Silica-induced caspase activation in mouse alveolar macrophages is dependent upon mitochondrial integrity and aspartic proteolysis.

Although silica has been documented to cause apoptotic cell death, the cellular pathways leading to caspase activation have not been extensively investigated. Here we demonstrate in a mouse macrophage cell line (MH-S cells) that alpha-quartz silica exposure (12.5 mug/cm2 to 50 mug/cm2) elicited activation of both caspase 3 and caspase 9, whereas anatase titanium dioxide (TiO2), a non-fibrogenic particle, did not. Silica exposure in vitro also induced apoptosis after 6 h, as measured by the appearance of subdiploid cell fragments in a flow cytometric analysis. Exposure to TiO 2 did not elicit significant apoptosis. Silica-induced apoptosis and caspase 3 activation were, in part, caspase 9 dependent, as determined by their sensitivity to either a general caspase inhibitor (Z-VAD-FMK) or a specific caspase 9 inhibitor (Z-LEHD-FMK). Silica exposure in vitro also elicited significant mitochondrial depolarization after 2 and 6 h of exposure. Cyclosporin A, an inhibitor of the mitochondrial permeability pore, partially decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, suggesting a role for mitochondrial dysfunction in these events. Pepstatin A, an inhibitor of cathepsin D, also decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, whereas leupeptin, an inhibitor of cathepsin B, had no effect. These data suggest that short-term silica exposure in vitro induces both caspase 3 and caspase 9 activity, which appears to participate in apoptosis. Activation of these caspases seems to be dependent, in part, on aspartic proteolysis and loss of mitochondrial integrity.

Cellular and molecular mechanisms regulating silica-induced adhesion molecule expression in mice.

Exposure of mice to silica particles generates an intense inflammatory response characterized by the influx of neutrophils and monocytes into the alveoli with resulting cell activation. These cell trafficking and effector functions are, in part, mediated by different families of adhesion molecules. One such adhesion protein, intercellular adhesion molecule-1 (ICAM-1), is expressed on a variety of cells including vascular endothelial cells, fibroblasts, types I and II alveolar epithelial cells, some lymphocytes, and monocytes/macrophages. Over the last several years, our laboratory has focused on silica-induced ICAM-1 expression in exposed mice (in vivo) and cells (in vitro) by examining the role and regulation of this adhesion protein on pulmonary cells. We have determined that intratracheal exposure of mice to silica (2 mg per mouse) elicits increased expression of ICAM-1 on pulmonary macrophages and type II epithelial cells within the lung parenchyma, on bronchoalveolar lavage macrophages, and as a soluble protein in lavage fluid. We hypothesize that increased ICAM-1 expression mayparticipate in silica-induced neutrophil influx into the alveoli, as well as in macrophage-derived phlogistic signals responsible for migration of neutrophils. ICAM-1 expression on silica-exposed mouse macrophages is enhanced by reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-alpha) and appears to be regulated through specific sequence elements within the ICAM-1 promoter.

Cytokine-induced stabilization of newly synthesized I(kappa)B-alpha.

NF-kappaB activation is triggered by the degradation of inhibitory proteins, such as I(kappa)B-alpha. I(kappa)B-alpha levels are only transiently lowered since one gene activated by NF-kappaB is I(kappa)B-alpha. We found that I(kappa)B-alpha was replenished rapidly in a human colon cell line (HT-29), even in the presence of degradation-inducing phosphorylation (at serine-32). This finding lead us to hypothesize that posttranscriptional mechanisms were also in place to facilitate I(kappa)B-alpha replenishment. Expression of I(kappa)B-alpha from the constitutive, non-NF-kappaB regulated cytomegalovirus promoter in HT-29 cells showed that TNF-alpha or IL-1beta treatment increased I(kappa)B-alpha levels in the absence of transcriptional activation. The TNF-alpha-induced increase in transgenic I(kappa)B-alpha appeared to result from the stabilization of newly synthesized I(kappa)B-alpha, since this increase was effectively preempted by a proteasome inhibitor (MG132) or by I(kappa)B-alpha stabilization through the deletion C-terminal destabilizing elements (without additive or synergistic effects). Analysis of a hepatoma cell line (Hepa 1-4C7) indicated that the I(kappa)B-alpha stabilization may be constitutive in these cells. NF-kappaB stimuli therefore appear to trigger negative feedback pathways in some cells that terminate a NF-kappaB response by increasing the stability of newly synthesized I(kappa)B-alpha.

