Grantee Research Project Results
2000 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: Rochester PM Center
Center Director: Oberdörster, Günter
Title: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
Investigators: Finkelstein, Jacob N. , Morrow, P. E. , Stripp, Barry , O'Reilly, Michael , Phipps, Richard
Current Investigators: Finkelstein, Jacob N.
Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2000 through May 31, 2001
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air
Objective:
The experiments proposed within this project are designed to address specific mechanistic hypotheses regarding the interactions between inhaled ultrafine particles and specific pulmonary cell populations. We will use cell lines and primary cells derived from rats to test the overall PM Center hypothesis that the unique biological characteristics of ultrafine particles in comparison to accumulation mode particles of similar composition lead to the observed increases in morbidity and mortality in susceptible populations exposed environmentally. The proposed in vitro experiments are intended to provide a link between the whole animal and controlled clinical (human) exposures, described in the other programs of this PM Center, by elucidating specific mechanisms that are triggered following particle cell contact and to test the specific hypothesis that many of the subsequent physiologic effects are the consequences of cellular oxidative stress. Similar to studies proposed in other projects in the Center, we plan to examine host factors, such as age and environmental factors, including the influence of co-exposure to gaseous oxidants or prior priming or activation by pre-exposure to other inflammatory stimuli such as endotoxin on the response of cells in culture. An important aspect of the proposed studies is our plan to examine the response to ultrafine particle interactions in epithelial, inflammatory, and interstitial cells. Work by a number of authors (Breen, et al., 1992; Crestani, et al., 1994; Driscoll, et al., 1996; Finkelstein, 1990; Finkelstein, et al., 1997) has suggested that production of inflammatory mediators following particle interaction is not limited to classic inflammatory cells, and that pulmonary parenchymal elements including epithelial cells (type II, Clara cells) and fibroblasts also may contribute to the milieu. Moreover, data suggesting rapid translocation of ultrafine particles following deposition increases the probability of multiple particle cell interactions.
A second element of our studies involves our ability to model, in vitro, the effects of age on specific cell populations and how aging may influence cellular response particles. A large body of data exists that demonstrates age-related changes in immune cell response and pulmonary function (Albright & Albright, 1994; Chorinchath, et al., 1996; Ding, et al., 1994; Gyetko & Toews, 1993; Hartwell, et al., 1995). These effects have been shown to be detectable in isolated cell populations obtained from aged animals and humans. We hypothesize that these effects, as they relate to changes in antioxidant capacity and cytokine production at the cellular level, contribute to increased particle sensitivity. We propose to evaluate oxidant signaling and cytokine gene expression in primary isolated cell populations from 20-month old rats as a model of in vitro age effects.
Progress Summary:
Research during the current year continues to develop in vitro models of particle cell interactions with the goal 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. Although a number of experimental difficulties have altered some of the approaches, we continue to make significant progress in our experimental goals.
One of our main goals for this year was to evaluate 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 22-27 month old rats to cells from 10-12 week old rats. When macrophages from young rats are treated with LPS, a clear dose response is obtained with MIP-2 as the endpoint. A similar dose response relationship was observed with 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 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 co-administration 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. Unfortunately, our inability to obtain aged rats from our supplier, due to a problem in their animal colony, has impaired our ability to investigate the mechanism behind these differences. Instead, we have begun to repeat these studies with cells prepared from aged mice. Our initial studies with mouse macrophages have shown a somewhat blunted response in these cells in comparison to rats. We plan to continue these studies and characterize this response until aged rats become available.
An important question regarding the in vitro studies is the choice of the appropriate endpoint to measure. Although 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 conducted 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 IL-6 was observed when epithelial cells were cultured in the presence of silica, used as a positive particle control, and LPS. However, no effect of the addition of Carbon particle on IL-6 protein or mRNA was observed in our mouse experiments. We will continue to consult with the in vivo animal studies to attempt to develop additional in vitro markers that could accurately predict effects of the inhalation studies.
