Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide

Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-alpha and IFN-gamma and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the responses. As expected, ODN 2216 was a potent inducer of IFN-alpha secretion by both bovine and ovine PBMC, but ODN 2007 also induced dose-dependent, CpG-specific IFN-alpha secretion by ovine PBMC. In contrast, a significant dose-dependent, CpG-specific IFN-gamma secretion response was only observed following ODN 2216 stimulation of bovine PBMC. Furthermore, both ODN 2216 and ODN 2007 induced CpG-specific non-MHC-restricted cytotoxicity with ovine but not bovine PBMC. Finally, there was not a single assay in which PBMC from all sheep or cattle responded at a detectable level. A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.     

Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus

The human Werner syndrome protein, WRN, is a member of the RecQ helicase family and contains 3′→5′ helicase and 3′→5′ exonuclease activities. Recently, we showed that the exonuclease activity of WRN is greatly stimulated by the human Ku heterodimer protein. We have now mapped this interaction physically and functionally. The Ku70 subunit specifically interacts with the N-terminus (amino acids 1–368) of WRN, while the Ku80 subunit interacts with its C-terminus (amino acids 940– 1432). Binding between Ku70 and the N-terminus of WRN (amino acids 1–368) is sufficient for stimulation of WRN exonuclease activity. A mutant Ku heterodimer of full-length Ku80 and truncated Ku70 (amino acids 430–542) interacts with C-WRN but not with N-WRN and cannot stimulate WRN exonuclease activity. This emphasizes the functional significance of the interaction between the N-terminus of WRN and Ku70. The interaction between Ku80 and the C-terminus of WRN may modulate some other, as yet unknown, function. The strong interaction between Ku and WRN suggests that these two proteins function together in one or more pathways of DNA metabolism.     

Effect of inhaled crystalline silica in a rat model: time course of pulmonary reactions

Numerous investigations have been conducted to elucidate mechanisms involved in the initiation and progression of silicosis. However, most of these studies involved bolus exposure of rats to silica, i.e. intratracheal instillation or a short duration inhalation exposure to a high dose of silica. Therefore, the question of pulmonary overload has been an issue in these studies. The objective of the current investigation was to monitor the time course of pulmonary reactions of rats exposed by inhalation to a non-overload level of crystalline silica. To accomplish this, rats were exposed to 15 mg/m3 silica, 6 h/day, 5 days/week for up to 116 days of exposure. At various times (5-116 days exposure), animals were sacrificed and silica lung burden, lung damage, inflammation, NF-KB activation, reactive oxygen species and nitric oxide production, cytokine production, alveolar type II epithelial cell activity, and fibrosis were monitored. Activation of NF-KB/DNA binding in BAL cells was evident after 5 days of silica inhalation and increased linearly with continued exposure. Parameters of pulmonary damage, inflammation and alveolar type II epithelial cell activity rapidly increased to a significantly elevated but stable new level through the first 41 days of exposure and increased at a steep rate thereafter. Pulmonary fibrosis was measurable only after this explosive rise in lung damage and inflammation, as was the steep increase in TNF-alpha and IL-1 production from BAL cells and the dramatic rise in lavageable alveolar macrophages. Indicators of oxidant stress and pulmonary production of nitric oxide exhibited a time course which was similar to that for lung damage and inflammation with the steep rise correlating with initiation of pulmonary fibrosis. Staining for iNOS and nitrotyrosine was localized in granulomatous regions of the lung and bronchial associated lymphoid tissue. Therefore, these data demonstrate that the generation of oxidants and nitric oxide, in particular, is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and fibrosis. This suggests an important role for nitric oxide in the initiation of silicosis.     

Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, beta-arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling. We find that the truncation of the mGluR1a C-terminal tail prevents mGluR1a phosphorylation and that GRK2 does not contribute to the phosphorylation of an mGluR1 splice variant (mGluR1b). However, mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation is attenuated following GRK2 expression. The expression of the GRK2 C-terminal domain to block membrane translocation of endogenous GRK2 increases mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation, presumably by blocking membrane translocation of GRK2. In contrast, expression of the kinase-deficient GRK2-K220R mutant inhibits inositol phosphate formation by these unphosphorylated receptors. Expression of the GRK2 N-terminal domain (residues 45-185) also attenuates both constitutive and agonist-stimulated mGluR1a, mGluR1a-866Delta, and mGluR1b signaling, and the GRK2 N terminus co-precipitates with mGluR1a. Taken together, our observations indicate that attenuation of mGluR1 signaling by GRK2 is phosphorylation-independent and that the interaction of the N-terminal domain of GRK2 with mGluR1 contributes to the regulation of mGluR1 G protein coupling.     

Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases

The bacterium, Vitreoscilla, can induce the synthesis of a homodimeric hemoglobin under hypoxic conditions. Expression of VHb in heterologous bacteria often enhances growth and increases yields of recombinant proteins and production of antibiotics, especially under oxygen-limiting conditions. There is evidence that VHb interacts with bacterial respiratory membranes and cytochrome bo proteoliposomes. We have examined whether there are binding sites for VHb on the cytochrome, using the yeast two-hybrid system with VHb as the bait and testing every Vitreoscilla cytochrome bo subunit as well as the soluble domains of subunits I and II. A significant interaction was observed only between VHb and intact subunit I. We further examined whether there are binding sites for VHb on cytochrome bo from Escherichia coli and Pseudomonas aeruginosa, two organisms in which stimulatory effects of VHb have been observed. Again, in both cases a significant interaction was observed only between VHb and subunit I. Because subunit I contains the binuclear center where oxygen is reduced to water, these data support the function proposed for VHb of providing oxygen directly to the terminal oxidase; it may also explain its positive effects in Vitreoscilla as well as in heterologous organisms.     

Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1

The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.     

Enhanced nitric oxide and reactive oxygen species production and damage after inhalation of silica

In previous reports from this study, measurements of pulmonary inflammation, bronchoalveolar lavage cell cytokine production and nuclear factor-kappa B activation, cytotoxic damage, and fibrosis were detailed. In this study, we investigated the temporal relationship between silica inhalation, nitric oxide (NO), and reactive oxygen species (ROS) production, and damage mediated by these radicals in the rat. Rats were exposed to a silica aerosol (15 mg/m(3) silica, 6 h/day, 5 days/wk) for 116 days. We report time-dependent changes in 1) activation of alveolar macrophages and concomitant production of NO and ROS, 2) immunohistochemical localization of inducible NO synthase and the NO-induced damage product nitrotyrosine, 3) bronchoalveolar lavage fluid NO(x) and superoxide dismutase concentrations, and 4) lung lipid peroxidation levels. The major observations made in this study are as follows: 1) NO and ROS production and resultant damage increased during silica exposure, and 2) the sites of inducible NO synthase activation and NO-mediated damage are associated anatomically with pathological lesions in the lungs.     

Iron regulates xanthine oxidase activity in the lung

The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreductase activity in cultured V79 cells was increased with exposure to ferric ammonium sulfate and inhibited by deferoxamine. Lung XO and total xanthine oxidoreductase activities were reduced in rats fed an iron-depleted diet and increased in rats supplemented with iron, without change in the ratio of XO to total oxidoreductase. Intratracheal injection of an iron salt or silica-iron, but not aluminum salts or silica-zinc, significantly increased rat lung XO and total xanthine oxidoreductase activities, immunoreactive xanthine oxidoreductase, and the concentration of urate in bronchoalveolar fluid. These results suggest the possibility that the production of uric acid, a major chelator of iron in extracellular fluid, is directly influenced by iron-mediated regulation of the expression and/or activity of its enzymatic source, xanthine oxidase.