2001 Progress Report: Immunomodulation by PM: Role of Metal Composition and Pulmonary Phagocyte Iron Status

EPA Grant Number: R827351C008
Subproject: this is subproject number 008 , established and managed by the Center Director under grant R827351
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: EPA NYU PM Center: Health Risks of PM Components
Center Director: N/A
Title: Immunomodulation by PM: Role of Metal Composition and Pulmonary Phagocyte Iron Status
Investigators: Cohen, Mitchell
Institution: New York University School of Medicine
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


Particulate matter (PM) has been shown in epidemiological analyses to induce/exacerbate infectious lung disease, and in toxicological studies to alter the manner by which the lung handles bacteria. The main objective of this research project is to validate the hypotheses that: (1) PM modulates lung phagocyte antibacterial function by altering cellular iron (Fe) status; (2) metals (rather than organics, endotoxin, etc.) in PM underlay any change in leukocyte Fe status; and (3) the relative content of Fe in PM governs these mechanisms. In PM with a high relative Fe content, the uptake/slow dissolution of PM insoluble Fe and an increase in cellular deposition of soluble Fe (due to lacto- [Lf] or transfer-rin [Tf] activity) will lead to Fe overload and decreased antibacterial function. In PM with relatively low Fe content, the presence of relatively greater levels (with respect to Fe) of competitors for intrapulmonary T-/LF-binding (e.g., aluminum [Al], manganese [Mn], and vanadium [V]) will cause an intracellular Fe deficit and reduced antibacterial function due to inhibited transport of endogenous Fe to the phagocytes.

In cooperation with the Los Angeles and Seattle Centers, daily PM2.5 samples were collected over a 3-month period. These sites were selected as (along with New York City [NYC]) having distinct patterns of proportionality of Fe to Al, Mn, and V in their PM. All samples are analyzed by x-ray flourescence (XRF) to determine relative and absolute contents of the four metals.

In vitro studies using a rat lung macrophage cell line (i.e., NR8383) examine the impact of the competitors on cell iron response protein activation and/or ferritin synthesis to determine if the presence of Al, Mn, and V has an impact on iron homeostasis in phagocytes. Although varying doses of each metal are used, relative amounts of each are reflective of those when intact PM is utilized. Analyses of these endpoints allow a determination as to whether the cells convert to states of Fe deficit or excess when Mn, Al, and V are present. The use of the "metals only" versus intact PM allows assessment as to whether (in)organic agents in PM might augment or inhibit the responses. Lastly, these studies permit the extrapolation of minimal effective doses (MED) of PM for use in in vivo studies.

In in vivo studies (performed for each set of regional samples), PM (using pooled fragments from multiple filters to achieve MED) is instilled into the lungs of adult F344 rats. After 1, 3, 6, and 24 hours, pulmonary alveolar macrophage (PAM) are recovered by lavage and analyzed to determine any time-related change (as well as type of change) in Fe status. Lavage fluid is assayed for total, apo-, and holo-Fe, Tf, and Lf. To assess if PM-related changes in PAM Fe status gives rise to modified antibacterial function, ex vivo analyses of uptake/intracellular killing of Listeria monocytogenes by PAM (isolated from PM-exposed hosts at time of maximal change in Fe status) are conducted.

Progress Summary:

· Daily regional PM2.5 samples have been collected and analyzed by XRF to determine relative and absolute Fe, Al, Mn, and V content. Results in Table 1 indicate that: (1) the samples from NYC, Los Angeles, and Seattle reflect disparate ambient metal compositions; (2) wide variations in absolute and relative Fe, Mn, Al, and V contents exist among the regions; and (3) there are significant regional differences in the relative ratios of each competitor to Fe. Based on these differences in absolute content and relative Fe content versus Mn, V, and Al, it should be possible to determine which competitor may be most important for inducing the hypothesized effect(s).

· Protocols to be used for the in vitro studies have been finalized. Pilot studies using the "best" regional filter samples have been initiated at the time of this report.

· In preparation for in vivo studies with regional PM samples, studies that examined the effects from Fe and Mn on rat lung Tf and Lf were initiated. Levels of both proteins were quantified 1 hr and 18 hrs after deposition of approximately 1 µg Mn2+ or Fe3+, or a mixture of 0.5 µg of both. Figure 1 shows a representative immunoblot used for the quantification of Lf in wells loaded with a total of 10 µg bronchioalveolar lavage (BAL) protein.

The results indicate that deposition of Mn2+ resulted in increases in the amount of Lf present at 1 hr and 18 hrs postdeposition compared to those in control rats. In rats that received Fe3+, Lf levels were initially unaffected, but decreased by 18 hrs. In rats that received Mn and Fe, Lf levels were initially unaffected, but increased by 18 hrs. The data suggest that: (1) Fe deposited in the lungs causes Lf levels to decrease over time (as protein acts first to sequester and then transport Fe to lung cells); (2) presence of Mn causes a continual increase in Lf availability (due to an effect that causes cells to (in)voluntarily release the protein and/or an inhibition of normal extracellular Lf clearance); and (3) the effect on Lf from Mn is dose related.

