Andrea K. Hubbard

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis

Exposure to particulate silica (most crystalline polymorphs) causes a persistent inflammation sustained by the release of oxidants in the alveolar space. Reactive oxygen species (ROS), which include hydroxyl radical, superoxide anion, hydrogen peroxide, and singlet oxygen, are generated not only at the particle surface, but also by phagocytic cells attempting to digest the silica particle. Two distinct kinds of surface centers-silica-based surface radicals and poorly coordinated iron ions-generate O(2)(*)(-) and HO(*) in aqueous solution via different mechanisms. Crystalline silica is also a potent stimulant of the respiratory burst in phagocytic cells with increased oxygen consumption and production of O(*)(-), H(2)O(2), and NO leading to acute inflammation and HO(*) generation in the lung. Oxidative stress elicited by crystalline silica is also evidenced by increased expression of antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD) and glutathione peroxidase, and the enzyme inducible nitric oxide synthase (iNOS). Generation of oxidants by crystalline silica particles and by silica-activated cells results in cell and lung injury, activation of cell signaling pathways to include MAPK/ERK kinase (MEK), and extracellular signal-regulated kinase (ERK) phosphorylation, increased expression of inflammatory cytokines (e.g., tumor necrosis factoralpha [TNFalpha], interleukin-1 [IL-1]), and activation of specific transcription factors (e.g., NFkappaB, AP-1). Silica can also initiate apoptosis in response to oxygen- and nitrogen-based free radicals, leading to mitochondrial dysfunction, increased gene expression of death receptors, and/or their ligands (TNFalpha, Fas ligand [FasL]).

Intercellular adhesion molecule-1 (ICAM-1) expression and cell signaling cascades

The collective interaction between cells is, in part, mediated by different families of adhesion molecules. Intercellular adhesion molecules (ICAMs) are structurally related members of the immunoglobulin supergene family and are ligands for the beta2 integrin molecules present on leukocytes. Of the five ICAMs identified, ICAM-1 is the most extensively studied. Although ICAM-1 is expressed constitutively at low levels on endothelial cells and on some lymphocytes and monocytes, its expression can be significantly increased in the presence of cytokines (TNFalpha, IL-1, IFNgamma) and reactive oxygen species. Depending upon cell type, ICAM-1 participates in trafficking of inflammatory cells, in cell:cell interactions during antigen presentation, in microbial pathogenesis, and in signal transduction through outside-in signaling events. Again, depending upon cell type examined, ICAM-1 engagement has been documented to activate specific kinases through phosphorylation, resulting in transcription factor activation and increased cytokine production, increased cell membrane protein expression, reactive oxygen species production, and cell proliferation.

The luminal short-chain fatty acid butyrate modulates NF-κB activity in a human colonic epithelial cell line

BACKGROUND & AIMS The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in regulating immune and inflammatory responses. Because butyrate deficiency has been associated with inflammatory bowel disease, we examined the effect of butyrate on NF-kappaB activity in the human HT-29 colonic cell line. METHODS The influence of butyrate (4 mmol/L) on NF-kappaB activity was determined using the gel mobility shift assay. The effect of butyrate on the expression of NF-kappaB subunits and inhibitory proteins was determined by immunoblotting. NF-kappaB-regulated gene expression was assayed by primer extension of intercellular adhesion molecule 1 and Mn superoxide dismutase messenger RNA, and by analysis of a transfected luciferase reporter. RESULTS Exposure of HT-29 cells to butyrate eliminated their constitutive NF-kappaB, p50 dimer activity. This inhibition corresponded with a reduction in p50 nuclear localization, without a reduction in expression. Butyrate also selectively modulated activation of NF-kappaB, suppressing its activation by tumor necrosis factor alpha and phorbol ester more than 10-fold, without affecting the activity induced by interleukin (IL)-1beta. Butyrate did, however, enhance formation of the stronger p65-p50 transcriptional activator in IL-1beta-stimulated cells. The changes in NF-kappaB activation did not correlate with changes in IkappaBalpha levels. Gene expression reflected DNA binding. The influence of butyrate on NF-kappaB may result in part from its ability to inhibit deacetylases because the specific deacetylase inhibitor trichostatin A has a similar effect. CONCLUSIONS These findings suggest that the influences of butyrate on colonic inflammatory responses may result in part from its influence on NF-kappaB activation. This activity of butyrate apparently involves its ability to inhibit deacetylases.

