factors

Interleukin-17A (IL-17A) and IL-17F have been shown to mediate a crucial crosstalk between the immune system and various epithelial tissues, stimulating various defensive mechanisms to bacterial infections. A number of studies have characterized the response to IL-17A and IL-17F of epithelial cells from airways, intestine, and skin, but not from the mammary gland. To evaluate the potential contribution of IL-17 to the immune defense of the mammary gland, we analyzed the effects of recombinant bovine IL-17A and IL-17F on primary bovine mammary epithelial cells (MEC) by quantitative PCR and ELISA. We found expression (mRNA) of the two components of the IL-17 receptor complex, IL-17RA and IL-17RC, in mammary tissue and MEC in vitro. The expression of a number of genes encoding cytokines, chemokines and proteins endowed with antibacterial activities was increased by IL-17A, and to a lesser extent by IL-17F, but the magnitude of responses was modest. As expected, responses were augmented by the combination of IL-17A or IL-17F with TNF-α. Interestingly, responses of a few of the tested genes, such as IL8, CCL20, iNOS, and CfB, were augmented by the combination of IL-17A with staphylococcal lipoteichoic acid or muramyl dipeptide, bacterial agonists of the innate immune system. This can be interpreted as indicating that IL-17A and IL-17F are tailored to exert their full potential in a septic environment. MEC responses were characterized by the expression of chemokines targeting not only neutrophils (CXCL3 and CXCL8) but also mononuclear leucocytes (CCL2, CCL20). Production of IL-6 was low and the inflammatory cytokines TNF-α and IL-1β were expressed (mRNA) but proteins were not secreted. Altogether, our results suggest that IL-17A and IL-17F have a potential to modulate the mammary gland immune response to mastitis-causing pathogens.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Cattle; Epithelial Cells; Female; Gene Expression Regulation; Humans; Immunity; Immunity, Innate; Interleukin-17; Interleukin-8; Lipopolysaccharides; Mammary Glands, Animal; NF-kappa B; Receptors, Interleukin-17; Receptors, Pattern Recognition; RNA, Messenger; Staphylococcus; Teichoic Acids; Tumor Necrosis Factor-alpha

Odontoblasts, dental pulp fibroblasts and immature dendritic cells (DCs) have been involved in the human dental pulp immune response to oral pathogens that invade dentine during the caries process. How they regulate the inflammatory response to Gram-positive bacteria remains nevertheless largely unknown. In this study we investigated the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (CXCL8) in these three cell types upon stimulation with lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria that activates the pattern recognition molecule Toll-like receptor 2 (TLR2). We observed that TNF-alpha gene expression was up-regulated in all LTA-stimulated cell types. IL-1beta gene expression was not or barely detectable in odontoblast-like cells and pulp fibroblasts when stimulated or not, but was expressed in immature DCs and increased upon stimulation. TNF-alpha and IL-1beta proteins were detected in DC culture supernatants but not in odontoblast-like cell and pulp fibroblast ones. CXCL8 gene and protein were clearly expressed and increased in the three cell types upon LTA stimulation. These data indicate that LTA-dependent TLR2 activation in odontoblasts and pulp fibroblasts, in contrast to immature DCs, does not lead to significant TNF-alpha and IL-1beta production, but that all three cell types influence the pulp inflammatory/immune response through CXCL8 synthesis and secretion.

Topics: Cell Differentiation; Cells, Cultured; Dendritic Cells; Dental Pulp; Fetal Blood; Fibroblasts; Gene Expression Profiling; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Humans; Immunity, Innate; Interleukin-1beta; Interleukin-8; Lipopolysaccharides; Molar, Third; Odontoblasts; Teichoic Acids; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

CXC chemokines are potential attractants for polymorphonuclear leucocytes (PMNs) and play an important role in resistance to infectious diseases, such as bovine mastitis. In this study, a bovine mammary epithelial cell line (MAC-T) and blood PMNs were stimulated with bacterial cell wall components of gram negative and gram positive bacteria, including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). The expression of two CXC chemokines, interleukin (IL)-8 and granulocyte chemotactic protein (GCP)-2 was analysed by real-time PCR. High concentrations (1 or 10 μg/mL) of LPS and LTA, but not PGN, significantly increased the expression of GCP-2 and IL-8 in both MAC-T and PMNs. Biopsies from mammary glands of cattle with clinical Escherichia coli mastitis also had increased expression of GCP-2. Using an in vitro transepithelial migration assay, recombinant human GCP-2 (rhGCP-2) showed weak chemoattractant effects on bovine blood PMNs. It was concluded that PMNs and MAC-T cells can express GCP-2 in response to certain bacterial cell components during the course of mastitis.

Topics: Animals; Cattle; Cell Line; Chemokine CXCL6; Chemotactic Factors; Epithelial Cells; Escherichia coli; Female; Humans; Interleukin-8; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Neutrophils; Peptidoglycan; Recombinant Proteins; Teichoic Acids

Bacterial infection with Staphylococcus aureus is a known trigger for worsening of atopic dermatitis (AD); the exact mechanisms by which bacterial infection worsens dermatitis are unknown.. We sought to characterize the amounts of the biologically active bacterial lipoprotein lipoteichoic acid (LTA) in infected AD lesions.. Eighty-nine children with clinically impetiginized lesions of AD were enrolled in this study. A lesion was graded clinically by using the Eczema Area and Severity Index (EASI), wash fluid obtained from the lesion for quantitative bacterial culture, and measurement of LTA and cytokines. The staphylococcal isolate was tested for antibiotic susceptibilities. The patients were treated with a regimen that included topical corticosteroids and systemic antibiotics, and the lesion was reanalyzed after 2 weeks.. S aureus was identified in 79 of 89 children enrolled in the study. The bacterial colony-forming unit (CFU) counts correlated with the EASI lesional score (P = .04). LTA levels as high as 9.8 mug/mL were measured in the wash fluid samples, and the amounts correlated with the lesional EASI scores (P = .01) and S aureus CFU (P < .001). Approximately 30% of clinically impetiginized AD lesions contained greater than 1 mug/mL LTA, amounts that exert effects on various cell types in vitro. Moreover, injection of skin tissue ex vivo with amounts of LTA found in AD lesions resulted in epidermal cytokine gene expression.. Pharmacologic levels of LTA are found in many infected atopic dermatitis lesions.

Topics: Child; Child, Preschool; Colony Count, Microbial; Dermatitis, Atopic; Eczema; Humans; Infant; Interleukin-8; Lipopolysaccharides; Severity of Illness Index; Skin; Staphylococcal Skin Infections; Staphylococcus aureus; Teichoic Acids; Tumor Necrosis Factor-alpha

Staphylococcus aureus is a significant human pathogen that can colonize the skin. Neutrophils are well known to be involved in clearance of the bacterium. This study focused on exploring the role that human keratinocytes have as first responders to bacterial challenges. IL-1alpha and IL-1beta increased mRNA production and protein secretion of the neutrophil chemotactic CXCL1, CXCL2, and IL-8 in keratinocytes. S. aureus and the bacterial cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) induced similar expression profiles in a Toll-like receptor (TLR)-2-dependent manner. Interestingly, the S. aureus-induced mRNA levels peaked at later time points than those induced by IL-1. The S. aureus-activated chemokine production was preceded by significant IL-1alpha and IL-1beta secretion. Expression of IL-1alpha was significantly higher than that of IL-1beta. Inhibition of IL-1RI using neutralizing antibodies revealed that S. aureus-derived LTA and PGN-induced chemokine expression requires IL-1RI engagement. Surprisingly, we further found that chemokine secretion is dependent upon endocrine IL-1alpha, but not IL-1beta, signaling. Our data show that the innate immune response of keratinocytes is regulated differently than those of other cell types. This may represent a fail-safe system that protects the host against genetic variation and immune evasion mechanisms developed by pathogens.

Topics: Antibodies, Neutralizing; Autocrine Communication; Cell Wall; Cells, Cultured; Chemokine CXCL1; Chemokine CXCL2; Chemotaxis, Leukocyte; Humans; Interleukin-1alpha; Interleukin-1beta; Interleukin-8; Keratinocytes; Lipopolysaccharides; Neutrophils; Oligonucleotide Array Sequence Analysis; Peptidoglycan; Receptors, Interleukin-1; RNA, Messenger; Signal Transduction; Staphylococcal Skin Infections; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptor 2

Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and potentiating signalling pathways associated with TLR. These results suggest that neuronal responses may influence innate host defence responses.

