Superoxide dismutase attenuates hyperoxia-induced interleukin-8 induction via AP-1.

Exposure of lung epithelial cells to hyperoxia results in the generation of excess reactive oxygen species (ROS), cell damage, and production of proinflammatory cytokines (interleukin-8; IL-8). Although activation of the NF-kappaB and c-Jun N-terminal kinase (JNK)/activator protein (AP)-1 transcription pathways occurs in hyperoxia, it is unclear whether activation of the AP-1 pathway has a direct impact on IL-8 production and whether overexpression of superoxide dismutase (SOD) can mitigate these proinflammatory processes. A549 cells were exposed to 95% O(2), and ROS production, AP-1 activation, and IL-8 levels were determined. Experimental groups included cells transduced with a recombinant adenovirus encoding CuZnSOD or MnSOD (two- to threefold increased activity) or transfected with a JNK1 small interfering RNA (RNAi). Hyperoxia resulted in significant increases in ROS generation, AP-1 activation, and IL-8 production, which were significantly attenuated by overexpression of either MnSOD or CuZnSOD. JNK1 RNAi also moderated IL-8 induction. The data indicate that activation of JNK1/AP-1 and subsequent IL-8 induction in hyperoxia are mediated by intracellular ROS, with SOD having significant protective effects.

Antioxidants improve antibacterial function in hyperoxia-exposed macrophages.

Hyperoxia and pulmonary infections are well known to increase the risk of acute and chronic lung injury in newborn infants, but it is not clear whether hyperoxia directly increases the risk of pneumonia. The purpose of this study was to examine: (1) the effects of hyperoxia and antioxidant enzymes on inflammation and bacterial clearance in mononuclear cells and (2) developmental differences between adult and neonatal mononuclear cells in response to hyperoxia. Mouse macrophages were exposed to either room air or 95% O2 for 24 h and then incubated with Pseudomonas aeruginosa. After 1 h, bacterial adherence, phagocytosis, and macrophage inflammatory protein (MIP)-1alpha production were analyzed. Bacterial adherence increased 5.8-fold (p < 0.0001), phagocytosis decreased 60% (p < 0.05), and MIP-1alpha production increased 49% (p < 0.05) in response to hyperoxia. Overexpression of MnSOD or catalase significantly decreased bacterial adherence by 30.5%, but only MnSOD significantly improved bacterial phagocytosis and attenuated MIP-1alpha production. When monocytes from newborns and adults were exposed to hyperoxia, phagocytosis was impaired in both groups. However, adult monocytes were significantly more impaired than neonatal monocytes. Data indicate that hyperoxia significantly increases bacterial adherence while impairing function of mononuclear cells, with adult cells being more impaired than neonatal cells. MnSOD reduces bacterial adherence and inflammation and improves bacterial phagocytosis in mononuclear cells in response to hyperoxia, which should minimize the development of oxidant-induced lung injury as well as reducing nosocomial infections.

Factors influencing T-lymphopoiesis after allogeneic hematopoietic cell transplantation.

In adult recipients of allogeneic hematopoietic cell transplants (HCT) studied at 1 year after grafting, there was a significant correlation between the counts of T cell receptor excision circle (TREC)-containing CD4 T cells (presumed recent thymic emigrants) and the counts of total T cells (r=0.65, P<0.001). Thus, the reconstitution of CD4 T cell pool depends on T cell generation from hematopoietic stem cells (T-lymphopoiesis). We evaluated factors that could affect T-lymphopoiesis. Low TREC-containing CD4 T cell counts were associated with older patient age (r=-0.41, P=0.01) but not with donor age, graft type (marrow vs. blood stem cells), CD34 cell dose, conditioning (with vs. without irradiation), acute graft-versus-host disease (aGVHD), or chronic graft-versus-host disease (cGVHD) in multivariate analysis. We conclude that patient age is the primary determinant of CD4 T-lymphopoiesis after allogeneic HCT.

IL-10 modulates placental responses to TLR ligands.

PROBLEM: 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. METHODS OF STUDY: 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. RESULTS: 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. CONCLUSIONS: Our results demonstrate that placental exposure to LTA and LPS appear to trigger distinct cytokine responses that can be modulated by IL-10.

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells.

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.

Perfluorochemical liquids enhance delivery of superoxide dismutase to the lungs of juvenile rabbits.

