Final Report: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression

EPA Grant Number: R827354C005
Subproject: 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: Oberd√∂rster, G√ľnter
Title: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
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)
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air

Objective:

A key component of our studies was to examine particle cell interactions in individual cell populations to begin to assess the role of ultrafine particles (UFP) in altering inflammatory gene expression by an oxidant-related mechanism. In our experiments, in collaboration with Core 4 (R827354C004) we were able to define susceptible populations on the basis of age as well as prior or concurrent infection.

Summary/Accomplishments (Outputs/Outcomes):

To test the hypothesis that increased susceptibility of aged animals is due to cell intrinsic differences in oxidant sensitivity, we evaluated the effect of age on the response of cells to particles. We compared macrophage production of cytokines following lipopolysaccharide (LPS) and particles from 22-27 month old rats to cells from 10-12 week old rats. Baseline (unstimulated) production of MIP-2 (and TNF) was elevated 30-50% in these cells as well as increased response to exogenous stimulus. Increased production of prostaglandin E2 (PGE2) by alveolar macrophages from “aged” animals, an endpoint chosen to better correlate with the animal studies (Core 4) and the human clinical studies (Core 3) was observed when cultured in the presence of LPS used as a positive particle control, and LPS plus particles confirming age effects for a number of endpoints

An important development during this project was the ability to use laboratory generated ultrafine particles containing various metals. The choice of the specific metal was based on the data provided by our Chemical UFP characterization (Core 1) that iron is among the most abundant metal constituents. This material was produced by our particle generation Core. We compared macrophage production of cytokines following LPS and particles (with C/Fe) incubation with cells from 20-22 month old and 8-10 week old mice. Baseline MIP-2 and TNF was significantly elevated in cells from “old” mice. After stimulation the old mice were also 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 leads 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 in assessing cellular response to PM is the production of prostaglandins (PGs). By measuring changes in PGs we could indirectly monitor activity of 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 lead 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 reinforces the hypothesis that age is related to increased PM susceptibility.

We also developed 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 developed a human lung cell line, A549, which was stably transfected with a reporter gene that in other studies has been shown to be responsive to oxidant stress. Using this transfected A549 cell line we were able to detect changes in gene expression at particle doses below 1 μg/cm2. This clearly puts us in the realistic range of ultrafine PM mass burdens. In future studies, together with our particle generation Facility Core we will determine if this relationship will be maintained for particles of different composition or with ambient particles collected using the Harvard ultrafine particle concentrator that we have available for our use. Our initial studies comparing cytokine analysis with luciferase activity show a reasonable correlation between these two measurements.

Particle Effects on Vascular Endothelium

Recent experiments, to better bridge the experiments that are being carried out in Clinical Studies Core and Animal Exposure Core, have focused on vascular endothelial models that could be useful in assessing particle induced changes in endothelial gene expression; and that may represent aspects of endothelial dysfunction. To more accurately reflect the complex nature of endothelial interactions with particles, we have used two complementary culture models.

Many of our experiments utilize a standard monolayer culture of primary vascular endothelial cells. A second model, a bilayer epithelial/endothelial co-culture system permits study of cell-cell interactions mediated by particles.

Monolayer cultures of human umbilical vein endothelial cells (HUVEC) were established and optimized with regard to media, serum and other culture conditions. Production of IL-6 and PGE2 when endothelial cells were cultured in the presence of LPS or TNF for 24 hours was used to establish the basic parameters. Our major objective with these cultures was to establish appropriate dose and time parameters of incubation with particles so that we could begin testing with UFP of various compositions. Based on our experiments with cultured epithelial cells we began our studies using laboratory generated particles containing 25% Fe. Particles were added to the cells at concentrations ranging from 0.47 to19.0 μg/cm2 and media collected at 6 and 24 hours. Particle induced cytotoxicity was measured by LDH release. Addition of particles in the presence of a priming dose of LPS stimulated the release (production) of both IL-6 and PGE2 at both 6 and 24 hours. The PGE2 response appears to be more sensitive as it is observed at doses as low as 0.47 μg/cm2 (which converts to a total mass dose of ~1.5 µg of particle).