NF-κB regulates transcription of the mouse telomerase catalytic subunit

Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most cells. Analysis of the mouse TERT promoter revealed a potential NF-κB binding site 350 base pairs upstream from the translational start site. An oligonucleotide from this region of the TERT promoter bound to proteins in a nuclear extract prepared from a mouse hepatoma cell line. These proteins were identified as NF-κB by a number of criteria: 1) the protein complex formed on the TERT oligonucleotide had an electrophoretic mobility similar to that formed on an NF-κB consensus oligonucleotide; 2) protein binding to this site was enhanced by NF-κB activators tumor necrosis factor-α, phorbol 12-myristate 13-acetate, and interleukin-1β; and 3) the complex was specific and could be supershifted with antibodies against the p50 or p65 NF-κB subunits. The NF-κB binding site from the mouse TERT promoter activated transcription when fused to a basal SV40 promoter and enhanced the activity of the native TERT promoter in mouse hepatoma cells stimulated with phorbol 12-myristate 13-acetate. Transcriptional activation by the TERT NF-κB site could also be enhanced by co-transfection with an NF-κB1 expression vector. NF-κB may therefore contribute to the activation of TERT expression observed in mouse tissue.

Growing old with nuclear factor-kB

Abstract The transcription factor nuclear factor–κB (NF-κB) is involved in the regulation of a broad spectrum of genes that play important roles in a myriad of physiological and pathological events ranging from the immune response to carcinogenesis. Interestingly, many processes in which NF-κB plays a central role have long been noted for their alteration with age. A number of research groups have reported rather dramatic changes in NF-κB activity as humans and animals age, with tissue-specific increases and decreases in NF-κB activity being reported. The extent to which changes in NF-κB activity drive aging and influence life span in humans and other mammals is not clear. However, given the dramatic impact that NF-κB can have on the function of numerous tissues and organs, understanding how NF-κB activity changes with age will undoubtedly enhance our understanding of the many diseases associated with growing old.

Regulation of ICAM-1 expression in mouse macrophages.

In a mouse model of silica (SI) induced lung injury, SI exposure increases expression of intercellular adhesion molecule-1 (ICAM-1) on lung (alveolar/interstitial) macrophages and alveolar type II epithelial cells. To investigate the regulation of SI induced ICAM-1 expression on mouse macrophages, freshly isolated macrophages (alveolar, peritoneal) and macrophage cell lines (MH-S, RAW 264.7) were evaluated for ICAM-1 expression elicited by the particle silica (α quartz; 20 μg/ml; 6 μg/cm2) or the inflammatory cytokine, TNFα (20 ng/ml). TNFα significantly increased ICAM-1 expression in all cell types whereas SI elicited an increase in peritoneal macrophages (PM) and the cell line, MH-S. This pattern of increased expression was confirmed by immunocytochemistry. To investigate the regulation of ICAM-1 expression, PM were incubated with SI, TNFα or media concomitantly with anti-TNFα antibody, the antioxidant, NAC, or the iNOS synthase inhibitor, L-NAME. Both anti-TNFα and NAC, but not L-NAME, inhibited elicited (TNFα, SI) as well as constitutive (media) ICAM-1 expression. These data demonstrate that both inflammatory cytokines and inorganic particles can increase ICAM-1 expression on mouse macrophages and that this expression is mediated, in part, by TNFα and reactive oxygen species.

Role of Mitogen-Activated Protein Kinases, Early Response Protooncogenes, and Activator Protein-1 in Cell Signaling by Asbestos.

Abstract Cell signaling by pathogenic minerals may initiate the transactivation of genes that are critical to carcinogenesis and fibroproliferative diseases of the lung and pleura. We have shown previously that stimulation of the mitogen-activated protein kinase (MAPK) cascade by asbestos fibers leads to phosphorylation events involved in transactivation of lun and Fos proteins that comprise the activator protein-1 (AP-1) transcription factor. Recently, we have also used AP-1 luciferase reporter transgenic mice and immunocytochemistry to show that transactivation of AP-1 occurs in bronchiolar and alveolar epithelial cells after inhalation of asbestos fibers. After inhalation of asbestos, epithelial cells of the lung also show increased immunoreactivity of pliosphorylated extracellular signal regulated kinases (ERKs 1/2) at sites of fibrogenesis. The availability of lung epithelial cell-specific promoters has allowed the creation of transgenic mice with mutations in the transactivation domains of key receptors and protein intermediates that comprise the MAPK signaling cascade. These rodent models may reveal whether cell signaling events initiated by mineral dusts in epithelial cells are critical to the development of cell proliferation, apoptosis, and lung disease.