Topics: Active Transport, Cell Nucleus; Arachidonate 5-Lipoxygenase; Cell Degranulation; Cell Line; Chemokines; Cytokines; Drug Synergism; Gene Expression; Humans; Interleukin-8; Leukotriene C4; Lipopeptides; Lipopolysaccharides; Mast Cells; Mitogen-Activated Protein Kinases; Phosphorylation; Substance P; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptors; Transcription Factors; Up-Regulation

Streptococcus pneumoniae LTA is a highly complex glycophospholipid that consists of nine carbohydrate residues: three glucose, two galactosamine and two 2-acetamino-4-amino-2,4,6-trideoxygalactose (AATDgal) residues that are each differently linked, one ribitol and one diacylated glycerol (DAG) residue. Suitable building blocks for the glucose and the AATDgal residues were designed and their synthesis is described in this paper. These building blocks permitted the successful synthesis of the core structure Glcβ(1-3)AATDgalβ(1-3)Glcα(1-O)DAG in a suitably protected form for further chain extension (1 b, 1 c) and as unprotected glycolipid (1 a) that was employed in biological studies. These studies revealed that 1 a as well as 1 lead to interleukin-8 release, however not via TLR2 or TLR4 as receptor.

Topics: Glycolipids; HEK293 Cells; Humans; Interleukin-8; Lipopolysaccharides; Molecular Structure; Streptococcus pneumoniae; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4

During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-alpha and IL-1beta. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 microg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-alpha, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.

Topics: Animals; Cattle; Cattle Diseases; Chemokine CCL2; Chemokine CXCL6; Dinucleoside Phosphates; Female; Gene Expression Regulation; Inflammation; Interleukin-8; Lipopolysaccharides; Mammary Glands, Animal; Oligodeoxyribonucleotides; Peptidoglycan; Teichoic Acids; Toll-Like Receptor 9

Intra-uterine infections increase production of pro-inflammatory cytokines. It is unclear whether different infectious agents determine the relative expression of pro-and anti-inflammatory cytokines.. We compared the placental inflammatory response induced by bacterial lipopolysaccharide (LPS, endotoxin from Gram-negative bacteria) with those induced by lipoteichoic acid (LTA, a cell wall component of Gram-positive bacteria). Placental explants from term delivery were treated with either LPS or LTA, in the presence or absence of IL-10, for 24 hrs. Cytokines, prostaglandin E(2) (PGE(2)) production and cyclo-oxygenase-2 (COX-2) expression were quantified.. Both LTA and LPS significantly induced several cytokines with LPS eliciting more potent effects. IL-6 and IL-8 were induced to comparable levels in response to both LTA and LPS whereas monocyte chemotactic protein-1 (MCP-1) production was induced more by LTA, demonstrating a differential placental response to a specific toll-like receptor (TLR) ligand. IL-10 treatment significantly reduced most pro-inflammatory cytokines as well as PGE(2) induced by both LPS and LTA. Interestingly, IL-10 down-regulated LTA-mediated MCP1 induction, but not that mediated by LPS. Moreover, IL-10 was more effective in down-regulating PGE(2) after LPS- when compared with LTA stimulation.. Our results demonstrate that placental exposure to LTA and LPS appear to trigger distinct cytokine responses that can be modulated by IL-10.

Topics: Chemokine CCL2; Cyclooxygenase 2; Dinoprostone; Female; Humans; Immunomodulation; Interleukin-10; Interleukin-6; Interleukin-8; Lipopolysaccharides; Organ Culture Techniques; Placenta; Pregnancy; Teichoic Acids; Toll-Like Receptors

Proinflammatory cytokines are centrally involved in tumor progression and survival in non-small cell lung cancer, and both the presence of infiltrating neutrophils and bacterial infection in the lung may indicate a poor prognosis. Against this background, we investigated the effect of the bacterial cell wall component lipopolysaccharide (LPS) on interleukin (IL)-6 and IL-8 synthesis in the non-small cell lung cancer line A549 and in A549-neutrophil cocultures. The LPS induced a dose-dependent and time-dependent release of IL-8 from A549 cells, whereas IL-6 could not be detected. Interestingly, in A549-neutrophil cocultures, IL-8 synthesis was massively amplified and IL-6 was also released, compared with the respective monocultures. The A549 cells were identified as the primary cellular source of these cytokines, as enhanced cytokine mRNA transcription was detected in this cell type, although not in neutrophils in the coculture system. Experiments done in transwells indicated that direct cell-cell contact was a prerequisite for the increased cytokine generation. Inhibition of tumor necrosis factor-alpha bioactivity by neutralizing antibodies and blocking cyclooxygenase-2 activity blunted the enhanced cytokine generation in the coculture system. Amplification of LPS-induced cytokine secretion could be reproduced when the small cell lung cancer cell line H69 was cocultured with neutrophils. When the Gram-positive cell wall component lipoteichoic acid was used instead of LPS, cytokine synthesis was also amplified in A549-neutrophil cocultures, to a similar extent to that observed with LPS. These data indicate that interaction between bacterial pathogens, neutrophils, and tumor cells might amplify the release of proinflammatory cytokines which may promote tumor growth in vivo.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Communication; Cell Line, Tumor; Cells, Cultured; Chemotaxis, Leukocyte; Coculture Techniques; Cyclooxygenase 2 Inhibitors; Cytokines; Disease Progression; Dose-Response Relationship, Drug; Humans; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Lipopolysaccharides; Lung Neoplasms; Neutrophils; Pneumonia, Bacterial; RNA, Messenger; Teichoic Acids; Time Factors; Transcriptional Activation

Pulpitis is characterized by the marked infiltration of inflammatory cells in response to an invasion of caries-related bacteria. It is well known that chemokines regulate the trafficking of lymphocytes, and CC chemokine ligand 20 (CCL20) has been recently shown to play a crucial role in the recruitment of memory T cells and immature dendritic cells into inflammatory lesions. We previously reported that CCL20 was mainly expressed in microvascular endothelial cells and macrophages that accumulated in inflamed pulp tissues and that its specific receptor, CCR6, was expressed on infiltrated lymphocytes. However, the mechanism of CCL20 expression remains unclear.. In this study, we investigated the expression of CCL20 in monocytes/macrophages, endothelial cells, and pulpal fibroblasts after stimulation with Streptococcus mutans, a representative of caries-related bacteria, or proinflammatory cytokines. CCL20 messenger RNA was detected by reverse transcription-polymerase chain reaction in inflamed pulp, but not in clinically normal pulp. By enzyme-linked immunosorbent assay, S. mutans induced a human monocytic cell line, differentiated macrophage-like THP-1 cells, and human umbilical vein endothelial cells (HUVEC) to produce an increased amount of CCL20. Lipoteichoic acid from S. mutans also elicited CCL20 production by HUVEC. Moreover, CCL20 production from pulpal fibroblasts was increased by stimulation with inetrleukin-1beta and tumor necrosis factor-alpha.. Our results indicate that CCL20 expression is induced by stimulation with caries-related bacteria that have invaded deeply into the dentinal tubules as well as by proinflammatory cytokines in the inflamed pulpal lesions. It may be involved in the progression of pulpitis via accumulation of inflammatory cells.

Topics: Adult; Aged; Cell Differentiation; Cell Line; Cells, Cultured; Chemokine CCL20; Cytokines; Dental Pulp; Endothelial Cells; Endothelium, Vascular; Female; Fibroblasts; Humans; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-8; Lipopolysaccharides; Macrophages; Male; Middle Aged; Monocytes; Pulpitis; Streptococcus mutans; Teichoic Acids; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.

Topics: Animals; Cattle; CD36 Antigens; Cell Line; Cloning, Molecular; Female; Flow Cytometry; Humans; Interleukin-8; Lipopolysaccharides; Mastitis, Bovine; Nitric Oxide; Peptidoglycan; Receptors, Pattern Recognition; Reverse Transcriptase Polymerase Chain Reaction; RNA; Streptococcal Infections; Streptococcus agalactiae; Teichoic Acids; Toll-Like Receptor 2; Transfection

Human endothelial cells (EC) express Toll-like receptor 4 (TLR4), a receptor for lipopolysaccharides (LPS), but little or no TLR2, a lipopeptide receptor. The aim of this study was to investigate to what extent inflammatory stimuli modify the expression by EC of TLR4 and TLR2, of the TLR2 co-receptors TLR1 and TLR6 and of the TLR2-accessory proteins CD14 and CD36. Stimulation of umbilical vein derived EC with TNF-alpha, LPS or IL-1beta for 24h induced a strong increase in TLR2 mRNA but not in TLR1, TLR4 and TLR6 mRNA. Inflammatory activation had little effect on CD14 mRNA, but decreased the expression of CD36 mRNA. TLR2 antigen was readily detected by flow cytometry on activated EC, but not on resting EC. A significant proportion of TLR2 was found to be located intracellularly. By using specific signalling pathway inhibitors we established that the induction of TLR2 by inflammatory stimuli was dependent on NF-kappaB, p38-MAP kinase and c-Jun kinase. IRAK-1 phosphorylation after treatment with 10mug/ml of lipoteichoic acid (LTA), a TLR2 agonist, was only observed in TNF-alpha-stimulated EC and not in resting EC. Furthermore, LTA potentiated the increase of the inflammatory markers E-Selectin or IL-8 in EC pre-treated with TNF-alpha, LPS or IL-1beta, but not in resting EC. These results imply that the up-regulated TLR2 is functionally active. Interestingly, LTA had no effect on TLR2 expression, nor maintained TLR2 expression, in activated EC. This suggests that lipopeptide responses of EC are dependent on the continued presence of inflammatory cytokines, provided by other cell types, or LPS. In conclusion, inflammatory stimuli induce a high TLR2 expression in EC, which in turn enables the cells to strongly respond to lipopeptides. The up-regulation of TLR2 may be of relevance for the vascular effects of Gram-positive bacteria.

