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2001 Progress Report: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
EPA Grant Number: R827354C005Subproject: this is subproject number 005 , established and managed by the Center Director under grant R827354
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Airborne PM - Rochester PM Center
Center Director: Oberdorster, Gunter
Title: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
Investigators: Finkelstein, Jacob N. , O'Reilly, Michael , Phipps, Richard
Current Investigators: Finkelstein, Jacob N.
Institution:
Current Institution: University of Rochester
EPA Project Officer: Stacey Katz/Gail Robarge,
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2001 through May 31, 2002
RFA: Airborne Particulate Matter (PM) Centers (1999)
Research Category: Particulate Matter
Description:
Objective:The objective of this research project is to develop in vitro models useful for understanding the mechanism of ultrafine particle induced gene expression in various cell types. Additionally, these models should prove useful in studies of other size fractions, and we are using them to attempt to differentiate between particles of differing toxicity and activation potential. A goal of these experiments is to define mechanisms of cellular activation, the effects of age or prior activation on cytokine gene activation, and differential responses of epithelial cells and macrophages to particles of different sizes.
Progress Summary:In an effort to better characterize the aged mice, and evaluate their utility as a model of human response, we measured baseline cytokine levels in their plasma. The cytokines we chose to measure were based on the recommendations of our advisory committee that we use similar markers that are used, or suggested for use, in the in vivo animal experiments and the human clinical studies. As shown in Figure 1, both tumor necrosis factor-alpha (TNFa) and interleukin-6 (IL-6), were significantly elevated in the aged mice.
Figure 1. Cytokine Levels in Plasma of Young and Old Mice
One of our main goals for this year was to evaluate the effect of age on the response of cells to particles. Our most recent studies in mice compared the effects of age on macrophage responses to particles and LPS. Similar to our data in rats, mouse macrophages showed an age dependent difference in cytokine production following stimulation with particles or lipopolysaccharide (LPS). In these studies, we chose to make use of a particle composed of both carbon and iron. This was chosen, in part based on the data provided by our analysis project R827354C001, which suggested that iron is among the most abundant metal constituents. Our studies with this particle will be used to assess the likelihood of using this particle in both the animal studies R827354C004, as well as potentially in the human clinical studies R827354C003.
In our initial studies, we compared macrophage production of cytokines following LPS and particle incubation, with cells from 8-10 week old "young mice" and 20-22 month old "old mice". When macrophages from young mice are treated with LPS, a clear dose response, with macrophage inflammatory protein-2 (MIP-2) as the endpoint, is obtained (see Figure 2). When a similar study was carried out using macrophages from old mice, a number of clear differences were observed. Interestingly, baseline (unstimulated) production of MIP-2 and tumor necrosis factor (TNF) was elevated 30-50 percent in these cells. In addition, response to LPS was enhanced at every dose.
Figure 2. Expression of MIP-2 by Macrophages After Stimulation by Endotoxin: Effect of Age
When particles and LPS were combined as a stimulus, an enhanced effect is observed only in the old cells, except at the highest dose of particles. Most significant, in the context of our investigation of age effects and the ability of particles to induce effects at low dose, was the fact that in the aged animals co-administration (HYPHENATE) of particles and LPS lead to synergistic effects at the lowest dose of particles. This result is somewhat similar to results obtained in the in vivo studies, in which enhanced response to combined insult was noted in aged rats.
An important question regarding the in vitro studies is the choice of the appropriate endpoint to measure. While production of TNFa or MIP-2 following interaction with particles is well described, their role in environmental particle induced systemic disease is less clear. Thus, some studies were carried out looking at additional endpoints. These were chosen on the basis of data obtained from the clinical studies, and the possibility that measurements could be made in the in vivo studies. Among the cytokines measured, the only one that showed some promise was IL-6. Production of interleukin-6 (IL-6) was observed when epithelial cells were cultured in the presence of silica, used as a positive particle control. However, no effect of the addition of carbon particle on IL-6 protein or messenger ribonucleic acid (mRNA) was observed in our mouse experiments. We will continue to consult with the in vivo animal studies in an attempt to develop additional in vitro markers that could accurately predict effects of the inhalation studies. One marker that has proven useful is the production of prostaglandins (PGs). By measuring changes in PGs, we could indirectly monitor activity of COX-2, the rate limiting enzyme, and determine the role of PG's in pulmonary and systemic inflammation. As shown in Figure 4, stimulation of young and old cells with a combination of particles and LPS lead to an increase in PG-E2 production. As with MIP-2 (and TNF), this was mainly observed in the cells from the old mice. This is consistent with our other age experiments and reinforces the theme of age related increased susceptibility.
