Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFa production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the I?Ba. Low basal levels of I?Ba explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-?B. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.

Attenuation of depression of muscle protein synthesis induced by lipopolysaccharide, tumor necrosis factor, and angiotensin II by beta-hydroxy-beta-methylbutyrate

beta-Hydroxy-beta-methylbutyrate (HMB; 50 microM) has been shown to attenuate the depression in protein synthesis in murine myotubes in response to lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) with or without interferon-gamma (IFN-gamma), and angiotensin II (ANG II). The mechanism for the depression of protein synthesis by all three agents was the same and was attributed to activation of double-stranded RNA-dependent protein kinase (PKR) with the subsequent phosphorylation of eukaryotic initiation factor 2 (eIF2) on the alpha-subunit as well as increased phosphorylation of the elongation factor (eEF2). Myotubes expressing a catalytically inactive PKR variant, PKRDelta6, showed no depression of protein synthesis in response to either LPS or TNF-alpha, confirming the importance of PKR in this process. There was no effect of any of the agents on phosphorylation of mammalian target of rapamycin (mTOR) or initiation factor 4E-binding protein (4E-BP1), and thus no change in the amount of eIF4E bound to 4E-BP1 or the concentration of the active eIF4E.eIF4G complex. HMB attenuated phosphorylation of eEF2, possibly by increasing phosphorylation of mTOR, and also attenuated phosphorylation of eIF2alpha by preventing activation of PKR. These results suggest that HMB may be effective in attenuating muscle atrophy in a range of catabolic conditions.

Pro-inflammatory cytokines IL6, TNF-α, IL1β, procalcitonin, lipopolysaccharide binding protein and C-reactive protein in infective endocarditis

Objective: To evaluate the serum levels and diagnostic value of cytokines and acute phase proteins in patients with infective endocarditis (IE). Patients and methods: Serum samples from 63 patients diagnosed with IE and 71 control patients were analysed for the following markers: interleukin-6 (IL6), tumour necrosis factor-α (TNF-α), interleukin 1-β (IL1β), procalcitonin (PCT), lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP). Results: Serum levels of IL6, IL1β and CRP were significantly elevated in patients with IE as compared to controls. PCT, TNF-α and LBP were not elevated. Conclusion: Serum CRP and IL6 are elevated in IE. IL 6 may aid in establishing the diagnosis. There was no correlation between IL 6 levels and CRP, causative microorganism, echocardiographic features or outcome. © 2007 The British Infection Society.

Paracellular permeability is increased by basal lipopolysaccharide in a primary culture of colonic epithelial cells; an effect prevented by an activator of Toll-like receptor-2

Lipopolysaccharide (LPS), which generally activates Toll-like receptor 4 (TLR4), is expressed on commensal colonic bacteria. In a number of tissues, LPS can act directly on epithelial cells to increase paracellular permeability. Such an effect in the colon would have an important impact on the understanding of normal homeostasis and of pathology. Our aim was to use a novel primary culture of colonic epithelial cells grown on Transwells to investigate whether LPS, or Pam(3)CSK( 4), an activator of TLR2, affected paracellular permeability. Consequently, [(14)C]-mannitol transfer and transepithelial electrical resistance (TEER) were measured. The preparation consisted primarily of cytokeratin-18 positive epithelial cells that produced superoxide, stained for mucus with periodic acid-Schiff reagent, exhibited alkaline phosphatase activity and expressed TLR2 and TLR4. Tight junctions and desmosomes were visible by transmission electron microscopy. Basally, but not apically, applied LPS from Escherichia coli increased the permeability to mannitol and to a 10-kDa dextran, and reduced TEER. The LPS from Helicobacter pylori increased paracellular permeability of gastric cells when applied either apically or basally, in contrast to colon cells, where this LPS was active only from the basal aspect. A pan-caspase inhibitor prevented the increase in caspase activity caused by basal E. coli LPS, and reduced the effects of LPS on paracellular permeability. Synthetic Pam(3)CSK(4) in the basal compartment prevented all effects of basal E. coli LPS. In conclusion, LPS applied to the base of the colonic epithelial cells increased paracellular permeability by a mechanism involving caspase activation, suggesting a process by which perturbation of the gut barrier could be exacerbated. Moreover, activation of TLR2 ameliorated such effects.