Topics: CD36 Antigens; Cell Membrane; E-Selectin; Endothelial Cells; Humans; Inflammation; Interleukin-1 Receptor-Associated Kinases; Interleukin-1beta; Interleukin-8; Intracellular Space; Lipopeptides; Lipopolysaccharide Receptors; Lipopolysaccharides; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; RNA, Messenger; Signal Transduction; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 6; Tumor Necrosis Factor-alpha; Up-Regulation

The various functions of gelsolin in extracellular compartments are not yet clearly defined but include actin scavenging and antiinflammatory effects. Gelsolin was recently reported to bind endotoxin (LPS) from various Gram-negative bacteria with high affinity. In this study we investigate whether gelsolin also interacts with bacterial wall molecules of Gram-positive bacteria such as lipoteichoic acid (LTA) and whether gelsolin's interaction with bacterial lipids from Gram-negative or Gram-positive bacteria affects their cellular inflammatory responses. A peptide based on the PPI binding site of gelsolin (160-169) binds purified LTA at the same molecular ratio that it binds phosphatidylinositol 4,5-bisphosphate. The OD of recombinant human plasma gelsolin was found to decrease following the addition of purified LTA, and the binding of gelsolin to LTA inhibits F-actin depolymerization by gelsolin. Simultaneously, the ability of LTA to activate translocation of NF-kappaB, E-selectin expression, and adhesion of neutrophils to LTA-treated human aortic endothelial cells was compromised by gelsolin. Gelsolin was able to partially inhibit LPS- or LTA-induced release of IL-8 from human neutrophils but was unable to prevent Gram-positive Bacillus subtilis or Gram-negative Pseudomonas aeruginosa growth and had no effect on the antibacterial activity of the cathelicidin-derived antibacterial peptide LL37. These data suggest that extracellular gelsolin is involved in the host immune recognition of LTA or LPS following release of these molecules from the bacterial outer membrane during cell division or attack by drugs and immune components.

Topics: Actins; Cell Adhesion; Cell Wall; Cells, Cultured; E-Selectin; Extracellular Fluid; Gelsolin; Humans; Immunity, Cellular; Inflammation Mediators; Interleukin-8; Lipopolysaccharides; Neutrophils; NF-kappa B; Peptide Fragments; Protein Binding; Staphylococcus aureus; Teichoic Acids

Rhinovirus infection is responsible for considerable morbidity and mortality as the major cause of exacerbations of asthma, and is also known to induce exacerbations of cystic fibrosis and chronic obstructive pulmonary disease. Exacerbations of these diseases are also frequently associated with bacterial and atypical bacterial infection. Alveolar macrophages are the major immune cells in the airways and are important in defence against bacterial infections.. The authors investigated whether rhinovirus modifies cytokine release, the pattern recognition receptor expression and phagocytosis by human alveolar macrophages in response to bacterial products.. Viable rhinovirus was detected in macrophages up to 3 days after exposure and viral RNA expression persisted for 10 days. Infectious but not UV inactivated rhinovirus increased tumour necrosis factor alpha (TNFalpha) and interleukin (IL)8 release by macrophages. In contrast, infectious rhinovirus impaired lipopolysaccharide and lipoteichoic acid induced TNFalpha and IL8 secretion by macrophages. Rhinovirus induced impairment of macrophage antibacterial immune responses did not involve IL10, prostaglandin E(2) or downregulation of Toll-like receptor 2. Furthermore, the macrophage phagocytic response to labelled bacterial particles, but not to latex beads, was impaired.. The authors have identified impairment of cytokine responses to bacterial lipopolysaccharide and lipoteichoic acid by alveolar macrophages in response to infectious rhinovirus. Virus induced impairment of antibacterial host defence has important implications in the pathogenesis of exacerbations of respiratory diseases.

Topics: Down-Regulation; Enzyme-Linked Immunosorbent Assay; Female; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Humans; Immunity, Cellular; Interleukin-8; Lipopolysaccharides; Macrophages, Alveolar; Male; Middle Aged; Phagocytosis; Picornaviridae Infections; Rhinovirus; Teichoic Acids; Tumor Necrosis Factor-alpha

The evaluation of the effects of Enterococcus hirae, an intestinal bacterium in the adjacent mucosa (mucosal bacterium), on tumour necrosis factor-alpha (TNF-alpha)-induced barrier impairment in human epithelial Caco-2 cells.. The filter-grown Caco-2 monolayers were used as an intestinal epithelial model system. In Caco-2 cells, heat-killed E. hirae ATCC 9790(T) suppressed the TNF-alpha-induced barrier impairment and increase in interleukin-8 (IL-8) secretion, but lipase- and mutanolysin-treated E. hirae ATCC 9790(T) did not have these effects. It was demonstrated that lipoteichoic acid (LTA) from E. hirae ATCC 9790(T) is responsible for Caco-2 cells' recovery from TNF-alpha-induced impairments. In addition, Caco-2 cells had the same response to Toll-like receptor 2 (TLR2) ligand, Pam(3)Cys-Ser-(Lys)(4) as they did to LTA. Increased expression of zonula occludens-1 was observed by the addition of E. hirae ATCC 9790(T) to TNF-alpha-treated Caco-2 cells, and decreased expression of myosin light chain kinase was observed by the addition of LTA and Pam(3)Cys-Ser-(Lys)(4); this, in turn, led to barrier enforcement.. Enterococcus hirae ATCC 9790(T) cell wall fractions, such as LTA, protect against intestinal impairment by regulation of epithelial tight junction via TLR2 signalling.. Enterococcus hirae could be useful in the treatment of inflammatory bowel disease, as well as other intestinal disorders.

Topics: Caco-2 Cells; Cell Wall; Enterococcus; Epithelial Cells; Humans; Interleukin-8; Lipopeptides; Lipopolysaccharides; Membrane Proteins; Myosin-Light-Chain Kinase; Peptides; Phosphoproteins; Teichoic Acids; Tight Junctions; Tumor Necrosis Factor-alpha; Zonula Occludens-1 Protein

Bacterially derived ligands, Pam3CSK4 and LPS, can directly impact adrenal glands steroidogenesis through microdomain-related TLR1/2 and 4, respectively, and indirectly via immune cell-derived cytokines. The bilateral immunoadrenal relationship plays an important role in the proper functioning of both systems. CXC chemokine-dependent immune cell infiltration into adrenocortical carcinomas (ACC), which correlates with poor prognosis, is a common phenomenon. Recently, IL8 was identified in ACC and NCI-H295R cells, and was found to contribute to ACC tumour growth. The aim of this study was to clarify the role of different TLR ligands in IL8 production in NCI-H295R cells. This is the first study to demonstrate the expression of several TLRs including TLR1, 3, 6, 7 and 9 in human adrenocortical cells by using the RT-PCR approach. Only stimulation with TLR1/6 together with TLR2 ligands resulted in IL8 peptide and mRNA induction in a dose and time-dependent manner. Our data suggest that gram-positive bacteria-related TLR1/2/6 ligands might contribute to adrenal gland tumorigenesis via IL8 production.

Topics: Adrenal Cortex Neoplasms; Humans; Interleukin-8; Ligands; Lipopeptides; Lipopolysaccharides; Peptides; Teichoic Acids; Toll-Like Receptors

Ambient particulate matter (PM) has been shown to be associated with mortality and morbidity. Diesel exhaust particles (DEP) contribute to ambient PM. Alveolar macrophages (AM) are important targets for PM effects in the lung. The effects of DEP exposure on human AM response to lipopolysachharide (LPS; from gram-negative bacteria) challenge in vitro were determined by monitoring the production of interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)). The roles of organic compounds and carbonaceous core of DEP in response to LPS were evaluated by comparing the DEPs effect to that of carbon black (CB), a carbonaceous particle with few adsorbed organic compounds. AMs were exposed in vitro to Standard Reference Material (SRM) DEP 2975, SRM DEP 1650, SRM 1975 (a dichloromethane extract of SRM DEP 2975) and CB particles for 24 h. DEPs induced a decreased secretion of IL-8, TNF-alpha and PGE(2) in response to a subsequent LPS stimulation. DEPs also show suppressive effect on the release of inflammatory mediators when stimulated with lipoteichoic acid, a product of gram positive bacteria. In summary, in vitro exposure of human AM to DEPs significantly suppress AM responsiveness to gram-negative and positive bacterial products, which may be a contributing factor to the impairment of pulmonary defense.

