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2002 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, 2002 through May 31, 2003
RFA: Airborne Particulate Matter (PM) Centers (1999)
Research Category: Particulate Matter
Description:
Objective:The objective of this research project is to address specific mechanistic hypotheses regarding the interactions between inhaled ultrafine particles (UFP) and pulmonary cell populations. We have used a number of cell lines and primary cells derived from rats and mice to test the overall Particulate Matter (PM) Center hypothesis that the unique physicochemical characteristics of UFP—in comparison to accumulation mode particles of similar composition—contribute to the observed increases in morbidity and mortality in susceptible populations exposed environmentally. We will define mechanisms that follow particle cell contact, and will test the specific hypothesis that many of the subsequent physiologic effects are the consequences of cellular oxidative stress. In addition, we plan to examine host and environmental factors, including age, the influence of coexposure to gaseous oxidants, or prior priming or activation by preexposure to other inflammatory stimuli. A key component of the proposed studies is our plan to examine these particle cell interactions in individual cell populations to begin to assess the role of epithelial, inflammatory, and interstitial cells in the systemic response to UFP. In our experiments, in collaboration with research project R827354C004, we have begun to define susceptible populations on the basis of age as well as prior or concurrent infection. In this way, the proposed in vitro experiments are intended to provide a link between the whole animal (R827354C004) and controlled clinical (human) exposures (R827354C003), described in the other programs of this PM Center. Using animal models developed in research project R827354C004 and marker endpoints identified in R827354C002 and R827354C003 we are able to elucidate specific mechanism that are triggered following particle cell contact.
Progress Summary:Progress for Years 1-3 of the Project
The initial phase of our investigations progressed along two simultaneous tracks, with the major effort in establishing the various cell models and beginning studies of their responses to particles in solution. Experiments were conducted to examine ultrafine carbon black particles as well as a number of studies combining exposures of particles, endotoxins, and ozone. We established conditions for lipopolysaccharide (LPS) induced chemokine expression in vitro. After 24 hours of exposure to either of these stimuli, production of macrophage inflammatory protein-2 (MIP-2) was enhanced twofold to eightfold more than unexposed controls. Interestingly, culture of these cells at the air/liquid interface suggested primary release of this chemokine into the airspace compartment. Similar experiments were performed with a macrophage cell line to determine the cellular specificity of this response. Having established appropriate culture conditions of media and adherence, we determined the appropriate dose response and time course relationship for LPS in these cells. Cytokine and chemokine expression was found to be stimulated twofold to thirtyfold, dependent on appropriate dose and time. Similar to our studies with the epithelial cells, ultrafine carbon alone did not lead to a stimulation of chemokine production. Addition of ultrafine carbon either before, during, or after exposure to LPS led to a suppression of chemokine production.
We also evaluated the effect of age on the response of cells to particles. In our initial studies, we compared macrophage production of cytokines following LPS and particles from 22- to 27-month-old rats to cells from 10- to 12-week-old rats. When macrophages from young rats are treated with LPS, a clear dose response, with TNF as the endpoint, was obtained. A similar dose response relationship was observed with ultrafine carbon particles alone. When the two stimuli are combined, no enhanced effect is observed except at the highest dose of particles. When a similar study was performed with macrophages from “old rats,” 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. Response to particles alone was similar to that observed in young cells. 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, coadministration of particles and LPS led to synergistic effects at the lowest dose of particles. This result was similar to results obtained in the in vivo studies in collaboration with research project R827354C004, where enhanced response to combined insult was noted in aged rats.
We had previously proposed using isolated cells from aged animals as a model to study age-related particle-induced gene activation. In collaboration with research project R827354C004, we began to further characterize the aged mice and evaluate the utility of isolated cells from these mice as a model of aging. Because one of the outcomes being measured was changes in cytokine gene expression, we first measured baseline cytokine levels in the plasma of aged mice. 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. Both TNFα and interleukin-6 (IL-6) were significantly elevated in the aged mice. We also continued to evaluate the effect of age on the response of cells to particles.
An important question that was addressed in collaboration with research projects R827354C002, R827354C003, and R827354C004 was the choice of appropriate endpoints. While production of TNFα or MIP-2 following interaction with particles may be well described, the role of these mediators in environmental particle-induced systemic disease is less clear. Thus, some studies were conducted to examine 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 promise was IL-6. Production of IL-6 was observed when epithelial cells were cultured in the presence of silica, used as a positive particle control, and LPS. However, the addition of ultrafine carbon particles appeared to have no effect on IL-6 protein or mRNA in our mouse experiments.
An important development was the use of laboratory-generated UFPs containing various metals. The choice of the specific metal was based on the data provided by our Chemical UFP characterization research project (R827354C001) that iron is among the most abundant metal constituents. This material was produced by our particle generation research project. We will assess the likelihood of using this particle in both the animal studies (R827354C004) as well as potentially in the human clinical studies (R827354C003).
We compared macrophage production of cytokines following LPS and particles (with C/Fe) incubation with cells from 20 to 22-month-old and 8 to 10-week-old mice. Baseline MIP-2 and TNF were significantly elevated in cells from “old” mice. After stimulation, the old mice also were found to be more responsive. 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 of particles and LPS led 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.