In this system we also assessed the response of these cells to laboratory generated carbon, similar to the material used in the human clinical studies, TiO2, and a laboratory generated Mn-oxide. In contrast to our experiments with epithelial cells, the endothelial cell cultures were moderately responsive to the carbon alone. After 24 hours of incubation, PGE2 production was increased 2-3 fold. In contrast C/Fe particles increased PGE2 by 5-6 fold. Additionally, in support of the role of oxidant stress in particle effects, pretreatment with either a soluble ( N-acetyl-cysteine) or lipophilic (BHA) antioxidant suppressed the production of PGE2 induced by PM.

Overall comparison of particle, dose, and time parameters suggest that PGE2 is the most reliable marker of endothelial activation. It was also noted that particle composition was a major response factor, with TiO2 being most active and Mn-oxide being most directly cytotoxic. 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. Using this second model we have begun to examine cytokine production in response to various stimuli, including particle and LPS. Among the cytokines we evaluated in this model were IL-6 and PGE2.

The endothelium appears to respond to lower particle mass burdens than does the epithelium. This may account for the enhanced sensitivity of the vascular endothelium in vivo. Additionally, aided by our Immunology and Vascular Core, we also measured production of prostaglandins in these culture supernatants. Since the majority of the prostaglandin was found below the membrane this would suggest it is derived from the endothelium. This is consistent with the in vivo results from Core 4 showing enhanced prostaglandin production following particle exposure in a sensitive animal model.

Our experiments have focused on a number of critical issues that relate to the overall goals of the Rochester PM Center. A key point of the studies was the emphasis on real-world particles in lieu of laboratory surrogates. The delay in the characterization of the HUCAPS concentrator in Rochester has delayed this effort somewhat. However, it has enabled us to continue mechanistic studies with defined composition particles that may ultimately be important in attributing effects seen in specific cell populations to unique sources. The one source of real world particles available for in vitro studies was the material collected as part of the MAPS, multi-Center multi-site particle collection effort begun at the end of the grant period.

Vascular endothelial cells (HUVEC) exposed to concentrated fine and ultrafine particles respond through increased production of IL-6. This cytokine was chosen as a potential sentinel as a result of experiments from the Animal and Clinical Studies Cores that suggested a possible acute phase response following particle inhalation. Interestingly, using this marker and cell type, we revealed a differential response from particles collected from certain sites. It is hypothesized that this relates to the abundance of vehicle emissions at these sites. More detailed analyses and source calculations are planned with the help of Core 1 as the compositional data are provided.

An additional important piece of data was revealed as a consequence of this study. When epithelial cells (A549 cells) were similarly exposed to these materials, no differential response based on site selection was noted. To verify this cell-specific difference and to relate this potential mechanistic difference to studies from Cores 3 and 4, we carried out a direct comparison of the response of the epithelium and vascular endothelium in a series of well characterized particles. We chose these carbon particles as they had previously been used for in vivo studies within the Center. While these studies are interesting and important, they do not necessarily address the most relevant question, that of the response of the microvascular endothelium, which is being investigated during the next project cycle.

Technical Report:

Full Final Technical Report (PDF, 9pp., 244KB, about PDF)