Topics: Cell Survival; Dinoprostone; Female; Humans; In Vitro Techniques; Inflammation Mediators; Interleukin-8; Lipopolysaccharides; Macrophages, Alveolar; Male; Teichoic Acids; Tumor Necrosis Factor-alpha; Vehicle Emissions

In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils.. Isolated human neutrophils from healthy volunteers.. Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies.. Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction.. The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Topics: Analysis of Variance; Cells, Cultured; Cytokines; Humans; Interleukin-10; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Neutrophils; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Gram-positive bacterial products such as peptidoglycan (PGN) and lipoteichoic acid (LTA) are potent stimulators of innate inflammatory responses. We previously reported that lipopolysaccharide (LPS), a major biologically active agent of gram-negative bacteria, induces a proinflammatory response via the Toll-like receptor (TLR) 4 in hepatic stellate cells (HSCs). Here we investigated the mechanism of proinflammatory action by PGN and LTA in activated human HSCs. Following treatment with either TNF-alpha or IL-1beta, expression of TLR2 and CD14 was determined by real-time PCR and Western blotting. NF-kappaB activation was assessed by NF-kappaB-driven luciferase assay and electrophoretic mobility shift assay. Interleukin-8 (IL-8) from culture supernatant was measured by ELISA. Activated human HSCs express TLR2 and CD14, which are receptors for PGN and LTA signaling. TNF-alpha and IL-1beta significantly upregulated the expression of TLR2 mRNA and protein in HSCs. PGN and LTA induced NF-kappaB activation and stimulated production of IL-8 in HSCs. Pretreatment with TNF-alpha or IL-1beta augmented NF-kappaB activation and IL-8 production in response to PGN or LTA. Both PGN- and LTA-induced NF-kappaB activation and IL-8 secretion were completely inhibited by anti-TLR2 blocking antibody (T2.5). These findings suggest that TNF-alpha or IL-1beta primed HSCs enhance the production of IL-8 in response to PGN and LTA through augmentation of the TLR2 system.

Topics: Adenoviridae; Biomarkers; Cell Culture Techniques; Cell Line, Transformed; Cytokines; Fibrosis; Genes, Reporter; Hepatocytes; Humans; Inflammation; Interleukin-1; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Liver; Luciferases; NF-kappa B; Peptidoglycan; Teichoic Acids; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha; Up-Regulation

Human intrahepatic biliary epithelial cells (HIBECs) may play active roles in both the innate and adaptive immune responses. Little is known, however, about the role of toll-like receptors (TLRs) on HIBECs in inflammatory cholangiopathies.. The expression of TLR1-9 and the biological responses to their ligands, lipopolysaccharide (LPS) or lipoteichoic acid (LTA), were studied in cultured HIBECs by reverse transcription-polymerase chain reaction, immunoblotting, and enzyme-linked immunosorbent assay.. HIBECs constitutively expressed transcripts encoding TLR1-6 and 9, as well as myeloid differentiation factor 88 (MyD88), MD2, and CD14. Stimulation of HIBECs with LPS resulted in translocation of NF-kappaB subunits from the cytoplasmic to the nuclear fraction, followed by increased secretion of a variety of chemokines/cytokines, including interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), and IL-6. Treatment with BAY11-7082 efficiently inhibited the LPS-induced transcription and secretion of these chemokines/cytokines. In HIBECs, the mitogen-activated protein kinases (MAPKs) were also activated by LPS stimulation. These results indicated that LPS activates HIBECs via a TLR4-MyD88-dependent pathway. Stimulation of HIBECs with LTA induced the secretion of a similar profile of cytokines/chemokines via a TLR2-MyD88-dependent pathway.. In HIBECs, at least TLR2 and 4 are capable of mediating innate immune system function in vitro. This result, in conjunction with our recent finding that TLR4 expression is increased in biliary epithelial cells in primary biliary cirrhosis, suggests the involvement of TLRs in the development of chronic inflammatory cholangiopathies.

Topics: Bile Duct Diseases; Bile Ducts, Intrahepatic; Cells, Cultured; Chemokine CCL2; Epithelial Cells; Gene Expression Regulation; Humans; Immunity, Innate; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Nitriles; RNA, Messenger; Signal Transduction; Sulfones; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.

Topics: Acute-Phase Proteins; Carrier Proteins; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Lung; Macrophages; Membrane Glycoproteins; Models, Biological; Mutation; Staphylococcus aureus; Surface Plasmon Resonance; Teichoic Acids; Tumor Necrosis Factor-alpha

Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors (PRR) of mammals that participate in the activation of innate immune responses against microbial infections. Among these receptors, TLR2 is essential for the recognition of conserved structural components of bacteria, protozoa and fungi. Until now, expression of TLR2 in dogs has not been investigated. In this work we describe a partial sequence of the gene coding for canine TLR2 and show that TLR2 mRNA is constitutively expressed in canine blood PMNs. We also show that stimulation of purified PMNs with lipoteichoic acid (LTA), a ligand of TLR2, leads to the release of proinflammatory chemokine IL-8. Furthermore, TLR2 protein is easily detectable by flow cytometry on the canine peripheral blood granulocyte and monocyte cell surface, and slightly on lymphocytes. These findings suggest that, also in dogs as in humans the initial antibacterial response of PMNs could be elicited through engagement of TLR2.

Topics: Animals; Base Sequence; Dogs; Flow Cytometry; Interleukin-8; Lipopolysaccharides; Membrane Glycoproteins; Molecular Sequence Data; Neutrophil Activation; Phagocytes; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, DNA; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptors

We have developed a method for obtaining pneumococcal lipoteichoic acid (LTA) with none, one, or two acyl chains. Anion-exchange chromatography at pH 9.5 yields pneumococcal LTA (labeled LTA-9.5) that has a mass spectrum identical to that of pre-ion-exchange LTA and loses 500 mass units after deacylation by alkali hydrolysis. Anion exchange at pH 10.5 produces LTA (labeled LTA-10.5) with mass peaks that are 264 mass units lower than those of pre-ion-exchange LTA, and deacylation of LTA-10.5 by alkali hydrolysis reduces the mass by only 239 mass units. This result indicates that LTA-10.5 has lost one of the two acyl chains, whereas LTA-9.5 has both acyl chains. When the biological properties of LTA-9.5 and LTA-10.5 are examined with mouse cells, only LTA-9.5 (and not LTA-10.5) is able to stimulate mouse cells to produce tumor necrosis factor alpha, interleukin-1beta, and nitric oxide. In contrast, both LTA-9.5 and LTA-10.5 can stimulate human cells. LTA became inactive when both acyl chains were removed. Thus, acyl chains are critical for LTA function, and small variations in acyl chains can alter biological properties of LTA.

Topics: Acylation; Animals; Chromatography, Ion Exchange; Humans; Interleukin-1; Interleukin-8; Lipopolysaccharides; Macrophages; Mice; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptococcus pneumoniae; Teichoic Acids

Heparin is one of the most effective drugs for preventing and treating thromboembolic complications in surgical patients. Recent evidence suggests that heparin enhances the proinflammatory responses of human peripheral blood monocytes to Gram-negative endotoxin (LPS). We have identified LPS-binding protein (LBP) as a novel heparin-binding plasma protein. The affinity of LPB to heparin was KD = 55 +/- 8 nM, as measured by surface plasmon resonance. Using a fluorescence-based assay, we showed that clinically used heparin preparations significantly enhance the ability of LBP to catalytically disaggregate and transfer LPS to CD14, the LPS receptor. The presence of clinically relevant heparin concentrations in human whole blood increased LPS-induced production of the proinflammatory cytokine IL-8. Fondaparinux, which is identical with the antithrombin III-binding pentasaccharide in heparin, did not bind to LBP or alter LBP function. Thus, this novel anticoagulant drug is a potential candidate for safe administration to patients who have endotoxemia and require anticoagulation.

Topics: Acute-Phase Proteins; Adjuvants, Immunologic; Antithrombin III; Biological Transport; Boron Compounds; Carrier Proteins; Enoxaparin; Fluorescent Dyes; Heparin; Humans; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Membrane Glycoproteins; Monocytes; Signal Transduction; Solubility; Sulfates; Teichoic Acids; Thrombin

To investigate i) whether the Dialyzable Leukocyte Extract (DLE) modulates the production of proinflammatory cytokines in leukocytes activated by the bacterial cell wall components lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN); ii) the effect of DLE on LPS-stimulated endothelial cells; and iii) whether the regulatory effect of DLE on inflammatory mediators is related to the modulation of Toll-like receptors (TLRs), NF-kappaB and cAMP signaling pathways.. Leukocytes were stimulated with LPS, LTA, and PGN in the presence of DLE. Endothelial cells were stimulated with LPS and treated with DLE. The levels of Tumor Necrosis Factor-alpha(TNFalpha), Interleukin-6 (IL-6), and IL-8 in culture supernatants were evaluated by ELISA. The expression of Toll-like receptor 2 (TLR2) and 4 (TLR4), NF-kappaB activity and cAMP levels were evaluated by flow cytometry, EMSA, and EIA, respectively.. The addition of DLE to leukocytes stimulated with cell wall constituents suppressed the production of TNFalpha. However, DLE induced IL-8 release in monocytes and enhanced IL-6 and IL-8 production by activated monocytes and endothelial cells. Also, DLE induced TLR2 and TLR4 expression, and increased cAMP levels, whereas NF-kappaB activity was inhibited.. The present data indicate the differential regulation by DLE of the production of TNFalpha, IL-6, and IL-8 cytokines, associated with effects on TLR2 and TLR4 expression and NF-kappaB and cAMP activities. We suggest a putative mechanism for the biological effects of DLE in activated leukocytes and endothelial cells.