Apoptosis induction in gastric mucous cells in vitro:lesser potency of Helicobacter pylori than Escherichia coli lipopolysaccharide, but positive interaction with ibuprofen

Non-steroidal anti-inflammatory drugs (NSAIDs) cause peptic ulcer disease, but whether they interact with Helicobacter pylori to promote damage is controversial. Moreover, the reported induction of apoptosis in gastric cells by H. pylori lipopolysaccharide (LPS) (10-9 g /ml) contrasts with studies showing low immunological potency of this LPS. Therefore, the effects of LPS from H. pylori NCTC 11637 and Escherichia coli 0111:B4 on apoptosis in a primary culture of guinea-pig gastric mucous cells were investigated in the presence and absence of the NSAID, ibuprofen. Cell loss was estimated by a crystal violet assay, and apoptosis determined from caspase activity and from condensation and fragmentation of nuclei. Exposure to E. coli LPS for 24 h caused cell loss and enhanced apoptotic activity at concentrations ≥ 10-9 g/ml, but similar effects were only obtained with H. pylori LPS at concentrations ≥10-6 g/ml. Although ibuprofen (250 μM) caused cell loss and apoptosis, addition of either E. coli or H. pylori LPSs further enhanced these effects. In conclusion, LPS and ibuprofen interact to enhance gastric cell loss and apoptosis. In such interactions, E. coli LPS is more potent than that of H. pylori. The low potency of H. pylori LPS may contribute to a chronic low-grade gastritis that can be enhanced by the use of NSAIDs. © W. S. Maney & Son Ltd.

Gravity spun polycaprolactone fibers for applications in vascular tissue engineering:proliferation and function of human vascular endothelial cells

Poly(ε-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under low-shear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 × 104 cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering. © Mary Ann Liebert, Inc.

Proteomic analysis of the anti-inflammatory action of minocycline

Minocycline possesses anti-inflammatory properties independently of its antibiotic activity although the underlying molecular mechanisms are unclear. Lipopolysaccharide (LPS)-induced cytokines and pro-inflammatory protein expression are reduced by minocycline in cultured macrophages. Here, we tested a range of clinically important tetracycline compounds (oxytetracycline, doxycycline, minocycline and tigecycline) and showed that they all inhibited LPS-induced nitric oxide production. We made the novel finding that tigecycline inhibited LPS-induced nitric oxide production to a greater extent than the other tetracycline compounds tested. To identify potential targets for minocycline, we assessed alterations in the macrophage proteome induced by LPS in the presence or absence of a minocycline pre-treatment using 2-DE and nanoLC-MS. We found a number of proteins, mainly involved in cellular metabolism (ATP synthase ß-subunit and aldose reductase) or stress response (heat shock proteins), which were altered in expression in response to LPS, some of which were restored, at least in part, by minocycline. This is the first study to document proteomic changes induced by minocycline. The observation that minocycline inhibits some, but not all, of the LPS-induced proteomic changes shows that minocycline specifically affects some signalling pathways and does not completely inhibit macrophage activation.

The innate immune system and the clearance of apoptotic cells

Apoptosis, programmed cell death, is used by multicellular organisms to remove cells that are in excess, damaged or diseased. Activation of the apoptosis programme generates "eat me" signals on the surface of the apoptotic cell that mediate recognition and clearance by the innate immune system. CD14, a pattern recognition receptor expressed on macrophages, is widely known for its ability to recognise the pathogen-associated molecular pattern lipopolysaccharide (LPS) and promote inflammation. However, CD14 has also been shown to mediate binding and removal of apoptotic cells in a process that is anti-inflammatory suggesting CD14 is capable of producing two distinct, ligand-dependent macrophage responses. Whilst the molecular basis for this dichotomy has yet to be defined it is clear that CD14 defines a point of interest on the macrophage surface where we may study ligand-specific responses of macrophages. Our work seeks to define the molecular mechanisms underlying the involvement of CD14 in the non-inflammatory clearance of apoptotic cells. Here we used three different differentiation strategies to generate macrophages from the monocytic cell line THP-1. The resultant macrophage models were characterised to assess the expression and function of CD14 within each model system. Whilst each macrophage model shows increased levels of surface CD14 expression, our results demonstrate significant differences in the various models’ abilities to respond to LPS and clear apoptotic cells in a CD14-dependent manner. TLR4 levels correlated positively with LPS responsiveness but not CD14-dependent apoptotic cell clearance or anti-inflammatory responses to apoptotic cells. These observations suggest CD14-dependent apoptotic cell clearance is not dependent on TLR4. Taken together our data support the notion that the CD14 ligand-dependent responses to LPS and apoptotic cells derive from changes at the macrophage surface. The nature and composition of the CD14-co-receptor complex for LPS and apoptotic cell binding and responses is the subject of further study.