Silica-induced activation of c-Jun-NH2-terminal amino kinases, protracted expression of the activator protein-1 proto-oncogene, fra-1, and S-phase alterations are mediated via oxidative stress.

Crystalline silica has been classified as a group 1 human carcinogen in the lung. However, its mechanisms of action on pulmonary epithelial cells which give rise to lung cancers are unclear. Using a nontransformed alveolar type II epithelial cell line (C10), we show that alpha-quartz silica causes persistent dose-related increases in phosphorylation of c-Jun-NH2-terminal amino kinases (JNKs) that are inhibited by antioxidants (P < or = 0.05). Increases in activator protein-1 (AP-1) binding to DNA and transactivation of AP-1-dependent gene expression by silica were accompanied by increases in steady-state mRNA levels of the AP-1 family members, c-jun, junB, fra-1, and c-fos at 8 h and elevated mRNA levels of fra-1 at 24 h (P < or = 0.05). Addition of tetramethylthiourea inhibited silica-associated increases infra-1 and proportions of cells in S-phase (P < or = .05). Our findings indicate that silica induces JNK activity, AP-1-dependent gene expression, ie., fra-1, and DNA synthesis via oxidative stress. Moreover, they suggest that silica may act mechanistically as a mitogen or tumor promoter, rather than a genotoxic carcinogen, in the development of lung cancers.

IkappaBbeta-related proteins in normal and transformed colonic epithelial cells.

The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes that can influence cell proliferation, apoptosis, and inflammatory responses. Since these events can contribute to carcinogenesis, we examined the expression of NF-kappaB inhibitory proteins (IkappaBs) in normal and transformed colonic epithelial cells. Immunohistochemical analysis of the mouse colon revealed a high level of IkappaBbeta expression in epithelial cells relative to the rest of the tissue, whereas IkappaBalpha was found primarily in cells of the lamina propria. Mouse colon tumors showed a similar cell-specific staining pattern. Immunoblot analysis of IkappaBbeta from mouse colonocytes and the human HT-29 colon cancer cell line indicated that most of the IkappaBbeta in these cells was similar to the C-terminal-truncated IkappaBbeta2 isoform. Cell fractionation studies were consistent with IkappaBbeta being a major regulator of p65-p50 NF-kappaB complexes in HT-29 cells. Interestingly, two larger proteins specifically recognized by IkappaBbeta antibodies (p106 and p112) were found in HT-29 cells and in colon tissue of carcinogen-exposed mice. The p106 and p112 proteins bound to NF-kappaB, and their levels changed during the transient interleukin-1beta activation of NF-kappaB in HT-29 cells. Evidence was obtained indicating that p106 and p112 are stably ubiquitinated forms of IkappaBbeta. We propose that deficiencies in the proteasomal degradation of IkappaBbeta lead to p106 and p112 accumulation, which in turn alter NF-kappaB regulation in colon cancer cells.

NF-kappa B 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-kappaB 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-kappaB 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-kappaB consensus oligonucleotide; 2) protein binding to this site was enhanced by NF-kappaB activators tumor necrosis factor-alpha, phorbol 12-myristate 13-acetate, and interleukin-1beta; and 3) the complex was specific and could be supershifted with antibodies against the p50 or p65 NF-kappaB subunits. The NF-kappaB 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-kappaB site could also be enhanced by co-transfection with an NF-kappaB1 expression vector. NF-kappaB may therefore contribute to the activation of TERT expression observed in mouse tissue.

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-kappaB 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.

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 (alpha quartz; 20 microg/ml; 6 microg/cm2) or the inflammatory cytokine, TNFalpha (20 ng/ml). TNFalpha 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, TNFalpha or media concomitantly with anti-TNFalpha antibody, the antioxidant, NAC, or the iNOS synthase inhibitor, L-NAME. Both anti-TNFalpha and NAC, but not L-NAME, inhibited elicited (TNFalpha, 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 TNFalpha and reactive oxygen species.

Effects of xenobiotics on macrophage function: evaluation in vitro.