Topics: Cells, Cultured; Cyclic AMP; Endothelial Cells; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Leukocytes; Lipopolysaccharides; Membrane Glycoproteins; Monocytes; NF-kappa B; Peptidoglycan; Receptors, Cell Surface; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Transfer Factor; Tumor Necrosis Factor-alpha

In Wegener's granulomatosis (WG), a pathogenetic role has been proposed for circulating anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti-PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell-wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti-PR3 antibodies induced the release of minor quantities of the chemokine interleukin-8 (IL-8), preincubation with anti-PR3 antibodies, but not with isotype-matched control immunogloblin G (IgG), resulted in a markedly enhanced IL-8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti-PR3 and peaked after 6 h. The anti-PR3-related priming was also observed for tumor necrosis factor alpha (TNF-alpha) and IL-6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA-IgG derived from WG serum but not with normal IgG. The priming effect of the anti-PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF-alpha. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti-PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14-dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.

Topics: Antibodies, Antineutrophil Cytoplasmic; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; Humans; Immunoglobulin G; Interleukin-6; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Monocytes; Neutrophils; Teichoic Acids; Time Factors; Tumor Necrosis Factor-alpha

Recognition of conserved bacterial structures called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), may lead to induction of a variety of "early immediate genes" such as chemokines. In the current study, we have in an ex vivo whole blood model studied the induction of the chemokines MIP-1alpha, MCP-1 and IL-8 by various PAMPs. The rate of appearance of Escherichia coli-Lipopolysaccharide (LPS) induced chemokines differed. The production of MIP-1alpha and IL-8 was after 1 h of stimulation significantly higher when compared to unstimulated whole blood, whereas MCP-1 was not significantly elevated until after 3 h. At peak levels the MIP-1alpha concentration induced by E. coli-LPS was 3-5-fold higher than MCP-1 and IL-8. By specific cell depletion, we demonstrated that all three chemokines were mainly produced by monocytes. However, the mRNA results showed that IL-8 was induced in both monocytes and granulocytes. The production of all three chemokines, induced by the E. coli-LPS and Neisseria meningitidis-LPS, was significantly inhibited by antibodies against CD14 and TLR4, implying these receptors to be of importance for the effects of LPS in whole blood. The chemokine production induced by lipoteichoic acid (LTA) and non-mannose-capped lipoarabinomannan (AraLAM) was, however, less efficiently blocked by antibodies against CD14 and TLR2. E. coli-LPS and LTA induced a dose-dependent increase of CD14, TLR2 and TLR4 expression on monocytes in whole blood. These data show that PAMPs may induce chemokine production in whole blood and that antibodies against PRRs inhibit the production to different extent.

Topics: Antibodies, Monoclonal; Antigens, Bacterial; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokines; Gene Expression; Granulocytes; Heparin; Humans; Interleukin-8; Leukocyte Reduction Procedures; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophage Inflammatory Proteins; Monocytes; Polysaccharides, Bacterial; Receptors, Pattern Recognition; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4

While transfection of tlr2 conveyed responsiveness to lipoteichoic acid (LTA), the Arg753Gln polymorphic gene could not. LTA induced a stronger chemokine and anti-inflammatory response than lipopolysaccharides did. Blood from heterozygous polymorphic and wild-type donors reacted uniformly to LTA and Staphylococcus aureus. Thus, one functional allele for Toll-like receptor 2 suffices for full cytokine response.

Topics: Chemokines; Heterozygote; Humans; In Vitro Techniques; Inflammation Mediators; Interferon-gamma; Interleukin-10; Interleukin-8; Lipopolysaccharides; Membrane Glycoproteins; Mutation, Missense; Polymorphism, Genetic; Receptors, Cell Surface; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptors; Transfection; Tumor Necrosis Factor-alpha

Despite the ability to participate in immune responses and the continuous presence of bacteria and bacterial products, functional responses of intestinal epithelial cells (IEC) seem to be muted. Previously, tolerance to Toll-like receptors (TLRs) ligands has been described in monocytic cells. However, mechanisms in the intestine are unknown.. The effect of purified lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on expression and function of TLRs in intestinal epithelial cells (Colo205, SW480, T84) was assessed by Northern and Western blot and FACS analysis, kinase activity assays, immunohistochemistry, and ELISA.. Expression of TLRs except 10 was detected in primary IEC and TLR1-10 in the cultured cells. Short-term stimulation with LPS or LTA activated proinflammatory signaling cascades in IEC, including phosphorylation of IRAK and MAP kinases and increased IL-8 secretion, whereas prolonged incubation resulted in a state of hyporesponsiveness with no reactivation of the cells by a second challenge with either substance detected. The cells remained responsive to tumor necrosis factor (TNF). Hyporesponsive cells showed no alteration in expression of TLR or signaling molecules but revealed a decrease in TLR surface expression and IRAK activity. Toll-interacting protein (Tollip) mRNA and protein expression were increased in hyporesponsive cells, and overexpression of Tollip in IEC resulted in a significantly decreased proinflammatory response.. Continuous presence of specific bacterial components results in a status of hyporesponsiveness in otherwise reactive IEC. Down-regulation of TLR surface expression and up-regulation of inhibitory Tollip with decreased phosphorylation of IRAK might all contribute to this hyporesponsiveness.

Topics: Bacteria; Bacterial Adhesion; Carrier Proteins; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Epithelial Cells; Gene Expression; Humans; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Intestinal Mucosa; Intracellular Signaling Peptides and Proteins; Ligands; Lipopolysaccharides; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Protein Kinases; Receptors, Cell Surface; Signal Transduction; Teichoic Acids; Toll-Like Receptor 1; Toll-Like Receptors

The respiratory epithelium plays a major role in the primary defense of the airways against infection. It has been demonstrated that bacterial products are involved in the induction of inflammatory reactions of the upper airways. Little is known about the effects of bacterial products on expression of the antimicrobial peptide hCAP-18/LL-37, the only human cathelicidin identified so far. The aim of this study was to investigate the effects of bacterial products from both gram-positive and gram-negative bacteria on the expression of hCAP-18/LL-37 by sinus epithelial cells using an air-exposed tissue culture model. Lipopolysaccharide and lipoteichoic acid both increased hCAP-18/LL-37 expression in cultured sinus epithelium as assessed by immunohistochemistry, where maximal stimulation occurred at 100 ng ml(-1) lipopolysaccharide or 10 microg ml(-1) lipoteichoic acid. The stimulatory effect of lipopolysaccharide and lipoteichoic acid was not restricted to expression of hCAP-18/LL-37, since also mucin expression and IL-8 release from cultured sinus epithelium cells were increased by lipopolysaccharide and lipoteichoic acid. This suggests that bacterial products may stimulate innate immunity in the upper airways.

Topics: Antimicrobial Cationic Peptides; Cathelicidins; Cells, Cultured; Epithelial Cells; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Immunity, Innate; Interleukin-8; Lipopolysaccharides; Mucins; Paranasal Sinuses; Teichoic Acids

Several case reports have described complete hematological remissions for patients with otherwise untreated acute myelogenous leukemia (AML) who receive hematopoietic growth factor therapy during complicating bacterial infections. This may be caused by indirect cytokine effects, but direct effects of infecting agents on the malignant cells are also possible because bacterial molecules can bind to specific receptors expressed by normal and malignant leukocytes. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria, and it can activate normal immunocompetent cells through binding to specific cell membrane receptors.. We investigated effects of LTA derived from Enterococcus faecalis on in vitro cultured (i) normal peripheral blood mononuclear cells (PBMC); (ii) remaining T cells derived from patients with hematologic malignancies and chemotherapy-induced leukopenia; and (iii) native human AML cells.. Increased interleukin 1beta (IL1beta) and IL8 release by in vitro cultured normal PBMC was observed after stimulation with LTA at concentrations > or =5 microg/mL; these levels were lower than for lipopolysaccharide (LPS)-stimulated cells and LTA antagonized LPS-induced cytokine release by normal PBMC. In most cases LTA did not alter T-cell proliferation for patients with chemotherapy-induced leukopenia. The LTA effects on AML blasts were investigated for 62 consecutive patients. LTA altered either cytokine (granulocyte-macrophage colony-stimulating factor + stem cell factor + IL3)-dependent proliferation or the release of IL1beta/IL8 for 23 patients; the effects were divergent but increased proliferation/cytokine levels were most commonly observed.. The LTA derived from E. faecalis can modulate the functional characteristics of normal leukocytes and native human AML blasts.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Dose-Response Relationship, Drug; Enterococcus faecalis; Female; Granulocyte Colony-Stimulating Factor; Granulocyte Precursor Cells; Hematologic Neoplasms; Humans; Interleukin-1; Interleukin-8; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; Leukopenia; Lipopolysaccharides; Lymphocyte Activation; Male; Middle Aged; T-Lymphocytes; Teichoic Acids; Tumor Cells, Cultured

