Effects of ambient air particles on nitric oxide production in macrophage cell lines

We assessed the in vitro toxicity of various particles on three murine macrophage cell lines, (J774A.1, WR19M.1, RAW264.7). The cells were exposed to aqueous suspensions (0-100 microg/30 mm2 well) of urban particulate matter (SRM-1648, SRM-1649, EHC-93), fine particulate matter (PM2.5), titanium dioxide (SRM-154b), and respirable cristobalite (SRM-1879) for 2 h and were then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and recombinant interferon-gamma (IFN, 100 U/ml). After overnight incubation with the particles and LPS/IFN, nitric oxide production was estimated from culture supernatant nitrite. Cell viability was determined by monitoring the rate of AlamarBlue reduction. The dose-effect relationships for nitrite and viability were modeled as a power function (Fold change = [Dose+1]beta), where beta represents the slope of the dose-response curve. Potency was defined as the rate of change in nitrite production corrected for cell viability (beta(POTENCY) = beta(NITRITE) - beta(VIABILITY)). Overall, the urban particles decreased nitric oxide production (beta(POTENCY) < 0), while exposure of the cells to fine particulate matter or cristobalite increased the production of nitric oxide (beta(POTENCY) > 0). Titanium dioxide (TiO2) was essentially inactive (beta(POTENCY) approximately to 0). The decrease in nitric oxide production seen in cells exposed to the urban particles was directly correlated to a decrease in the expression of inducible nitric oxide (iNOS) as determined by Western blot analysis. The results indicate that particles are modulators of nitric oxide production in murine macrophages and may directly disrupt expression of iNOS during concomitant pathogen exposure. Pathways leading to enhanced NO production causing cell injury, and to decreased NO release resulting in lower bacterial clearance, may both be relevant to the health effects of ambient particles.     

A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model.     

Acetylcholinesterase activation of peritoneal macrophages is independent of catalytic activity

Summary 1. In diverse tissues, acetylcholinesterase appears to play a critical role in the functional state of cells completely dependent of cholinergic transmission. However, very little is known about the mechanisms and actual molecular structures mediating the fundamental interactions between this protein and the cellular membrane.2. In this study, peritoneal macrophages were used as a model system to study the possible interaction between acetylcholinesterase, acting in a non-cholinergic capacity, and the cellular membrane.3. When acetylcholinesterase was incubated with macrophages harvested from rat peritoneum, the rate of oxygen consumption was increased in a concentration-dependent manner that was independent of mitochondrial block with sodium cyanide. Furthermore, heat inactivation of enzymatic activity or application of BW 284C51 at a concentration which totally blocks catalytic activity did not eliminate the effect.4. In contrast, incubation with bovine serum albumin or butyrylcholinesterase actually retarded oxygen consumption.5. The effect of acetylcholinesterase depended on the presence of divalent cations and was inhibited by mannan andd-mannose, but notd-galactose. It is concluded that acetylcholinesterase can induce a respiratory burst in macrophages independent of its conventional catalytic site but involving either the mannose receptor of the monocyte-derived macrophage or a possible sugar binding site on acetylcholinesterase itself.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA_359−379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra- and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 × 10⁻³ μg/ml and a limit of quantification of 24.5 × 10⁻³ μg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution (r = 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase (P < 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.     

Monocyte/macrophage proteomics: recent findings and biomedical applications

Macrophages, originating from the migration and differentiation of circulating monocytes into virtually all tissues, are extremely flexible and plastic cells that play vital homeostatic roles, but also contribute to the pathophysiology of many human diseases. For these reasons, they are intensively studied by different approaches, recently including proteomics. Macrophage cells can be taken from a range of different sources, including blood monocytes and macrophages from tissues. Macrophages can also be generated by in vitro culture from blood monocytes, and cell lines derived from this lineage can be used. Similarly, many different proteomic techniques can be used, ranging from classic approaches based on 2D gel electrophoresis to more recent high-throughput gel-free techniques essentially based on mass spectrometry. Here, we review the application of such techniques to the study of monocytes/macrophages, and summarize some results potentially relevant to two paradigmatic conditions – atherosclerosis and disorders of iron metabolism.     

Macrophage surface glycoproteins binding to galectin-3 (Mac-2-antigen)

Galectin-3 (formerly called Mac-2 antigen) is a ∼30 kDa carbohydrate-binding protein expressed on the surface of inflammatory macrophages and several macrophage cell lines. We have purified from lysates of the murine macrophage cell line WEHI-3 glycoproteins that bind to a galectin-3 affinity column. Several of these receptors are labelled after biotinylation of intact cells showing their location at the cell surface. N-terminal aminoacid sequencing of intact galectin-3-binding glycoproteins isolated from preparative SDS-gels or of chemically derived fragments showed several homologies with known proteins and identification was confirmed by immunoprecipitation with specific antibodies. The glycoproteins were shown to be: the α-subunit(CD11b) of the CD11b/CD18 integrin(Mac-1 antigen); the lysosomal membrane glycoproteins LAMPs 1 and 2 which are known in part to be expressed at cell surfaces; the Mac-3 antigen, a mouse macrophage differentiation antigen defined by the M3/84 monoclonal antibody and related immunochemically to LAMP-2; the heavy chain of CD98, a 125 kDa heterodimeric glycoprotein identified by the 4F2/RL388 monoclonal antibodies respectively on human and mouse monocytes/macrophages and on activated T cells. Further studies showed that CD11b/CD18, CD98 and Mac-3 are major surface receptors for galectin-3 on murine peritoneal macrophages elicited by thioglycollate. Abbreviations: PBS, phosphate buffered saline; CNBR, cyanogen bromide; PMSF, phenyl methyl sulphonyl fluoride This revised version was published online in November 2006 with corrections to the Cover Date.     

Metabolic Induction of Trained Immunity through the Mevalonate Pathway

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