Journal Articles on this Report : 7 Displayed | Download in RIS Format

Other subproject views: All 9 publications 8 publications in selected types All 7 journal articles
Other center views: All 104 publications 98 publications in selected types All 90 journal articles
Type Citation Sub Project Document Sources
Journal Article Elder ACP, Gelein R, Azadniv M, Frampton M, Finkelstein J, Oberdorster G. Systemic effects of inhaled ultrafine particles in two compromised, aged rat strains. Inhalation Toxicology 2004;16(6-7):461-471. R827354 (Final)
R827354C003 (Final)
R827354C004 (2003)
R827354C004 (Final)
R827354C005 (Final)
R826784 (Final)
R828046 (Final)
R832415 (2010)
R832415 (2011)
R832415 (Final)
R832415C003 (2011)
R832415C004 (2011)
R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor&Francis-Abstract
    Exit
  • Journal Article Elder ACP, Gelein R, Oberdorster G, Finkelstein J, Notter R, Wang Z. Efficient depletion of alveolar macrophages using intratracheally inhaled aerosols of liposome-encapsulated clodronate. Experimental Lung Research 2004;30(2):105-120. R827354 (Final)
    R827354C003 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R827354C005 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
    R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
    Exit
  • Journal Article Elder A, Gelein R, Finkelstein J, Phipps R, Frampton M, Utell M, Kittelson DB, Watts WF, Hopke P, Jeong C-H, Kim E, Liu W, Zhao W, Zhuo L, Vincent R, Kumarathasan P, Oberdorster G. On-road exposure to highway aerosols. 2. Exposures of aged, compromised rats. Inhalation Toxicology 2004;16(Suppl 1):41-53. R827354 (Final)
    R827354C003 (Final)
    R827354C004 (2003)
    R827354C004 (Final)
    R827354C005 (Final)
    R828046 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C003 (2011)
    R832415C004 (2011)
    R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
    Exit
  • Journal Article Elder A, Johnston C, Gelein R, Finkelstein J, Wang Z, Notter R, Oberdorster G. Lung inflammation induced by endotoxin is enhanced in rats depleted of alveolar macrophages with aerosolized clodronate. Experimental Lung Research 2005;31(6):527-546. R827354 (Final)
    R827354C004 (Final)
    R827354C005 (Final)
    R828046 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
    R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor&Francis-Abstract
    Exit
  • Journal Article Elder A, Gelein R, Silva V, Feikert T, Opanashuk L, Carter J, Potter R, Maynard A, Ito Y, Finkelstein J, Oberdorster G. Translocation of inhaled ultrafine manganese oxide particles to the central nervous system. Environmental Health Perspectives 2006;114(8):1172-1178. R827354 (Final)
    R827354C004 (Final)
    R827354C005 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2011)
    R832415C005 (2011)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Journal Article Singal M, Finkelstein JN. Use of indicator cell lines for determining inflammatory gene changes and screening the inflammatory potential of particulate and non-particulate stimuli. Inhalation Toxicology 2005;17(9):415-425. R827354 (Final)
    R827354C005 (Final)
    R832415 (2010)
    R832415 (Final)
    R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
    Exit
  • Journal Article Singal M, Finkelstein JN. Amorphous silica particles promote inflammatory gene expression through the redox sensitive transcription factor, AP-1, in alveolar epithelial cells. Experimental Lung Research 2005;31(6):581-597. R827354 (Final)
    R827354C005 (Final)
    R832415 (2010)
    R832415 (Final)
    R832415C005 (2011)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
    Exit
  • Supplemental Keywords:

    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, atmospheric, biostatistics, health effects, particle size, particulates, risk assessment, sensitive populations, cytokine production, altered gene expression, cardiopulmonary responses, fine particles, human health effects, lung, morbidity, ambient air monitoring, ambient air, cardiovascular vulnerability, pulmonary disease, susceptible populations, animal model, ambient monitoring, particle exposure, environmental health effects, pulmonary, lung inflamation, particulate exposure, coronary artery disease, PM, urban air pollution, inhalation toxicology, aerosol, cardiopulmonary, mortality, human health, urban environment, aerosols, cardiovascular disease, cell kinetic models, human health risk, metals, ultrafine particles

    Relevant Websites:

    Full Final Technical Report (PDF, 9pp., 244KB, about PDF)
    http://www2.envmed.rochester.edu/envmed/PMC/indexPMC.html Exit

    Progress and Final Reports:

    Original Abstract
  • 1999 Progress Report
  • 2000 Progress Report
  • 2001 Progress Report
  • 2002 Progress Report
  • 2003 Progress Report
  • 2004 Progress Report

  • Main 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