Macrophages participate in a variety of inflammatory and immunologic functions (e.g., phagocytosis, cytokine production, killing of microbes and tumor cells, and processing and presentation of antigen to T lymphocytes). Because these cells are widely distributed in the circulation and throughout tissue, the effects of xenobiotics on macrophage function can be significant. Measures in vitro of altered function elicited by xenobiotic exposure can include changes in expression of cell surface proteins, in production of oxygen and nitrogen free radicals, in production of cytokines [interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor alpha (TNFalpha)], in expression of adhesion molecules (ICAM-1), in phagocytosis and intracellular killing, and in antigen presentation.

Localization of intercellular adhesion molecule-1 (ICAM-1) in the lungs of silica-exposed mice.

Intercellular adhesion molecule-1 (ICAM-1) is expressed on a variety of cells including endothelial cells, alveolar epithelial cells, and alveolar macrophages. Endothelial/epithelial cell ICAM-1 participates in the migration of leukocytes out of the blood in response to pulmonary inflammation, whereas alveolar macrophage ICAM-1 may represent cell activation. Our previous studies have shown that there is increased expression of ICAM-1 in lung tissue during acute inflammation following intratracheal injection with silica particles (2 mg/mouse). This increased expression was shown to play a role, in part, in the migration of neutrophils from the circulation into the tissue parenchyma. The aim of the current work is to localize expression of ICAM-1 during acute inflammation in lungs of mice exposed to either silica or the nuisance dust, titanium dioxide. In silica-exposed mice, a significant increase in ICAM-1 was detected on day-1 and localized by immunohistochemistry to aggregates of pulmonary macrophages and to type II epithelial cells. Areas of the lung with increased ICAM-1 expression also showed increased tumor necrosis factor alpha expression. Immunocytochemical staining of bronchoalveolar lavage (BAL) cells demonstrated increased ICAM-1 expression associated with alveolar macrophages 3, 5, and 7 days following silica exposure. Finally, soluble ICAM-1 levels in the BAL fluid were significantly increased in mice exposed to silica on the same days. Titanium dioxide exposure elicited a minimal increase in expression of ICAM-1 in the lungs. These data demonstrate that exposure to the toxic particle silica specifically increases ICAM-1 expression localized to pulmonary macrophages and type II epithelial cells.

Silica exposure increases expression of pulmonary intercellular adhesion molecule-1 (ICAM-1) in C57Bl/6 mice.

Although the pathogenic mechanisms underlying silica-induced lung damage are well described, few studies have examined the expression and role of adhesion molecules in lung injury induced by this particle. Here we report that intratracheal instillation of silica crystals (alpha quartz) (SI) into the lungs of C57Bl/6 mice results in a significant increase in levels of intercellular adhesion molecule-1 (ICAM-1) in lung tissue and in lung lavage fluid. This increased expression of ICAM-1 appeared to be associated with later (> or = 24 h) cell influx and lung injury rather than in the initiation of these events. Exposure of mice to the nontoxic particle titanium dioxide did not elicit increased expression of ICAM-1 in lung tissue or lavage fluid. Passive administration of rat anti-mouse ICAM-1 monoclonal antibody significantly decreased the influx of neutrophils (PMNs) into the alveoli and the levels of lung tissue ICAM-1 and yet had no effect on measures of lung injury or increased collagen deposition. These data suggest that increased ICAM-1 expression in lung tissue following exposure to silica plays a partial role in the trafficking of neutrophils into the airways.

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.

Immunogenicity studies of a synthetic antigen of alpha methyl dopa.

Since the idiosyncratic liver toxicity of methyl dopa (L-alpha-methyl-3,4- dihydroxy-phenylalanine) may be due to immune mediated mechanisms, immunologic tools are needed to detect methyl dopa induced antibody and antigen. Hapten (methyl dopa)--carrier (albumin) conjugates were synthesized to generate antibodies reactive with this drug. Studies were also conducted to test the immunogenicity of this hapten-carrier conjugate and its cross reactivity with methyl catechol and levodopa. Methyl dopa (MD), levodopa (LD) or methyl catechol (MC) were conjugated to rabbit serum albumin (RSA) under high pH (base) conditions or by a tyrosinase (tyr) catalyzed reaction. Under the base conjugation conditions, MD-RSA, LD-RSA and MC-RSA conjugates were produced at higher hapten: carrier ratios than conjugates produced by the enzyme catalyzed reaction. Rabbits were subsequently immunized with either MD-RSA(base) or MD-RSA(tyr). Antibodies elicited by MD-RSA(base) had marked reactivity to the carrier protein, albumin, whereas antibodies elicited by MD-RSA(tyr) did not. In addition, reactivity of anti-MD antibody was equal to or greater with MC-RSA than reactivity with either MD-RSA or LD-RSA. This work suggests that the conjugation method using the tyr catalyzed reaction produces the optimal immunogen with minimal modification of the carrier protein. In addition, the catechol moiety of MD, MC and LD appears to be the immunogenic epitope on these haptens.

