Binding of antigen to IgE-receptor complexes on the surface of RBL-2H3 rat basophilic leukemia cells is the first event leading to the release of cellular serotonin, histamine, and other mediators of allergic, asthmatic, and inflammatory responses. We have used dinitrophenol-conjugated bovine serum albumin (DNP-BSA) as well as the fluorescent antigen, DNP-B-phycoerythrin, and the electron-dense antigen, DNP-BSA-gold, to investigate dynamic membrane and cytoskeletal events associated with the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. These multivalent antigens bind rapidly to cell surface IgE-receptor complexes. Their distribution is initially uniform, but within 2 min DNP-BSAgold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca 2÷. The F-actin content of the detergent-extracted cell matrices analyzed by SDS PAGE decreases during the first 10-30 s of antigen binding and then increases by 1 min to almost double the control levels. A rapid and sustained increase is also observed when total F-actin is quantified by flow cytometry after binding of rhodaminephalloidin. The antigen-stimulated increase in F-actin coincides with (and may cause) the transformation of the cell surface from a finely microvillous to a highly folded or plicated topography. Other early membrane responses include increased cell spreading and a 2-3-fold increase in the uptake of fluorescein-dextran by fluid pinocytosis. The surface and Factin changes show the same dependence on DNP-protein concentration as stimulated [3HI serotonin release; and both the membrane responses and the release of mediators are terminated by the addition of the non-cross-linking monovalent ligand, DNP-lysine. These data indicate that the same antigen-stimulated transduction pathway controls both the membrane/cytoskeletal and secretory events. However, the membrane and actin responses to IgEreceptor cross-linking are independent of extracellular Ca 2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca 2+ and is mimicked by the Ca 2+ ionophore A23187.The release of histamine, serotonin, and other inflammatory mediators from mast cells and basophils is the precipitating event in a variety of acute allergic, asthmatic, and inflammatory reactions (38). This release occurs by the fusion of cytoplasmic granules with each other and with the plasma membrane, leading to the discharge of granule matrix and soluble mediators (13). Current studies of the sequence of biochemical and morphological events leading to degranulation are focused on the rat basophil-like cell line, . In this cell line degranulation is measured by the release of [3H]serotonin or histamine after cross-linking of surface IgE-receptor complexes with multivalent antigen. Biochemical studies have shown that antigen binding stimulates phosphatidylinositol turnover and generates two second messengers, diacylglycerol to activate pro...

Cigarette smoke (CS)-induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome-dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke.

We are interested in the cytotoxic and proinflammatory effects of particulate pollutants in the respiratory tract. We demonstrate that methanol extracts made from diesel exhaust particles (DEP) induce apoptosis and reactive oxygen species (ROS) in pulmonary alveolar macrophages and RAW 264.7 cells. The toxicity of these organic extracts mimics the cytotoxicity of the intact particles and could be suppressed by the synthetic sulfhydryl compounds, N-acetylcysteine and bucillamine. Because DEP-induced apoptosis follows cytochrome c release, we studied the effect of DEP chemicals on mitochondrially regulated death mechanisms. Crude DEP extracts induced ROS production and perturbed mitochondrial function before and at the onset of apoptosis. This mitochondrial perturbation follows an orderly sequence of events, which commence with a change in mitochondrial membrane potential, followed by cytochrome c release, development of membrane asymmetry (annexin V staining), and propidium iodide uptake. Structural damage to the mitochondrial inner membrane, evidenced by a decrease in cardiolipin mass, leads to O⨪2 generation and uncoupling of oxidative phosphorylation (decreased intracellular ATP levels). N-Acetylcysteine reversed these mitochondrial effects and ROS production. Overexpression of the mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress apoptosis. Taken together, these results suggest that DEP chemicals induce apoptosis in macrophages via a toxic effect on mitochondria.