One marker that has proven useful is the production of prostaglandins (PG). By measuring changes in PG, we could indirectly monitor activity of cyclooxygenase-2 (COX-2), the rate limiting enzyme, and also determine the role of PGs in pulmonary and systemic inflammation. Stimulation of young and old cells with a combination of ultrafine C/Fe particles and LPS led to an increase in PGE2 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 it reinforces the theme of age-related increased susceptibility.
We have continued to use the cell lines we have developed to pursue our mechanistic studies, and to test the oxidant stress/signaling 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 C/Fe particle-induced gene expression. The addition of the p38 kinase inhibitor PD98059 effectively inhibited both LPS and particle-induced MIP-2 expression. In contrast, SB203580, which blocks p44 mitogen-activated protein kinase, had little effect. We plan to continue to use this cell line as well as others to test the response of cells to particles, and to develop an understanding of the potential interactions between particles and endotoxin.
Summary of Year 4 Progress
Recent work has continued the refinement of in vitro models of particle cell interactions with the objective of defining 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, and the development of assays for the specific endpoints.
We continue to maintain our focus in the area of understanding how age affects the interaction 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, coadministration of particles and LPS led to synergistic effects at the lowest dose of particles of 0.47 μg/cm 2.
Our current results show both an age-dependent change in cytokine production as well as a response at low, environmentally relevant doses. We have continued our studies on dose effects by refining our indicator cell line to be useful in detecting particle-induced effects at a wide range of doses.
Using the transfected A549 cell line developed by our laboratory, we are able to detect changes in gene expression at particle doses below 1 μg/cm 2. This clearly puts us in the realistic range of nanoparticle mass burdens. We anticipate working with our particle generation research group to determine if this relationship would be maintained for particles of different composition or with particles collected using the Harvard UFP concentrator that we have available for our use once research project R827354C001 studies of the concentrator performance have been successfully completed. We also are working with our Immunology Core to attempt to make additional cytokine measurements on these cells to confirm this result. Our initial studies comparing cytokine analysis with luciferase activity show a reasonable correlation between these two measurements.
During the past year, we have worked closely with research project R827354C004 (in vivo animal exposures) on a number of experiments. As described in that research project, a large real-time “on-road” exposure study was conducted by exposing aged primed rats to the actual highway particle aerosol. After exposure, when animals were sacrificed, we recovered the lavage cells and placed them in culture, in the presence or absence of a laboratory test particle (mixed C/Fe). Our initial analysis of the media from these incubations did not reveal any enhancement of the cytokine response following exposure to the highway aerosol. The only factor that was found to alter macrophage cytokine production was prior exposure to the priming agent, either LPS or influenza virus.
In an effort to better bridge the experiments that are being carried out in R827354C003 (human clinical studies), addressing the effects of UFP on the vascular endothelium, we have begun to develop a vascular endothelial model that could be useful in assessing particle-induced changes in endothelial gene expression. Rather than using a typical monolayer culture, we have chosen to develop an epithelial/endothelial coculture system.
Using this model, we have begun to characterize cytokine production in response to various stimuli, including particle and LPS. We chose to use a number of different endpoints to better correlate our results with those of research projects R827354C003 and R827354C004.
IL-6 is among the cytokines we evaluated in this model. Production of IL-6 was observed when endothelial cells were cultured in the form of a classic monolayer in the presence of LPS or TNF. We also determined if coculturing these cells with A549 pulmonary epithelial cells would alter their ability to be stimulated by LPS or by particles. Both cell types appear to be responsive to particles and LPS with apparently different concentration dependence.
The endothelium appears to respond to lower particle mass burdens than the epithelium. This may account for the enhanced sensitivity of the vascular endothelium in vivo. Additionally, aided by our Immunology Core, we measured production of prostaglandins in these culture supernatants. Because the majority of the prostaglandin was found below the membrane, it appears to be derived from the endothelium. This is consistent with the in vivo results from research project R827354C004 showing enhanced prostaglandin production following particle exposure in a sensitive animal.
Future Activities:We will continue to characterize 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 parenchymal cells, fibroblasts, and epithelial cells. We also will investigate other markers of response. Measurement of prostaglandin production, and COX-2 activation will be evaluated with respect to its usefulness as a marker. Studies have shown COX-2 to be important in the induction of the inflammatory response and systemic responses.
Also, 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 UFP containing elemental carbon and iron and organic carbon compounds, and will 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:urban air pollution, atmosphere, metals, air, health, atmospheric sciences, biochemistry, environmental chemistry, epidemiology, molecular biology, health risk assessment, risk assessments, incineration, combustion, combustion engines, air toxics, tropospheric ozone, PM2.5, particulates, ultrafine particles, particulate matter, particle exposure, particle size, aerosol, ambient air, ambient air monitoring, ambient air quality, animal model, atmospheric, cardiopulmonary, cardiopulmonary responses, cardiovascular disease, cardiovascular vulnerability, coronary artery disease, cytokine production, fine particles, human exposure, human health, human health effects, environmental health effects, inhalation toxicology, lung, lung inflammation, metals, morbidity, mortality, pathophysiological mechanisms, pulmonary, pulmonary disease, stratospheric ozone, sensitive populations, susceptible populations,
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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
Relevant Websites:
http://www2.envmed.rochester.edu/envmed/
Progress and Final Reports:
1999 Progress Report
2000 Progress Report
2001 Progress Report
Original Abstract
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