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.

Silica-induced pulmonary inflammation and fibrosis in mice is altered by acute exposure to nitrogen dioxide.

The biologic impact of consecutive exposures to two environmental pollutants was examined in mice exposed to silica crystals (SI) by intratracheal (IT) injection followed by an inhalation exposure to nitrogen dioxide (NO2). C57Bl/6 mice received an IT injection of 2 mg SI or sterile saline (SAL) followed by a 2-h inhalation exposure to NO2 at 20 ppm either within 2 h of or 24 h after SI instillation. During acute inflammation (d 3 postsilica), mice exposed to NO2 at either time showed a dramatic and significant reduction in the number of lavaged alveolar neutrophils (PMN) when compared to silica/air-exposed mice. Animals exposed to NO2 24 h after silica also evidenced significant decreases in levels of lavage albumin and lactate dehydrogenase (LDH) 3 d after silica, as well as significant decreases in hydroxyproline content of the lung 30 and 60 d postsilica injection when compared to silica/air-exposed animals. NO2 administration 24 h after silica appeared to shift the appearance of PMN in the lung from d 3 to d 14, but did not otherwise alter chronic cellular inflammation. These data suggest that the marked neutrophil response and collagen deposition induced by SI can be modulated by NO2 exposure and that the time of oxidant gas exposure after silica administration is critical to this modulation.

Ethanol-induced suppression of cell-mediated immunity in the mouse.

Among its many effects on multiple organ systems, ethanol abuse also elicits potent immunomodulatory effects as well. Increased susceptibility to disease as well as increased severity in infection is well documented in alcoholics and may be related to an altered immune response. A mouse model has been developed in which C57Bl/6 mice are fed a nutritionally adequate liquid diet in which ethanol provides 27% of total calories (3.75% ethanol) in order to examine the effects of short-term, low dose ethanol administration on cell mediated immunity. All mice were evaluated for percentage of plasma ethanol, impaired motor skills and impaired delayed type hypersensitivity to sheep erythrocytes. Animals fed the ethanol diet for 5, 10, or 15 days evidenced plasma ethanol concentrations between 0.13% and 0.23% as well as significantly impaired motor skills. Mice maintained on the ethanol diet 8 days prior to erythrocyte sensitization and on the 6 days between sensitization and challenge demonstrated a significant reduction of 58% in footpad swelling. Moreover, mice fed ethanol for only the 6 days between erythrocyte sensitization and challenge also demonstrated a significant decrease of 35% in this delayed type immune response. However, animals administered ethanol prior to sensitization and then switched to control diet between sensitization and challenge evidenced a normal cell-mediated immune response to the sheep erythrocytes. Thus, it appears that the critical time of low dose ethanol-induced immunosuppression is between immunogen sensitization and challenge.

Screening for antibodies associated with halothane hepatitis.

The diagnosis of halothane hepatitis (HH) may be assisted by detection of antibodies reacting to trifluoroacetylated proteins (anti-TFA antibodies). An enzyme-linked immunosorbent assay (ELISA) utilizing trifluoroacetylated rabbit serum albumin (TFA-RSA) as antigen detected anti-TFA antibodies in 67% of sera from patients for whom a clinical diagnosis of HH was made. Anti-TFA antibodies were detected in 33% of sera when using an ELISA with liver microsomal protein from halothane-treated rabbits as antigen. Absorption of the sera with untreated rabbit liver microsomal protein before using the microsomal protein ELISA resulted in detection of anti-TFA antibodies in 42% of sera. Using the presumptive hapten N-epsilon-trifluoroacetyl-1-lysine to block antibody binding in an ELISA resulted in positive detection in 50% of sera: the results did not always agree with the other ELISA methods. The TFA-RSA ELISA was the most sensitive method and, combined with the TFA-lysine blocking ELISA, resulted in 92% of sera from HH patients testing positive for HH-associated antibodies.