A comparison between humans and rabbits was performed based on stimulation of whole blood with well-known pyrogens from Gram-negative and Gram-positive bacteria, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. The reactivity was measured as release of IL-1 beta and IL-8 by ELISA. The reactivity of the two species towards LPS was found to be similar, whereas their reactivity towards LTA differed considerably. Differences between the levels of IL-1 beta and IL-8 release were observed in both species. This finding suggests that the In vitro Pyrogen Test (IPT) which uses human blood to detect contaminations, e.g. of injectable drugs, might predict the human reaction to the contamination better than the "gold standard" rabbit pyrogen test.

Topics: Animals; Blood; Enzyme-Linked Immunosorbent Assay; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Interleukin-1; Interleukin-8; Lipopolysaccharides; Pyrogens; Rabbits; Teichoic Acids

Intestinal myofibroblasts are known to respond to inflammatory signals and may play a role in Crohn's disease-associated fibrosis. However, putative involvement by myofibroblasts in innate immune responses as part of intestinal host defense has not been characterized. We therefore analyzed expression and regulation of toll-like receptors (TLRs) in colonic human myofibroblasts (CCD-18) and primary human colonic myofibroblasts in comparison with human lung myofibroblasts (CCD-37).. Expression of TLRs (1-10) and NOD 1 and 2 was assessed before and after stimulation with either lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by using a custom microarray, reverse-transcription polymerase chain reaction, Northern blot and Western blot analysis, and immunohistochemistry. Activation of signaling pathways, translocation of p65, and secretion of interleukin (IL)-8 were determined.. Messenger RNAs encoding for TLR1-9, as well as NOD1 and NOD2, were amplified from cultured and primary human intestinal myofibroblasts. After stimulation with LPS or LTA, a 1.5-4.2-fold up-regulation of TLRs (2, 3, 4, 6, 7) and elements of the signaling cascade (MyD88, TIR domain-containing adapter protein [TIRAP]) was observed. CCD-18 and CCD-37 cells expressed TLR 2 and 4 protein, which were located primarily on the cell membrane. Stimulation with LTA or LPS resulted in activation of the mitogen-activated protein kinases pathway, nuclear translocation of p65, and significantly increased IL-8 secretion.. Bacterial components directly activate intestinal myofibroblasts expressing TLRs. These cells may therefore participate in innate immune responses by sensing and responding to bacterial products that have penetrated into the subepithelial compartment.

Topics: Cells, Cultured; Colon; Fibroblasts; Humans; Immunity, Innate; Inflammatory Bowel Diseases; Interleukin-8; Intestinal Mucosa; Lipopolysaccharides; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Oligonucleotide Array Sequence Analysis; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teichoic Acids; Toll-Like Receptor 1; Toll-Like Receptor 2; Toll-Like Receptors

This study investigated the effect of various structural components of Gram-positive (lipotheichoic acid and protein A) and Gram-negative (porins and lipopolysaccharide) bacteria on human dermal fibroblasts. Fibroblasts are important effector cells which have a potential role in augmenting the inflammatory response in various diseases. In this study we present a profile of TNF-alpha, IL-6 and IL-8, the expression of intercellular adhesion molecules (ICAM-1) and the activation of transcriptional nuclear factor NF-kB and AP-1 in human dermal fibroblasts stimulated by bacterial surface components. Compared to the controls, increased ICAM-1, IL-6 and IL-8 gene expression after stimulation of LPS and porins at 2 and 4 h was more evident than that obtained following stimulation of LTA and PA. Gene expression was also associated with the production of cytokine proteins in culture supernatants. TNF-alpha gene expression remained undetectable. Moreover, LPS and porin treatments determined IkBalpha phosphorylation and degradation in human dermal fibroblasts and the subsequent activation of nuclear factors NF-kB and AP-1. These data suggest the importance of such stimuli in the first step of the inflammatory process, as well as the important role played by fibroblasts in skin inflammatory disease.

Topics: Adult; Autocrine Communication; Cell Adhesion; Cytokines; Female; Fibroblasts; Gene Expression Regulation, Bacterial; Humans; I-kappa B Proteins; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Lipopolysaccharides; Lymphocytes; Middle Aged; NF-kappa B; NF-KappaB Inhibitor alpha; Paracrine Communication; Phosphorylation; Porins; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Skin; Staphylococcal Protein A; Staphylococcus aureus; Teichoic Acids; Transcription Factor AP-1; Transcription, Genetic; Tumor Necrosis Factor-alpha

An earlier study reported that human gingival epithelial cells in primary culture and oral epithelial cell lines KB and HSC-2 cells were devoid of membrane CD14 (mCD14) and did not show enhanced production of interleukin (IL)-8 or granulocyte macrophage-colony stimulating factor (GM-CSF) upon stimulation with bacterial cell-surface components such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN) and synthetic muramyldipeptide (MDP) even in the presence of serum. The present study demonstrated that after treatment with interferon (IFN)-gamma for 3 days, these cells secreted IL-8 and GM-CSF in response to the bacterial components. Treatment with IFN-gamma enhanced Toll-like receptor (TLR) 2, TLR4, MD-2 and MyD88 mRNA expression as determined by reverse transcriptase PCR. Anti-TLR2 and anti-TLR4 monoclonal antibodies (MAbs) inhibited the IL-8 production induced by PGN and LTA as well as LPS, respectively, in IFN-gamma-primed oral epithelial cells, whereas neither MAb inhibited IL-8 production induced by MDP. These findings suggested that IFN-gamma primed oral epithelial cells to produce cytokines upon stimulation with various bacterial components by up-regulation of the TLR system.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adaptor Proteins, Signal Transducing; Adjuvants, Immunologic; Antigens, Differentiation; Antigens, Surface; Child; Cytokines; Drosophila Proteins; Epithelial Cells; Female; Gingiva; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-8; Lipopolysaccharides; Lymphocyte Antigen 96; Membrane Glycoproteins; Myeloid Differentiation Factor 88; Peptidoglycan; Receptors, Cell Surface; Receptors, Immunologic; Reverse Transcriptase Polymerase Chain Reaction; RNA; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors

Staphylococcus aureus strains lacking agr- and sarA-dependent gene products or specific MSCRAMM (microbial surface components recognizing adhesive matrix molecules) adhesins were compared for the ability to activate inflammatory responses in the lung. The mutants were evaluated for virulence in a mouse model of pneumonia and by quantifying their ability to stimulate interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in respiratory epithelial cells. In a neonatal mouse, only strains with intact agr and sarA loci were consistently associated with invasive, fatal pulmonary infection (P < 0.001) and sarA was specifically required to cause bacteremia (P < 0.001). The agr and/or sarA mutants were, nonetheless, fully capable of producing pneumonia and were as proficient as the wild-type strain in stimulating epithelial IL-8 expression, a polymorphonuclear leukocyte chemokine, in airway cells. In contrast, agr and especially sarA mutants induced less epithelial GM-CSF expression, and MSCRAMM mutants lacking fibronectin binding proteins or clumping factor A, a ligand for fibrinogen, were unable to stimulate epithelial GM-CSF production. The ability to induce IL-8 expression was independent of the adherence properties of intact bacteria, indicating that shed and/or secreted bacterial components activate epithelial responses. While conserved staphylococcal components such as peptidoglycan are sufficient to evoke inflammation and cause pneumonia, the agr and sarA loci of S. aureus are critical for the coordination of invasive infection of the lungs.

Topics: Animals; Bacterial Adhesion; Bacterial Proteins; Cell Line, Transformed; Coagulase; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-8; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mutation; Pneumonia, Staphylococcal; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Trans-Activators; Transcription Factors; Virulence

Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.