We also have begun to develop reagents and approaches that would allow extension of our in vitro studies to human cells, while also developing a test of our oxidant stress hypothesis. We have developed a human lung cell line, A549, that was stably transfected with a reporter gene and that other studies have shown was responsive to oxidant stress. Preliminary experiments confirm particle-induced increase in reporter gene activity with a peak at 6 hours posttreatment. We will continue to use this human cell line to investigate response to multiple exposures as well as antioxidants.
References:
Albright JW, Albright JF. Aging alters of the competence immune system to control parasitic infection. Immunology Letters 1994;40:279-285.
Breen E, Shull S, Burne S, Absher M, Kelley J, Phan S, Cutroneo KR. Bleomycin Regulation of transforming growth factor-beta messenger RNA in rat lung fibroblasts. American Journal of Respiratory Cell and Molecular Biology 1992;6:146-152.
Chorinchath BB, Kong LY, Mao L, McCallum RE. Age-associated differences in tnf-alpha and nitric oxide production in endotoxic mice. Journal of Immunology 1996;156(4):1525-1530.
Crestani B, Cornillet P, Dehoux M, Rolland C, Guenounou M, Aubier M. Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products. Journal of Clinical Investigation 1994;94:731-740.
Ding A, Hwang S, Schwab R. Effect of aging on murine macrophages. Diminished response to IFN-gamma for enhanced oxidative metabolism. Journal of Immunology 1994;153(5):2146-2152.
Driscoll KE, Howard BW, Carter JM, Asquith T, Johnston C, Detilleux P, Kunkel SL, Isfort RJ. Alpha-quartz-induced chemokine expression by rat lung epithelial cells: effects of in vivo and in vitro particle exposure [see comments]. American Journal of Pathology 1996;149:1627-1637.
Finkelstein JN. Physiologic and toxicologic responses of alveolar type II cells. Toxicology 1990;60:41-52.
Finkelstein JN, Johnston C, Barrett T, Oberdorster G. Particulate-cell interactions and pulmonary cytokine expression. Environmental Health Perspectives 1997;105(Suppl 5):1179-1182.
Gyetko MR, Toews GB. Immunology of the aging lung. Clinics in Chest Medicine 1993;14:379-391.
Hartwell DW, Fenton MJ, Levine JS, Beller DI. Aberrant cytokine regulation in macrophages from young autoimmune-prone mice-evidence that the intrinsic defect in mrl macrophage IL-1 expression is transcriptionally controlled. Molecular Immunology 1995;32(10):743-751.
Future Activities:
In the coming year, we plan to 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 as well. 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 have begun to examine the cytokine response to particles containing carbon and iron. In our standard exposure model with mouse cell lines, enhanced cell death was seen but cytokine production was similar to that of pure carbon.
Journal Articles:
No journal articles submitted with this report: View all 9 publications for this subprojectSupplemental Keywords:
pollution prevention, atmosphere, particulates, metals, sensitive population., RFA, Scientific Discipline, Health, Air, Toxicology, particulate matter, Environmental Chemistry, Health Risk Assessment, air toxics, Risk Assessments, Biochemistry, Atmospheric Sciences, Molecular Biology/Genetics, ambient air quality, cytokine production, particle size, particulates, sensitive populations, biostatistics, atmospheric, health effects, risk assessment, altered gene expression, cardiopulmonary responses, fine particles, human health effects, morbidity, ambient air monitoring, lung, cardiovascular vulnerability, pulmonary disease, susceptible populations, animal model, ambient air, environmental health effects, particle exposure, ambient monitoring, particulate exposure, lung inflamation, pulmonary, coronary artery disease, inhalation toxicology, urban air pollution, PM, mortality, urban environment, aerosol, cardiopulmonary, human health, aerosols, cardiovascular disease, ultrafine particles, pathophysiological mechanisms, metals, cell kinetic modelsRelevant Websites:
http://www2.envmed.rochester.edu/envmed/pmc/indexpmc.html Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R827354 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
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2004 Progress Report
- 2003 Progress Report
- 2002 Progress Report
- 2001 Progress Report
- 1999 Progress Report
- Original Abstract
7 journal articles for this subproject
Main Center: R827354
106 publications for this center
91 journal articles for this center