Lead at 2.5 and 5.0 µM induced aberrant MH-II surface expression through increased MII exocytosis and increased autophagosome formation in Raw 267.4 cells.

Aberrant major histocompatibility complex class II (MHC-II) surface expression on antigen presenting cells (APCs) is associated with dysregulated immune homeostasis. Lead (Pb) is known to increase MHC-II surface expression on murine peritoneal macrophages ex vivo at concentrations exceeding 25 µM. Little data exist examining this effect at physiologically relevant concentrations. To address this deficit, we examined the effects of Pb on MHC-II surface expression, secondary T-cell activation markers (CD80, CD86, CD40), cell viability, cellular metabolic activity, and ß-hexosaminidase activity in RAW 267.4 macrophage cell lines, with changes in cell ultrastructure evaluated by electron and confocal microscopy. Pb induced an increase in MHC-II, CD86, and lysosome-associated LAMP-1 and LAMP-2 surface mean expression during one doubling cycle (17 h), which was mirrored by increased ß-hexosaminidase activity. Although cell viability was unaffected, cellular metabolism was inhibited. Electron microscopy revealed evidence of lipid vacuolization, macroautophagy and myelin figure formation in cells cultured with either Pb or LPS. Confocal microscopy with antibodies against LC3B showed a punctate pattern consistent with the presence of mature autophagosomes. Collectively, these data suggest that 2.5-5.0 µM Pb increased MHC-II surface expression by inhibiting metabolic activity, inducing autophagy, and increasing MHC-II trafficking in a macrophage cell line.

5-Lipoxygenase expression in benign and malignant canine prostate tissues.

5-Lipoxygenase (5-LO) is overexpressed in human prostate carcinomas (PCs), and its inhibition decreases proliferation and induces apoptosis in prostate cancer cell lines. We hypothesized that 5-LO would be overexpressed in canine PC compared with benign prostate tissue and may be important in the pathogenesis of the disease. Immunoblot analysis of canine PC and benign prostatic hyperplasia (BPH) tissues demonstrated 5-LO expression in both. 5-LO immunohistochemical staining was not significantly different within the stromal or epithelial components of canine primary PC, BPH or suppurative prostatitis, suggesting that differential expression of this enzyme does not occur in these conditions. The percentage of tumour cells expressing 5-LO was significantly lower in metastatic PC lesions compared with primary PC (P < 0.0001). This decreased expression may indicate down-regulation or altered expression of the enzyme with progression of canine PC to a metastatic phenotype.

Black walnut extract-induced laminitis in horses is associated with heterogeneous dysfunction of the laminar microvasculature.

REASONS FOR PERFORMING STUDY: Equine laminitis purportedly involves haemodynamic dysfunction at the level of the laminar vasculature. However, to date, no studies have been performed characterising the function of laminar arteries and veins during the prodromal stages of equine laminitis. HYPOTHESIS: That the prodromal stages of laminitis are associated with contractile dysfunction of the equine laminar vasculature. OBJECTIVE: To assess contractile function of laminar arteries and veins to phenylephrine (PE) and 5-hydroxytryptamine (5-HT). METHODS: Horses were administered black walnut heartwood extract (BWHE) or water (control horses) via nasogastric intubation. After euthanasia, laminar vessels (100-800 microm internal diameter) were isolated and mounted on small vessel myographs to assess contractile function. RESULTS: Contractile responses to PE or 5-HT were identical in laminar arteries isolated from either control horses or those administered BWHE. In contrast, responses to PE or 5-HT were significantly reduced in laminar veins isolated from BWHE-administered horses when compared with laminar veins isolated from control horses. CONCLUSIONS AND POTENTIAL RELEVANCE: These results are consistent with the prodromal stages of laminitis being associated with selective dysfunction of laminar veins. Further studies are required to discern the precise nature of this dysfunction and its potential relevance to the pathogenesis of acute laminitis in the horse and possible therapeutic targets for treatment.

Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression.

Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.

The role of reactive oxygen and nitrogen species in airway epithelial gene expression.

The body first encounters deleterious inhaled substances, such as allergens, industrial particles, pollutants, and infectious agents, at the airway epithelium. When this occurs, the epithelium and its resident inflammatory cells respond defensively by increasing production of cytokines, mucus, and reactive oxygen and nitrogen species (ROS/RNS). As inflammation in the airway increases, additional infiltrating cells increase the level of these products. Recent interest has focused on ROS/RNS as potential modulators of the expression of inflammation-associated genes important to the pathogenesis of various respiratory diseases. ROS/RNS appear to play a variety of roles that lead to changes in expression of genes such as interleukin-6 and intercellular adhesion molecule 1. By controlling this regulation, the reactive species can serve as exogenous stimuli, as intercellular signaling molecules, and as modulators of the redox state in epithelial cells. Unraveling the molecular mechanisms affected by ROS/RNS acting in these capacities should aid in the understanding of how stimulated defense mechanisms within the airway can lead to disease.