Modulation of silica-induced lung injury by reducing lung non-protein sulfhydryls with buthionine sulfoximine

A role for non-protein sulfhydryl moieties (NPSH) (e.g., glutathione) in silica (SI)-induced cellular inflammation and fibrosis was examined in C57Bl/6 mice depleted of lung NPSH by buthionine sulfoximine (BSO). Lung NPSH levels in the BSO-treated groups were reduced to approx. 50% of the non-BSO-treated animals. In BSO-treated SI-injected (2 mg/mouse) animals, the number of pulmonary alveolar macrophages (PAM) lavaged from the lungs was significantly increased on day 1 and decreased on day 7. Moreover, BSO-treated SI-exposed mice evidenced significantly more lavage protein and albumin on days 1 and 3, respectively, than non-BSO-treated SI-exposed mice. SI-induced collagen deposition, however, was decreased by 18% in the BSO-treated animals. These data suggest that lung NPSH lessens the potential of silica to elicit acute lung injury.

Effect of nitrogen dioxide on ovalbumin-induced allergic airway disease in a murine model.

The effect of exposure to irritant air pollutants on the development of allergic airway disease is poorly understood. This study examines the effects of the lower respiratory tract irritant, NO 2 , on the outcome of ovalbumin (OVA)-induced allergic airway disease. Male and female C57Bl/6 mice were sensitized by weekly intraperitoneal (ip) OVA injections for 3 wk followed by daily 1-h OVA aerosol inhalation challenge for 3 or 10 d. Initially, mice were exposed daily for 3 d to air or 0.7 or 5 ppm NO 2 for 2 h following each OVA aerosol challenge. OVA exposure resulted in pronounced lower airway inflammation, as evidenced by a significant increase in bronchoalveolar lavage (BAL) total cellularity and eosinophil levels. BAL eosinophil levels were significantly lower in OVA-NO 2 compared to OVA-air animals. The reduction was similar at both NO 2 exposure concentrations. In a subsequent study, sensitized animals were exposed for 3 or 10 d to aerosolized OVA followed by air or 0.7 ppm NO 2 . BAL eosinophils were again reduced at 3 d by OVA-NO 2 exposure compared to OVA-air mice. At 10 d the eosinophilia was virtually abolished. This reduction in OVA-induced cellular inflammation by NO 2 was confirmed by histopathological analysis. Contrary to expectations, exposure to NO 2 during the aerosol challenge to OVA dramatically diminished the outcome of allergic disease in lungs as measured by airway cellular inflammation.

Acute NO2 exposure alters inflammatory cell activation and particle clearance in silica-injected mice

Previous work indicates that exposure to nitrogen dioxide (NO2) at different stages of silica-induced acute inflammation alters the outcome of lung injury. C57BI/6 mice were injected intratracheally (IT) with 2.0 mg silica particles (SI) and subsequently exposed to 20 ppm NO2 (or filtered air) within 2 or 24 h after SI. The present study demonstrates that exposure of mice to NO2 within 2 h after silica injection during acellular lung injury (increased alveolar protein, albumin, lactate dehydrogenase) resulted in increases in intraalveolar and interstitial polymorphonuclear leukocytes (PMNs) as well as more advanced granulomas on d 14, 30, and 60. In contrast, exposure of mice to NO2 24 h after silica during marked lung injury and inflammatory cell influx resulted in a less severe inflammatory reaction with fewer interstitial and alveolar PMNs and decreased size and number of pulmonary granulomas. NO2 exposure 2 or 24 h after SI appeared to increase in the lavage fluid levels of lysosomal enzymes and at the same time decrease levels of PMN chemotactins. Moreover, exposure to NO2 24 h after SI significantly decreased SI accumulation in the mediastinal lymph nodes. These data suggest that NO2 modulation of SI lung injury may be due, in part, to changes in inflammatory cell activation/influx and/or altered particle disposition within the lung. These effects are dependent upon the inflammatory status of SI exposed lungs at the time NO2 is administered.