Grantee Research Project Results
1999 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, 1999 through May 31, 2000
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 that are 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 increase 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 responses 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 has progressed along two simultaneous tracks. The major effort has gone into establishing the various cell models and beginning studies of their responses to particles in solution. While these studies were underway, preliminary work characterizing the in vitro aerosol exposure system was begun in collaboration with the Exposure Core. This system is designed to permit exposure to particles in the airborne state. We tested the performance of this chamber utilizing ultrafine Pt particles and measuring their deposition on wet filter surfaces in place of a cell layer in the transwell chambers over an exposure period of 6 hours. An average deposition of approximately 300 ng per transwell surface was found, which would be equivalent to a dose/cell in the respiratory tract after an in vivo inhalation exposure of 20 µg/m3 over a 24-hour period.
Studies have begun to address responses of cells and cell lines to carbon black particles as well as a number of studies combining exposures of particles, endotoxin, and ozone. Studies with epithelial cell lines have established conditions for ozone or LPS-induced chemokine expression in vitro. After 24 hours of exposure to either of these stimuli, production of MIP-2 was enhanced two- to eight-fold over unexposed controls. Interestingly, culture of these cells at the air/liquid interface, a necessary prerequisite for our aerosol studies, suggested primary release of this chemokine into the airspace compartment. Incubation of these cells with carbon particles alone did not appear to lead to increased cytokine expression, in fact inhibition of basal expression was noted. Moreover, addition of varying concentrations of carbon to cells in the presence of an additional stimulus also resulted in inhibition. 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 2- to 30-fold, dependent on appropriate dose and time. Similar to our studies with the epithelial cells, carbon alone did not lead to a stimulation of chemokine production. In fact, addition of carbon, either before, during, or after exposure to LPS, led to a suppression of chemokine production.
To attempt to understand the rationale behind this response, we incubated cells with LPS followed by addition of carbon black and TiO2. In this experiment, particles of both fine and ultrafine size were included to determine if the effect in previous studies was due to ultrafine particle toxicity. Results of this experiment confirmed the inhibitory effect of ultrafine carbon and showed a reduced inhibitory effect of the larger size particle. Most interesting was the response to TiO2. When added directly to cells, ultrafine TiO2 directly stimulated MIP-2 production and did not inhibit the LPS-induced stimulation. This suggests a clear difference in the ability of particles to induce cellular response that we will continue to investigate. We also have begun to evaluate the effect of age on the response of cells to particles. We have compared macrophage production of cytokines following LPS from 22-27 month old rats to cells from 10-12 week old rats. Although the responses appear to be different between the ages, a clear trend has not been established.
References:
Albright JW, Albright JF. Aging alters the competence of the 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):5-1182.
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;32:743-751.
Future Activities:
We will continue evaluating oxidant signaling and cytokine gene expression in primary isolated cell populations from 20-month old rats as a model of in vitro age effects. We also will continue to evaluate the effect of age on the response of cells to particles.
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, Health, Scientific Discipline, Air, particulate matter, Toxicology, air toxics, Environmental Chemistry, Health Risk Assessment, 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
- 2000 Progress Report
- Original Abstract
7 journal articles for this subproject
Main Center: R827354
106 publications for this center
91 journal articles for this center