Topics: Chemokine CXCL5; Chemokines, CXC; Enterocolitis; Gram-Negative Bacteria; HT29 Cells; Humans; Interleukin-8; Intestinal Mucosa; Lactobacillus; Lactobacillus acidophilus; Lipopolysaccharide Receptors; Lipopolysaccharides; Teichoic Acids; Tumor Necrosis Factor-alpha

An analog of 1alpha,25-dihydroxyvitamin D3, 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components. Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects with Salmonella lipopolysaccharide (LPS) or lipoteichoic acid (LTA) from Staphylococcus aureus, both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti-CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed with MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed with LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic cells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Adaptor Proteins, Signal Transducing; Antigens, Differentiation; Cells, Cultured; Cytokines; Drosophila Proteins; Drug Synergism; Humans; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Membrane Glycoproteins; Monocytes; Myeloid Differentiation Factor 88; Receptors, Cell Surface; Receptors, Immunologic; RNA, Messenger; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors

The incidence of severe invasive disease caused by serogroup A streptococci (GAS) is increasing, and to elucidate the role of streptococcal cell wall components in the inflammatory response, human whole blood was stimulated with lipoteichoic acid (LTA, 0.005-50 microg/mL) and peptidoglycan (10 and 100 microg/ml) from Streptococcus pyogenes. Both stimulants increased dose dependently the leukocyte release of cytokines many thousand fold: tumor necrosis factor alpha (0 to 158,000+/-4,900 pg/mL), interleukin (IL)-1beta (85+/-56 to 31,000+/-4,600 pg/mL), IL-6 (30+/-11 to 34,800+/-15,000 pg/mL), and IL-8 (300+/-150 to 29,000+/-14,000 pg/mL). Intracellular leukocyte levels of reactive oxygen species (ROS) as measured by flow cytometry increased 15-20 fold, from 25 to 400-500 mean fluorescence intensity. Aminoethyl-isothiourea (AE-ITU), a relatively selective inhibitor of the inducible nitric oxide synthase (iNOS) and a ROS scavenger, reduced the cytokine production by 70-100%, and intracellular leukocyte ROS levels by 50-70% (all P < 0.05). The non-selective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) did not affect intracellular ROS levels, but it caused a moderate selective inhibition of IL-8 production. Leukocyte NO production (measured up to 36 h) was not enhanced by LTA, peptidoglycan, inactivated streptococci, or cytokine combinations. The mechanisms for the anti-inflammatory effects of AE-ITU may be through a reduction of intracellular ROS levels, or through a direct effect on signal transduction, whereas NO modulation is an unlikely mechanism.

Topics: Cell Survival; Cell Wall; Cytokines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; In Vitro Techniques; Inflammation; Interleukin-1; Interleukin-6; Interleukin-8; Leukocytes; Lipopolysaccharides; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Peptidoglycan; Reactive Oxygen Species; Streptococcus pyogenes; Teichoic Acids; Thiourea; Tumor Necrosis Factor-alpha

Serum concentrations of catecholamines are high in patients with sepsis or acute respiratory distress syndrome (ARDS). Because chemokines mediate the recruitment of neutrophils into inflammatory sites, we addressed the question of whether dopamine (DA) is able to influence chemokine production in endothelial cells under basal and proinflammatory conditions. To this end, lung microvascular endothelial cells (LMVEC) were stimulated or not for 24 h with the bacterial toxins lipopolysaccharide (LPS) (1 microg/ml) or lipoteichonic acid (LTA) (10 microg/ml) in the presence or absence of various concentrations of DA (1-100 microg/ml). Whereas under basal and stimulatory conditions, the addition of DA to endothelial cells dose-dependently increased IL-8 production, the production of ENA-78 and Gro-alpha was significantly inhibited (P < 0.01). This effect could still be demonstrated when the cells were stimulated for up to 3 h with LPS before DA administration. Similar findings were detected for the mRNA expression of these chemokines. The influence of DA on chemokine production was not receptor mediated and could be prevented by antioxidants or radical scavengers. Moreover, addition of H(2)O(2) to endothelial cells gave results similar to those observed with DA stimulation, suggesting a pivotal role for reactive oxygen species in DA-mediated modulation of chemokine production in endothelial cells. Our data thus demonstrate that DA administration results in the induction of oxidative stress, with profound effects on endothelial chemokine production.

Topics: Cardiotonic Agents; Cell Polarity; Chemokine CXCL1; Chemokine CXCL5; Chemokines; Chemokines, CXC; Chemotactic Factors; Dopamine; Endothelium, Vascular; Gene Expression; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lipopolysaccharides; Lung; Microcirculation; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Respiratory Distress Syndrome; Teichoic Acids

Despite the fact that gram-positive infections constitute around 50% of all cases leading to septic shock, little is yet known about the mechanisms involved. This study was carried out to find out more about the effects of cell wall components peptidoglycan (PepG) and lipoteichoic acid (LTA) of the gram-positive bacterium Streptococcus pyogenes in the pig. Specific pathogen-free pigs (20 kg bodyweight) were pretreated with metyrapone (a cortisol-synthesis inhibitor) and then were given 2-h infusions of 160 microg/kg of PepG (n = 5), 160 microg/kg LTA (n=5), or a combination of both (LTA + PepG, 160 microg/kg each, n = 5). Four hours after start of the infusions, the PepG, LTA, and LTA + PepG groups showed decreases in mean arterial pressure (change of -11%, -25%, and -47% from baseline, respectively), dynamic lung compliance (-18%, -24%, and -38%), arterial oxygen tension (-10%, -16%, and -37%), changes in blood leukocyte numbers (+11%, -27%, and -67%), and increases in pulmonary vascular resistance index (+7%, +106%, and +307% from baseline) and metabolic acidosis (base excess values decreased with 1.8, 2.3 and 8.1 units). The differences between the PepG and LTA + PepG groups were statistically significant (P < 0.05, Kruskal-Wallis tests), but not between LTA and LTA + PepG groups. However, no changes in systemic nitric oxide (NO) production could be detected, which is much in contrast to studies on lower order animals. Moreover, comparison of the results obtained using this model with those obtained in a model of endotoxin-induced septic shock showed distinct difference in the mechanisms by which gram-positive and gram-negative bacterial components exert their actions. For example, a marked fall in systemic blood pressure and dynamic lung compliance is seen in both models, but in the present gram-positive sepsis model, much less interleukin-8 and tumor necrosis factor-alpha are produced. In conclusion, this study showed that PepG and LTA act synergistically to cause respiratory failure and septic shock in the pig. The infusion of the combination of PepG and LTA in the pig could serve as a new, well-controlled model for studies of gram-positive sepsis.

Topics: Animals; Drug Synergism; Endothelin-1; Endotoxins; Female; Hydrocortisone; Interleukin-8; Leukocyte Count; Lipopolysaccharides; Lung Compliance; Male; Metyrapone; Nitric Oxide; Oxygen; Peptidoglycan; Pulmonary Circulation; Sepsis; Shock, Septic; Specific Pathogen-Free Organisms; Streptococcus pyogenes; Swine; Teichoic Acids; Tumor Necrosis Factor-alpha; Vascular Resistance

We examined leukocyte counts and ex vivo cytokine response of whole blood to lipopolysaccharide (LPS) or lipoteichoic acid (LTA) in patients under low-dose molgramostim therapy. Patients were injected subcutaneously daily for ten days with 1 microg/kg (n=9) or 2 microg/kg (n=14) molgramostim. Leukocytosis was observed in all patients, but only the eosinophil fraction was significantly increased in relation to other leukocyte populations. Ex vivo IFN-gamma release was decreased and IL-10 and IL-1ra secretion were increased in response to LPS or LTA. Thus, in non-neutropenic patients, leukocytosis can already be initiated by low doses of molgramostim. The ex vivo cytokine data suggest that these doses prime blood towards a systemic anti-inflammatory response.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Basal Cell; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Head and Neck Neoplasms; Humans; Interferon-gamma; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Interleukin-6; Interleukin-8; Leukocytes; Lipopolysaccharides; Male; Middle Aged; Recombinant Proteins; Sialoglycoproteins; Teichoic Acids; Time Factors

CD14 has been implicated as a receptor of lipoteichoic acid (LTA) and other bacterial components as well as lipopolysaccharide (LPS). Since the structures of LTAs from various gram-positive bacteria are heterogeneous, we analyzed the effects of LTAs on the secretion of interleukin-8 (IL-8) by high- and low-CD14-expressing (CD14(high) and CD14(low)) human gingival fibroblasts (HGF). While Bacillus subtilis LTA had an IL-8-inducing effect on CD14(high) HGF which was considerably weaker than that of LPS, Streptococcus sanguis and Streptococcus mutans LTAs had practically no effect on the cells. B. subtilis LTA had only a weak effect on CD14(low) HGF, as did LPS. S. sanguis and S. mutans LTAs at a 1,000-fold excess each completely inhibited the IL-8-inducing activities of both LPS and a synthetic lipid A on CD14(high) HGF. The effect of LPS was also inhibited by the presence of an LPS antagonist, synthetic lipid A precursor IVA (LA-14-PP), with a 100-fold higher potency than S. sanguis and S. mutans LTAs and by anti-CD14 monoclonal antibody (MAb). S. sanguis and S. mutans LTAs, LA-14-PP, and anti-CD14 MAb had no significant effect on phorbol myristate acetate-stimulated IL-8 secretion by HGF. These LTAs also inhibited the IL-8-inducing activity of B. subtilis LTA on CD14(high) HGF, as did LA-14-PP and anti-CD14 MAb. The antagonistic and agonistic functions of LTAs were also observed with human monocytes. Binding of fluorolabeled LPS to human monocytes was inhibited by S. sanguis LTA, although the inhibition was 100 times weaker than that of LPS itself, and anti-CD14 MAb inhibited fluorolabeled LPS and S. sanguis LTA binding. Binding of LTAs to CD14 was also observed with nondenaturing polyacrylamide gel electrophoresis. These results indicate that LTAs act as antagonists or agonists via a CD14-dependent mechanism, probably due to the heterogeneous structure of LTAs, and that an antagonistic LTA might be a useful agent for suppressing the periodontal disease caused by gram-negative bacteria.