Previous studies suggest acute lung injury (ALI) in premature newborns is associated with relative deficiency of antioxidant enzymes that may be ameliorated by recombinant human superoxide dismutase (rhSOD). Perfluorochemicals (PFCs) are distributed homogeneously and support gas exchange in diseased lungs. We investigated whether PFCs could provide an effective delivery system for rhSOD. Juvenile rabbits were lung-lavaged, treated with surfactant, and randomized: group I: fluorescently labeled rhSOD (5 mg/kg in 2 mL/kg saline); group II: fluorescently labeled rhSOD (5 mg/kg in 18 mL/kg PFC). Animals were ventilated with oxygen for 4 h; the lungs were harvested for analysis of SOD distribution and oxidative injury. Cardiopulmonary indices remained stable and similar between groups. Qualitative assessment (QA) showed a more homogeneous lung SOD distribution in group II and a better histologic profile. QA of lung SOD distribution showed significant increase in SOD concentrations in group II (7.37 +/- 1.54 microg/mg protein) compared with group I (1.65 +/- 0.23 microg/mg protein). Oxidative injury as assessed by normalized protein carbonyl was 149.1 +/- 26.8% SEM in group II compared with 200.5 +/- 7.3% SEM in group I. Plasma SOD was significantly higher in group II. Administration of rhSOD with or without PFCs does not compromise cardiovascular function or impede lung recovery after ALI. PFCs enhance rhSOD delivery to the lungs by 400% while decreasing lung oxidative damage by 25% compared with rhSOD alone. These data suggest that PFCs optimize lung rhSOD delivery and might enhance the beneficial effects of rhSOD in preventing acute and chronic lung injury.

Superoxide dismutase moderates basal and induced bacterial adherence and interleukin-8 expression in airway epithelial cells.

Bacterial infection of the tracheobronchial tree is a frequent, serious complication in patients receiving treatment with oxygen and mechanical ventilation, resulting in increased morbidity and mortality. Using human airway epithelial cell culture models, we examined the effect of hyperoxia on bacterial adherence and the expression of interleukin-8 (IL-8), an important mediator involved in the inflammatory process. A 24-h exposure to 95% O(2) increased Pseudomonas aeruginosa (PA) adherence 57% in A549 cells (P < 0.01) and 115% in 16HBE cells (P < 0.01) but had little effect on Staphylococcus aureus (SA) adherence. Exposure to hyperoxia, followed by a 1-h incubation with SA, further enhanced PA adherence (P < 0.01), suggesting that hyperoxia and SA colonization may enhance the susceptibility of lung epithelial cells to gram-negative infections. IL-8 expression was also increased in cells exposed to both hyperoxia and PA. Stable or transient overexpression of manganese superoxide dismutase reduced both basal and stimulated levels of PA adherence and IL-8 levels in response to exposure to either hyperoxia or PA. These data indicate that hyperoxia increases susceptibility to infection and that the pathways are mediated by reactive oxygen species. Therapeutic intervention strategies designed to prevent accumulation of intracellular reactive oxygen species may reduce opportunistic pulmonary infections.

Interleukin-7 improves CD4 T-cell reconstitution after autologous CD34 cell transplantation in monkeys.

In mice, interleukin-7 (IL-7) hastens T-cell reconstitution and might cause autoimmune diseases, lymphoma, and osteoporosis. We assessed the effect of IL-7 on T-cell reconstitution and toxicity in baboons that underwent total body irradiation followed by autologous transplantation of marrow CD34 cells. Three baboons received placebo and 3 baboons received recombinant human IL-7 (rhIL-7, 75 microg/kg twice a day subcutaneously) between 6 and 10 weeks after transplantation. The mean increase in blood absolute CD4 T-cell counts was 0.9-fold in the placebo-treated animals versus 9.0-fold in those treated with IL-7 (P =.02). The increase observed in the IL-7-treated animals appeared attributable to peripheral expansion rather than de novo generation. The IL-7-treated animals had greater mean increases in the volumes of the spleen (2.0-fold with placebo versus 4.5-fold with IL-7, P =.02) and lymph nodes (1.8-fold with placebo versus 4.1-fold with IL-7, P =.10) but not the thymus (3.4-fold with placebo versus 1.1-fold with IL-7, P =.18). Side effects of IL-7 included thrombocytopenia and possibly neutropenia and hemolytic anemia. One IL-7-treated animal failed to thrive due to a disease resembling graft-versus-host disease. No animals developed lymphoma. Bone density was not decreased. In conclusion, IL-7 raises CD4 T-cell counts in irradiated primates. It remains to be determined whether this is associated with clinical benefit.

Development of human protein reference database as an initial platform for approaching systems biology in humans.

Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.