Figure 3. Expression of MIP-2 by Macrophages After Stimulation by Endotoxin and Particles
Figure 4. Expression of PG-E2 by Macrophages After Stimulation by Endotoxin and Particles
During this past project year we have continued to use the cell lines we developed for more detailed mechanistic studies of our oxidant stress hypothesis. This cell line has proven to be responsive to particles, and we have initiated studies looking at the possible signaling pathways that may be involved in particle induced stimulation. Using inhibitors of signaling pathways, we have begun to investigate the mechanism of particle induced gene expression. As shown in Figure 5, addition of the p38 kinase inhibitor PD98059 effectively inhibited both LPS and particle induced MIP-2 expression. In contrast, SB203580, which blocks p44 MAP kinase, had little effect. We plan to continue use of this cell line, as well as others, to test the response of cells to particles, and develop an understanding of the potential interactions between particles and endotoxin.
Figure 5. Effect of Kinase Inhibitors on MIP-2 Expression by Epithelial Cells After Particle Stimulation
In addition to the human cell line, we recently have developed a stably transfected mouse epithelial line using the same reporter construct. We currently are evaluating the response to particles in these cells and their relative sensitivity to priming.
Future Activities:In the coming year, we plan to continue characterizing the difference in response to stimuli, alone, and in combination as a function of age. We also expect to extend these studies from macrophages to respiratory epithelial, vascular endothelial cells, and fibroblasts. We also will investigate other markers of response. Measurement of prostaglandin production and cyclo-oxygenase-2 (COX-2) activation will be continued, with a special emphasis on production by these additional cell types. Studies have shown COX-2 to be important in the induction of the inflammatory response and systemic responses.
We also plan to use microarray analysis to screen young and old cells for differential gene expression changes with and without stimulation.
Further, in support of the in vivo projects, we will evaluate in vitro effects of particles of differing composition. We will continue to examine the cytokine response to particles containing carbon and iron, and begin studies of concentrated real world particles.
Journal Articles:No journal articles submitted with this report: View all 9 publications for this subproject
Supplemental Keywords:ultrafine, particle, lipopolysaccharide, LPS, gene, animal model, human model. , Air, Scientific Discipline, Health, RFA, Molecular Biology/Genetics, Toxicology, Risk Assessments, Health Risk Assessment, air toxics, Atmospheric Sciences, Biochemistry, particulate matter, Environmental Chemistry, aerosols, cardiopulmonary, risk assessment, susceptible populations, ultrafine particles, altered gene expression, urban environment, aerosol, ambient air quality, cardiovascular disease, cardiovascular vulnerability, coronary artery disease, health effects, mortality, lung inflamation, inhalation toxicology, ambient air, fine particles, environmental health effects, cardiopulmonary responses, human health risk, cytokine production, particle exposure, biostatistics, human health effects, particulates, sensitive populations, ambient monitoring, lung, metals, ambient air monitoring, pathophysiological mechanisms, atmospheric, pulmonary, urban air pollution, human health, cell kinetic models, morbidity, particle size, PM, pulmonary disease, animal model
Progress and Final Reports:
1999 Progress Report
2000 Progress Report
Original Abstract
2002 Progress Report
2003 Progress Report
2004 Progress Report
Final Report
Main Center Abstract and Reports:
R827354 Airborne PM - Rochester PM Center
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827354C001 Characterization of the Chemical Composition of Atmospheric Ultrafine Particles
R827354C002 Inflammatory Responses and Cardiovascular Risk Factors in Susceptible Populations
R827354C003 Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects
R827354C004 Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
R827354C005 Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
R827354C006 Development of an Electrodynamic Quadrupole Aerosol Concentrator
R827354C007 Kinetics of Clearance and Relocation of Insoluble Ultrafine Iridium Particles From the Rat Lung Epithelium to Extrapulmonary Organs and Tissues (Pilot Project)
R827354C008 Ultrafine Oil Aerosol Generation for Inhalation Studies