The role of reactive oxygen and nitrogen species in the response of airway epithelium to particulates.

Epidemiologic and occupational studies indicate adverse health effects due to inhalation of particulate air pollutants, but precise biologic mechanisms responsible have yet to be fully established. The tracheobronchial epithelium forms the body's first physiologic barrier to such airborne pollutants, where ciliary movement functions to remove the offending substances caught in the overlying mucus layer. Resident and infiltrating phagocytic cells also function in this removal process. In this paper, we examine the role of reactive oxygen and nitrogen species (ROS/RNS) in the response of airway epithelium to particulates. Some particulates themselves can generate ROS, as can the epithelial cells, in response to appropriate stimulation. In addition, resident macrophages in the airways and the alveolar spaces can release ROS/RNS after phagocytosis of inhaled particles. These macrophages also release large amounts of tumor necrosis factor alpha (TNF-alpha), a cytokine that can generate responses within the airway epithelium dependent upon intracellular generation of ROS/RNS. As a result, signal transduction pathways are set in motion that may contribute to inflammation and other pathobiology in the airway. Such effects include increased expression of intercellular adhesion molecule 1, interleukin-6, cytosolic and inducible nitric oxide synthase, manganese superoxide dismutase, cytosolic phospholipase A2, and hypersecretion of mucus. Ultimately, ROS/RNS may play a role in the global response of the airway epithelium to particulate pollutants via activation of kinases and transcription factors common to many response genes. Thus, defense mechanisms involved in responding to offending particulates may result in a complex cascade of events that can contribute to airway pathology.

Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators.

Deleterious environmental stimuli cause the airway epithelium to respond with increased secretions of mucus, reaction of oxygen/nitrogen species, changes in ciliary beating, and the influx of inflammatory cells. The epithelium is a target for factors released by infiltrating inflammatory cells, and has recently been shown to serve as an effector of such inflammation. Molecular mechanisms regulating production of secondary inflammatory mediators (cytokines, lipid mediators, and reactive oxygen/nitrogen species) have yet to be fully described. This report reviews the production of secondary mediators by epithelial cells and by airway epithelium. Lipid mediators are enzymatically produced by the airway epithelium in response to primary mediators. Molecular mechanisms regulating the production of cyclo-oxygenase, lipoxygenase and prostaglandin synthase are discussed, along with the potential of lipid mediators to produce inflammation. The molecular regulation of nitric oxide production is also described in the context of its role as a signalling molecule in pathways regulating secretion of mucus, ciliary motion, and intercellular adhesion molecule-1 (ICAM-1) expression. The production of cytokines by the airway epithelium is shown to play a role in causing inflammation associated with respiratory diseases. Particular attention is paid to molecular mechanisms governing the expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8).

Tumor necrosis factor alpha (TNF alpha)-induced ICAM-1 surface expression in airway epithelial cells in vitro: possible signal transduction mechanisms.

Within the past several years research on the interaction of cytokines and adhesion molecules with airway epithelium in diseases has allowed us to develop a better understanding of the disease process. The cytokine, TNF alpha and the adhesion molecule ICAM-1 are important mediators in the pathogenesis of airway diseases such as asthma, chronic bronchitis, and adult respiratory distress syndrome. Effects of TNF alpha on ICAM-1 surface expression was investigated in both primary cultures of normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cell line BEAS-2B. TNF alpha (0.015-150 ng/mL) significantly enhanced ICAM-1 surface expression (measured by flow cytometry) in a dose and time-dependent manner, with peak expression seen at 24 hours. This response was negated by heat inactivation of the TNF alpha prior to incubation. TNF alpha-induced ICAM-1 expression also was inhibited by pre- and coincubation of TNF alpha with 3 micrograms/mL soluble TNF-R1 or by the PKC inhibitor, Calphostin C (0.1 and 0.5 microM). The ROI scavengers, dimethylthiourea (4 mM), and dimethyl sulfoxide (0.001%), enhanced TNF alpha-induced ICAM-1 expression. Collectively, these results indicate that TNF alpha-induced ICAM-1 surface expression is a specific receptor-mediated response (TNF-R1), which is mediated by mechanisms dependent on PKC and intracellular reactive oxygen species.