Objective Mechanisms of air pollution-induced exacerbation of cardiovascular disease are currently unknown, thus we examined the roles of vascular endothelin-1 (ET-1) and reactive oxygen species (ROS) in regulating mediators of vascular remodeling, namely matrix metalloproteinases (MMPs), following exposure to vehicle engine emissions. Methods and Results ApoE-/- mice were exposed by inhalation to filtered air or gasoline engine exhaust (GEE, 1:12 dilution) 6 h/d for 1 or 7 days. Concurrently, mice were treated with either ETA receptor antagonist BQ-123 (100 ng/kg/day) via osmotic minipumps, Tempol (∼41 mg/kg/day, orally), or vehicle. GEE-exposure increased vascular MMP-2 and -9, endothelin-1 (ET-1), tissue inhibitor of metalloproteinases (TIMP)-2 mRNA and ROS levels. Aortic MMP protein and plasma MMP-9 were similarly upregulated. GEE-mediated increases in vascular ROS were attenuated by Tempol-treatment, as were MMP-2 and TIMP-2; whereas BQ-123 ameliorated GEE-induced vascular expression of MMP-9, MMP-2, ROS, and ET-1. In a parallel study, diesel exhaust exposure in volunteer human subjects induced significant increases in plasma ET-1 and MMP-9 expression and activity. Conclusions These findings demonstrate that acute exposure to vehicular source air pollutants results in upregulation of circulating and vascular factors associated with progression of atherosclerosis, mediated in part through activation of ET-1 - ETA receptor pathways.

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Mice develop pulmonary emphysema after chronic exposure to cigarette smoke (CS). In this study, the influence of gender, exposure duration, and concentration of CS on emphysema, pulmonary function, inflammation, markers of toxicity, and matrix metalloproteinase (MMP) activity was examined in A/J mice. Mice were exposed to CS at either 100 or 250 mg total particulate material/m 3 (CS-100 or CS-250, respectively) for 10, 16, or 22 weeks. Evidence of emphysema was first seen in female mice after 10 weeks of exposure to CS-250, while male mice did not develop emphysema until 16 weeks. Female mice exposed to CS-100 did not have emphysema until 16 weeks, suggesting that disease development depends on the concentration and duration of exposure. Airflow obstruction and increased pulmonary compliance were observed in mice exposed to CS-250 for 22 weeks. Decreased elasticity was likely the major contributor to airflow obstruction because substantial remodeling of the conducting airways, beyond mild mucous cell hyperplasia, was lacking. Exposure to CS increased the number of macrophages, neutrophils, lymphocytes (B cells and activated CD4-and CD8-positive T cells), and activity of MMP-2 and-9 in the bronchoalveolar lavage fluid (BALF). Treatment with antioxidants N-acetylcysteine or epigallocatechin gallate (EGCG) did not decrease emphysema severity, but EGCG slightly decreased BALF inflammatory cell numbers and lactate dehydrogenase activity. Inflammation and emphysema persisted after a 17-week recovery period following exposure to CS-250 for 22 weeks. The similarities of this model to the human disease make it promising for studying disease pathogenesis and assessing new therapeutic interventions.

BackgroundExposure to air pollution and, more specifically, particulate matter (PM) is associated with adverse health effects. However, the specific PM characteristics responsible for biological effects have not been defined.ObjectivesIn this project we examined the composition, sources, and relative toxicity of samples of PM with aerodynamic diameter ≥2.5 μm (PM2.5) collected from sites within the Southeastern Aerosol Research and Characterization (SEARCH) air monitoring network during two seasons. These sites represent four areas with differing sources of PM2.5, including local urban versus regional sources, urban areas with different contributions of transportation and industrial sources, and a site influenced by Gulf of Mexico weather patterns.MethodsWe collected samples from each site during the winter and summer of 2004 for toxicity testing and for chemical analysis and chemical mass balance–based source apportionment. We also collected PM2.5 downwind of a series of prescribed forest burns. We assessed the toxicity of the samples by instillation into rat lungs and assessed general toxicity, acute cytotoxicity, and inflammation. Statistical dose–response modeling techniques were used to rank the relative toxicity and compare the seasonal differences at each site. Projection-to-latent-surfaces (PLS) techniques examined the relationships among sources, chemical composition, and toxicologic end points.Results and conclusionsUrban sites with high contributions from vehicles and industry were most toxic.

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