CD14 and innate immune clearance of apoptotic cells

Rapid elimination of cells undergoing programmed cell death (apoptosis) is vital to maintain tissue homeostasis. The phagocytic removal of apoptotic cells (AC) is mediated by innate immune molecules, professional phagocytes and amateur phagocytes that recognise "eat me" signals on the surface of the AC. CD14, a pattern recognition receptor expressed on macrophages, is widely known for its ability to recognise the pathogen-associated molecular pattern lipopolysaccharide (LPS) and promote inflammation. CD14 also mediates the binding and removal of AC, a process that is considered to be anti-inflammatory therefore suggesting CD14 is capable of producing two distinct ligand-dependent responses. Our work seeks to define the molecular mechanisms underlying the involvement of CD14 in the non-inflammatory clearance of AC. Here we describe three different differentiation strategies used to generate macrophages from the monocytic cell line THP-1. Whilst CD14 expression was increased in each macrophage model we demonstrate significant differences in the various macrophage models' abilities to respond to LPS and clear AC. We show that CD14 expression correlates with CD14-dependent AC clearance and anti-inflammatory responses to AC. However LPS responsiveness correlates, as expected, with TLR4 but not CD14 expression. These observations suggest CD14-dependent AC clearance is not dependent on TLR4. Taken together our data support the notion that CD14 ligand-dependent responses to LPS and AC are derived from changes at the macrophage surface. The nature and composition of the CD14-co-receptor complex for LPS and AC binding and consequent responses is the subject of further study.

Antibody detection of Burkholderia pseudommallei and Burkholderia mallei

Monoclonal and polyclonaI antibodies have been produced for use in immunological assays for the detection of Burkholderia pseudomallei and Burkholderia mallei. Monoclonal antibodies recognising a high molecular weight polysaccharide material found in some strains of both species have been shown to be effective in recognising B. pseudomallei and B. mallei and distinguishing them from other organisms. The high molecular weight polysaccharide material is thought to be the capsule of B. pseudomallei and B. mallei and may have important links with virulence. B. pseudomallei and B. mallei are known to be closely related, sharing many epitopes, but antigenic variation has been demonstrated within both the species. The lipopolysaccharide from strains of B. pseudomal/ei and B. mallei has been isolated and the silver stain profiles found to be visually very similar. A monoclonal antibody raised to B. mallei LPS has been found to recognise both B. mallei and B. pseudomallei strains. However, in a small number of B. pseudomallei strains a visually atypical LPS profile has been demonstrated. A monoclonal ant ibody rai sed against this atypical LPS showed no recognition of the typical LPS profile of either B. mallei or B. pseudomallei. This atypical LPS structure has not been reported and may be immunologically distinct from the typical LPS. Molecular biology and antibody engineering techniques have been used in an attempt to produce single-chain antibody fragments reactive to B. pseudomallei. Sequencing of one of the single-chain antibody fragments produced showed high homology with murine immunoglobulin genes, but none of the single-chain antibody fragments were found to be specific to B. pselldomallei.

A study of potential serum markers for a diagnosis of gram-positive and gram-negative bacterial sepsis

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

Liposomes as carriers for polymyxins in the treatment of cystic fibrosis lung infections

Cystic fibrosis (CF) is the most common autosomal recessive disorder affecting Caucasian populations. The pathophysiology of this disorder predisposes the lungs of affected patients to chronic infection, typically by Pseudomonas aeruginosa, which is the main cause of morbidity and mortality. Recently, attention has focused on aerosolised polymyxins, which are given prophylactically in an effort to limit infection and subsequent lung damage. This class of antimicrobial compounds is highly active against P. aeruginosa and possess the advantage that resistance rarely develops. However, the rapid lung clearance of antibiotics is a well documented phenomenon and it was postulated that polymyxin treatment could be further improved by liposomal encapsulation. As part of the development of liposomal polymyxin B, analytical methodology (radiolabelling, HPLC and protein assay) applicable to liposomal formulations was established. Liposomes were prepared by the dehydration-rehydration method and encapsulation efficiencies were determined for a number of phospholipid compositions. Vesicles were characterised with respect to size, zeta potential, morphology and release characteristics. The surface hydrophobicity of vesicles was quantified by hydrophobic interaction chromatography and it was found that this method produced comparable results to techniques conventionally used to assess this property. In vivo testing of liposomal polymyxins demonstrated that encapsulation successfully prevented the rapid pulmonary clearance of PXB. Antimicrobial activity of liposomal formulations was quantified and found to be dependent on both the vesicle surface characteristics and their release profile. Investigation of the interaction of PXB with lipopolysaccharide was undertaken and results demonstrated that PXB caused significant structural distortion of the lipid A region. This may be sufficient to abrogate the potentiating action of LPS in the inflammatory cascade.

Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.