A prominent feature of various inflamed and diseased tissue is the presence of low oxygen tension (hypoxia). Effector cells of the innate immune system must maintain their viability and physiologic functions in a hypoxic microenvironment. Monocytes circulating in the bloodstream differentiate into macrophages. During this process, cells acquire the ability to exert effects at hypoxic sites of inflammation. The transcription factor hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability. In this study, we demonstrated that lipopolysaccharide (LPS) induces HIF-1 activation by enhancing both HIF-1alpha protein expression through a translation-dependent pathway and HIF-1alpha transcriptional activity in THP-1 human myeloid cells that have undergone macrophage differentiation but not in undifferentiated monocytic THP-1 cells. LPS-induced HIF-1 activation was blocked by treatment with antioxidant (N-acetylcysteine or thioredoxin-1), NADPH oxidase inhibitor (diphenyleneiodonium), indicating that reactive oxygen species generated in response to LPS are essential in this process. LPS-mediated activation of HIF-1 was independent of NF-kappaB activity. LPS-induced ROS generation and HIF-1 activation required the expression of Toll-like receptor 4 or myeloid differentiation factor (MyD) 88, thus providing a molecular basis for the selective activation of HIF-1 in differentiated THP-1 cells.

Monocytes/macrophages of the myeloid lineage are the main cellular effectors of innate immunity. Hypoxia-inducible factor 1 (HIF-1) is essential for myeloid cell activation in response to inflammatory stimuli. However, it has not been established whether HIF-1 activity is induced during differentiation from monocyte to macrophage. We demonstrate that macrophage differentiation of THP-1 cells or monocytes from peripheral blood induces increased expression of both HIF-1alpha and HIF-1beta as well as increased HIF-1 transcriptional activity leading to increased expression of HIF-1 target genes. The increased HIF-1 activity in differentiated THP-1 cells resulted from the combined effect of increased HIF-1alpha mRNA levels and increased HIF-1alpha protein synthesis. Differentiation-induced HIF-1alpha protein and mRNA and HIF-1-dependent gene expression was blocked by treating cells with an inhibitor of the protein kinase C or MAP kinase signaling pathway. THP-1 cell differentiation was also associated with increased phosphorylation of the translational regulatory proteins p70 S6 kinase, S6 ribosomal protein, eukaryotic initiation factor 4E, and 4E binding protein 1, thus providing a possible mechanism for the modulation of HIF-1alpha protein synthesis. RNA interference studies demonstrated that HIF-1alpha is dispensable for macrophage differentiation but is required for functional maturation.

Since nanoparticles easily agglomerate to form larger particles, it is important to maintain the size of their agglomerates at the nano-level to evaluate the harmful effect of the nanoparticles. We prevented agglomeration of nickel oxide nanoparticles by ultrasound diffusion and filtration, established an acute exposure model using animals, and examined inflammation and chemokine expression. The mass median diameter of nickel oxide nanoparticle agglomerates suspended in distilled water for intratracheal instillation was 26 nm (8.41 nm weighted average surface primary diameter). Male Wistar rats received intratracheal instillation of nickel oxide nanoparticles at 0.1 mg (0.33 mg/kg) or 0.2 mg (0.66 mg/kg), and were dissected 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The control group received intratracheal instillation of distilled water. Three chemokines (cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2alphabeta, and CINC-3) in the lung tissue and bronchoalveolar lavage fluid (BALF) were determined by quantitative measurement of protein by ELISA. Both CINC-1 and CINC-2alphabeta concentration was elevated from day 3 to 3 months in lung tissue and from day 3 to 6 months in BALF. On the other hand, CINC-3 was elevated on day 3 in both lung tissue and BALF, and then decreased. The total cell and neutrophil counts in BALF were increased from day 3 to 3 months. In lung tissue, infiltration of mainly neutrophils and alveolar macrophages was observed from day 3 to 6 months in alveoli. These results suggest that CINC was involved in lung injury by nickel oxide nanoparticles.

BackgroundMacrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner.Methods/Principal FindingsThe effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly.Conclusion/SignificanceMIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF's potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1α protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.

Focusing on the "size" impact of particles, the objective of this study was to analyze morphological and qualitative changes over time in the development of inflammation and collagen deposition in lung tissue after intratracheal instillation of two sizes of nickel oxide in rats, in comparison with the results of instillation of crystalline silica and titanium dioxide. The fine-sized nickel oxide sample (nNiOm: median diameter of agglomerated particles 0.8 microm) was prepared from crude particles of nickel oxide (median diameter of primary particle 27 nm) by liquid-phase separation. Another samples of micrometer-sized nickel oxide (NiO: median diameter of particles 4.8 microm), crystalline silica (Min-U-SIL-5; geometric mean diameter 1.6 microm, geometric standard deviation [GSD] 2.0), and TiO(2) (geometric mean diameter 1.5 microm, GSD 1.8) were also used. Well-sonicated samples of 2 mg per 0.4 ml saline or saline alone (control) were intratracheally instilled into Wistar rats (males, 10 wk old). Bronchoalveolar lavage fluid (BAL)F and lung tissue were examined at 3 days, 1 wk, 1 mo, 3 mo, and 6 mo after instillation, from 5 rats of each group. Histopathological findings showed that the infiltration of macrophages or polymorphonuclear cells and the alveolitis in rats treated with nNiOm were remarkable over time and similar to the effects of crystalline silica. The numbers of total cells in BALF and the percentage of plymorphonuclear leukocytes (PMNs) also increased in the nNiOm group and silica group. The point counting method (PCM) showed a significant increase of inflammatory area, with the peak at 3 mo after instillation in the nNiOm group. In contrast, NiO treatment showed only a slight inflammatory change. Collagen deposition in two regions in the lung tissue (alveolar duct and pleura) showed an increasing collagen deposition rate in nNiOm at 6 mo. Our results suggest that submicrometer nano-nickel oxide is associated with greater toxicity, as for crystalline silica, than micrometer-sized nickel oxide. Biological effects of factors of particle size reduction, when dealing with finer particles such as nanoparticles, were reconfirmed to be important in the evaluation of respirable particle toxicity.