Topics: Antibodies, Monoclonal; Bacillus subtilis; Fibroblasts; Gingiva; Humans; Interleukin-1; Interleukin-8; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Monocytes; Streptococcus mutans; Streptococcus sanguis; Teichoic Acids

Cell adhesion molecule expression (CAM) and IL-8 release in lung microvascular endothelium facilitate neutrophil accumulation in the lung. This study investigated the effects of lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, alone and with LPS or TNF-alpha, on CAM expression and IL-8 release in human lung microvascular endothelial cells (HLMVEC). The concentration-dependent effects of Staphylococcus aureus (S. aureus) LTA (0.3-30 microg/ml) on ICAM-1 and E-selectin expression and IL-8 release were bell shaped. Streptococcus pyogenes (S. pyogenes) LTA had no effect on CAM expression, but caused a concentration-dependent increase in IL-8 release. S. aureus and S. pyogenes LTA (30 microg/ml) abolished LPS-induced CAM expression, and S. aureus LTA reduced LPS-induced IL-8 release. In contrast, the effects of S. aureus LTA with TNF-alpha on CAM expression and IL-8 release were additive. Inhibitory effects of LTA were not due to decreased HLMVEC viability, as assessed by ethidium homodimer-1 uptake. Changes in neutrophil adhesion to HLMVEC paralleled changes in CAM expression. Using RT-PCR to assess mRNA levels, S. aureus LTA (3 microg/ml) caused a protein synthesis-dependent reduction (75%) in LPS-induced IL-8 mRNA and decreased the IL-8 mRNA half-life from >6 h with LPS to approximately 2 h. These results suggest that mechanisms exist to prevent excessive endothelial cell activation in the presence of high concentrations of bacterial products. However, inhibition of HLMVEC CAM expression and IL-8 release ultimately may contribute to decreased neutrophil accumulation, persistence of bacteria in the lung, and increased severity of infection.

Topics: Cell Adhesion Molecules; Drug Interactions; E-Selectin; Endothelium, Vascular; Half-Life; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Lipopolysaccharides; Lung; Lung Diseases; Microcirculation; Neutrophil Infiltration; RNA Stability; RNA, Messenger; Sepsis; Streptococcus; Streptococcus pyogenes; Teichoic Acids; Tumor Necrosis Factor-alpha

To examine the changes in neutrophil deformability, aggregation, and adherence in response to stimulation with proinflammatory cytokines and bacterial toxins.. Prospective, randomized trial.. Research laboratory.. Neutrophils isolated from healthy volunteers.. Neutrophils were exposed to tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-8, their combination, endotoxin (LPS), lipoteichoic acid (LTA), and staphyloccocal enterotoxin B (SEB). Neutrophil deformability was measured as percent neutrophils filtered through 5-microm diameter filters. Aggregation was measured using a platelet aggregometer. Adherence was determined by examining the binding of neutrophils to albumin-coated latex beads.. Exposure to TNF-alpha and IL-1beta led to significant decreases in neutrophil filterability, which was attenuated by cytochalasin D pretreatment. LPS and LTA also decreased deformability, suggesting that these toxins directly stimulated neutrophils independent of cytokines. IL-8 and SEB did not significantly affect neutrophil deformability. TNF-alpha and LPS were associated with significant neutrophil aggregation, which was inhibited by pretreatment with anti-CD18 antibodies. Neutrophil aggregation was not affected by IL-1beta, LTA, or SEB. TNF-alpha, IL-8, and LPS increased neutrophil adherence, which also was attenuated by pretreatment with anti-CD18 antibodies. IL-1beta, LTA, and SEB did not significantly affect neutrophil adherence.. Cytokines and bacterial toxins differ in their effects on neutrophil deformability, aggregation, and adherence. Of the cytokines examined, TNF-alpha appears to have the greatest direct effects on neutrophil rheology. Similarly, endotoxin appears to have greater direct effects on neutrophil rheology than the Gram-positive bacterial toxins, LTA, and staphylococcal enterotoxins.

Topics: Cell Adhesion; Cell Aggregation; Enterotoxins; Humans; Interleukin-1; Interleukin-8; Lipopolysaccharides; Neutrophils; Prospective Studies; Rheology; Salmonella; Staphylococcus; Teichoic Acids; Tumor Necrosis Factor-alpha

Our purpose was to determine whether cultured human decidual cells produce chemokines in response to different strains of group B streptococci and purified bacterial cell wall components.. Human decidual cells were cultured from term placentas by standard techniques. Different strains of group B streptococci were isolated from neonates with early-onset group B streptococci sepsis. Confluent cell monolayers were incubated with these different strains of group B streptococci and various concentrations of purified bacterial cell wall components (including lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A) for 16 hours at 37 degrees C. Culture supernatants were collected and assayed for macrophage inflammatory protein-1 alpha and interleukin-8. Statistical analysis was by analysis of variance.. We found that cultured human decidual cells produced significant amounts of the two chemokines macrophage inflammatory protein-1 alpha and interleukin-8 in a strain-specific fashion to the various different strains of group B streptococci tested, from 215% to 421% over baseline production (p < 0.05 by analysis of variance). Also, we found that incubation of decidual cells with various concentrations of lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A resulted in significant concentration-dependent increases in decidual cell macrophage inflammatory protein-1 alpha and interleukin-8 production (p < 0.05.). Decidual cells produced significant amounts of the chemokines macrophage inflammatory protein-1 alpha and interleukin-8 in response to intact group B streptococci in a strain-specific fashion and in response to various concentrations of different bacterial cell wall components. Because chemokines are important mediators signaling migration of different immune effector cells into areas of inflammation, we suggest that decidual cell chemokine production in response to bacteria and bacterial cell wall components may be a key early event in the pathogenesis of infection-associated preterm labor.

Topics: Analysis of Variance; Cell Wall; Cells, Cultured; Chemokine CCL4; Cytokines; Decidua; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Lipid A; Lipopolysaccharides; Macrophage Inflammatory Proteins; N-Acetylneuraminic Acid; Pregnancy; Streptococcus agalactiae; Teichoic Acids

Topics: Adult; Cytokines; Humans; In Vitro Techniques; Interleukin-1; Interleukin-6; Interleukin-8; Lipopolysaccharides; Polysaccharides, Bacterial; Streptococcal Infections; Streptococcus agalactiae; Teichoic Acids; Tumor Necrosis Factor-alpha

Invasion by gram-positive and gram-negative bacterial organisms is characterized immunopathologically by the activation of mononuclear phagocytic cells, leading to the elaboration of macrophage-derived regulatory and chemotactic factors, and the resultant influx of inflammatory leukocytes. Little is known regarding the mechanisms by which gram-positive organisms initiate macrophage activation and subsequent inflammation. In this investigation, we postulated that lipoteichoic acid (LTA) purified from two different gram-positive bacterial species was an important signal for the expression of chemotactic cytokines from human peripheral blood monocytes (PBM). In initial experiments, we demonstrated that cell-associated interleukin-8 (IL-8) was expressed by mononuclear phagocytes present in inflamed areas of endocardium in cases of acute Staphylococcus aureus endocarditis. We next demonstrated that LTA purified from either Staphylococcus aureus or Streptococcus pyogenes induced the time- and dose-dependent expression of IL-8 mRNA and protein from human PBM. The expression of IL-8 mRNA from LTA- but not lipopolysaccharide (LPS)-treated PBM was superinduced by concomitant treatment with cycloheximide, indicating that the expression of IL-8 mRNA from LTA-treated PBM was negatively controlled by repressor proteins. Furthermore, mRNA stability studies indicated that IL-8 mRNA was less stable in the presence of LTA than in the presence of LPS. Our findings indicate that LTA can induce the secretion of the polymorphonuclear leukocyte chemotactic factor IL-8 and that LTA may be an important cellular mediator of inflammatory cell recruitment that characterizes immune responses to gram-positive bacterial infections.

Topics: Cycloheximide; Endocarditis, Bacterial; Gene Expression; Humans; In Vitro Techniques; Interleukin-8; Lipopolysaccharides; Monocytes; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Streptococcus pyogenes; Teichoic Acids