The local anesthetic lidocaine induces cell death by altering reactive oxygen species (ROS) generation and mitochondrial electron transport chain function. Because hypoxia-inducible factor 1 (HIF-1) is involved in determining oxygen metabolism and mitochondria function, we investigated the involvement of HIF-1 activity in lidocaine-induced cell death. We investigated the role of HIF activation on lidocaine-induced caspase activation and cell death in renal cell-derived RCC4 cells lacking functional von Hippel-Lindau (VHL) protein. We demonstrate that HIF-1 suppressed oxygen consumption and facilitated glycolysis in a pyruvate dehydrogenase kinase-1-dependent manner and that activation of HIF-1 conferred resistance to lidocaine-induced cell death. We also demonstrated that exogenous HIF-1 activation, through HIFα-hydroxylase inhibition or exposure to hypoxic conditions, alleviates lidocaine toxicity by suppressing mitochondria function and generating ROS, not only in RCC4 cells, but also in the neuronal SH-SY5Y cells. In conclusion, we demonstrate that HIF-1 activation due to VHL deletion, treatment with small molecule HIFα-hydroxylase inhibitors, and exposure to hypoxic conditions suppresses mitochondrial respiratory chain function and confers resistance to lidocaine toxicity.Local anesthetics, including lidocaine, affect the intra-and extra-cellular signaling pathways of both neuronal and non-neuronal cells, resulting in long-term modulation of biological functions such as cell growth and death 1 . Although the primary target of lidocaine is voltage-gated sodium channels, the targets and mechanisms in the context of cell growth and death are unknown. Studies indicate that mitochondria are one of the critical targets of lidocaine [2][3][4] . Similarly, we previously reported that reactive oxygen species (ROS) derived from mitochondria play an essential role in lidocaine-induced apoptosis and treatment with the antioxidants N-acetyl cysteine (NAC) and Trolox effectively prevents apoptosis 4 . The mitochondrial oxidative phosphorylation (OXPHOS) system plays a critical role in modulating ATP supply in the human body. Deficits derived from genetic and pharmacologic treatments cause functional disorders and cell death. The OXPHOS machinery consists of four enzyme complexes in the respiratory chain that transport electrons obtained from the oxidation of carbohydrates and fats to molecular oxygen (O 2 ); a fifth enzyme complex uses the energy derived from this process to drive ATP synthesis. O 2 is the electron acceptor, resulting in the production of H 2 O in a process that is catalyzed by complex IV of the mitochondrial electron transport chain (ETC). Because the process is not completely efficient, electron transport to O 2 may occur in complexes I or III, resulting in generation of ROS that can oxidize cellular proteins, lipids, and nucleic acids [5][6][7] . The generated ROS causes cell dysfunction or death when cells are exposed to drugs affecting electron transport in the ETC.

The objective of this study was to examine what kinds of cytokines are related to lung disorder by well-dispersed nanoparticles. The mass median diameter of nickel oxide in distilled water was 26 nm. Rats intratracheally received 0.2 mg of nickel oxide suspended in distilled water, and were sacrificed from three days to six months. The concentrations of 21 cytokines including inflammation, fibrosis and allergy-related ones were measured in the lung. Infiltration of alveolar macrophages was observed persistently in the nickel oxide-exposed group. Expression of macrophage inflammatory protein-1alpha showed a continued increase in lung tissue and broncho-alveolar lavage fluid (BALF) while interleukin-1alpha (IL-1alpha), IL-1beta in lung tissue and monocyte chemotactic protein-1 in BALF showed transient increases. Taken together, it was suggested that nano-agglomerates of nickel oxide nanoparticles have a persistent inflammatory effect, and the transient increase in cytokine expression and persistent increases in CC chemokine were involved in the persistent pulmonary inflammation.

BackgroundWe used fullerenes, whose dispersion at the nano-level was stabilized by grinding in nitrogen gas in an agitation mill, to conduct an intratracheal instillation study and an inhalation exposure study. Fullerenes were individually dispersed in distilled water including 0.1% Tween 80, and the diameter of the fullerenes was 33 nm. These suspensions were directly injected as a solution in the intratracheal instillation study. The reference material was nickel oxide in distilled water. Wistar male rats intratracheally received a dose of 0.1 mg, 0.2 mg, or 1 mg of fullerenes and were sacrificed after 3 days, 1 week, 1 month, 3 months, and 6 months. In the inhalation study, Wistar rats were exposed to fullerene agglomerates (diameter: 96 ± 5 nm; 0.12 ± 0.03 mg/m3; 6 hours/days for 5 days/week) for 4 weeks and were sacrificed at 3 days, 1 month, and 3 months after the end of exposure. The inflammatory responses and gene expression of cytokine-induced neutrophil chemoattractants (CINCs) were examined in rat lungs in both studies.ResultsIn the intratracheal instillation study, both the 0.1 mg and 0.2 mg fullerene groups did not show a significant increase of the total cell and neutrophil count in BALF or in the expression of CINC-1,-2αβ and-3 in the lung, while the high-dose, 1 mg group only showed a transient significant increase of neutrophils and expression of CINC-1,-2αβ and -3. In the inhalation study, there were no increases of total cell and neutrophil count in BALF, CINC-1,-2αβ and-3 in the fullerene group.ConclusionThese data in intratracheal instillation and inhalation studies suggested that well-dispersed fullerenes do not have strong potential of neutrophil inflammation.