Table 1. Comparison of PM2.5 Samples From the Three Regional PM Centers. Values are Mean ± Standard Error.

Total µg PM2.5/24-hr Period
315.93 ± 26.56
a429.04 ± 47.33
b,d139.27 ± 15.55
Ng Fe/m3
179.14 ± 20.60
b56.93 ± 4.95
b72.77 ± 12.24
Ng Al/m3
53.94 ± 4.84
60.60 ± 10.33
c33.47 ± 4.44
Ng Mn/m3
11.47 ± 1.93
b0.00 ± 0.00
a4.64 ± 1.10
Ng V/m3
8.79 ± 0.70
a5.67 ± 1.12
b2.64 ± 0.51
Ng Fe in 24 hr
3869.43 ± 445.06
b1366.67 ± 118.93
B1055.08 ± 177.34
Ng Al in 24 hr
1165.13 ± 104.56
1454.69 ± 248.09
a,d486.00 ± 64.73
Ng Mn in 24 hr
247.79 ±
41.75 b0.00 ± 0.00
b67.18 ± 15.77
Ng V in 24 hr
189.81 ± 15.14
136.12 ± 26.87
b,c38.20 ± 7.31
Fe as % of total mass
1.481 ± 0.294
0.458 ± 0.087
0.778 ± 0.115
Al as % of total mass
0.401 ± 0.048
0.388 ± 0.068
0.370 ± 0.069
Mn as % of total mass
0.089 ± 0.018
b0.000 ± 0.000
0.053 ± 0.016
V as % of total mass
0.070 ± 0.007
b0.033 ± 0.008
b0.029 ± 0.008
Al:Fe ratio
0.372 ± 0.042
b1.146 ± 0.222
d0.441 ± 0.082
Mn:Fe ratio
0.045 ± 0.005
b0.001 ± 0.001
d0.056 ± 0.006
V:Fe ratio
0.086 ± 0.012
0.109 ± 0.027
c0.039 ± 0.007
a,b Value is significantly different from that in New York at ap < 0.05 or bp < 0.01
c,d Value is significantly different from that in Los Angeles at cp < 0.05 or dp < 0.01

Figure 1. Immunoblot Detection of BAL Lf 1 and 18 Hours After Metal Deposition.

Future Activities:

The future activities of this research project will be to:

· Finish XRF analyses of the few remaining regional samples not yet completed.

· Select representative regional filters to be used for in vitro studies, analyze all outcomes in context of the relationship between amounts of Fe and the three competitors present on the filters, and analyze to account for potential synergistic/antagonistic effects from other filter constituents.

· Establish (by extrapolating dose to culture surface area to that for the total alveolar surface area of rat lung) MEDs used for the in vivo studies of each regional sample.

· Perform in vivo studies for each regional sample, and analyze outcomes in context of overall relative amounts of Fe, Al, Mn, and V, and relative amounts of each competitor with respect to Fe present on the filters.

· Continue the inter-Center cooperation initiated with this project; this includes the ongoing collection of PM samples at each site and sharing of harvested materials with Investigators (i.e., Drs. Luchtel and Liu in Seattle, and Dr. Sioutas in Los Angeles) for incorportation into their projects.

Journal Articles:

No journal articles submitted with this report: View all 5 publications for this subproject

Supplemental Keywords:

particulate matter, PM, exposure, epidemiology, toxicology, metals, pulmonary macrophage, pulmonary neutrophil, leukocyte, Los Angeles, California, CA, Seattle, Washington, WA, x-ray fluorescence, XRF, bronchioalveolar lavage, BAL., RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Air, ENVIRONMENTAL MANAGEMENT, particulate matter, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Analytical Chemistry, Environmental Monitoring, Physical Processes, Atmospheric Sciences, Risk Assessment, ambient air quality, atmospheric particulate matter, particulates, lung injury, metal absorption, air toxics, atmospheric particles, chemical characteristics, toxicology, ambient air monitoring, acute lung injury, airborne particulate matter, environmental risks, exposure, epidemelogy, Sulfur dioxide, air pollution, pneumonia, leukocyte function, aerosol composition, atmospheric aerosol particles, human exposure, PM, exposure assessment, human health risk, metals

Progress and Final Reports:

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

  • Main Center Abstract and Reports:

    R827351    EPA NYU PM Center: Health Risks of PM Components

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827351C001 Exposure Characterization Error
    R827351C002 X-ray CT-based Assessment of Variations in Human Airway Geometry: Implications for Evaluation of Particle Deposition and Dose to Different Populations
    R827351C003 Asthma Susceptibility to PM2.5
    R827351C004 Health Effects of Ambient Air PM in Controlled Human Exposures
    R827351C005 Physicochemical Parameters of Combustion Generated Atmospheres as Determinants of PM Toxicity
    R827351C006 Effects of Particle-Associated Irritants on the Cardiovascular System
    R827351C007 Role of PM-Associated Transition Metals in Exacerbating Infectious Pneumoniae in Exposed Rats
    R827351C008 Immunomodulation by PM: Role of Metal Composition and Pulmonary Phagocyte Iron Status
    R827351C009 Health Risks of Particulate Matter Components: Center Service Core
    R827351C010 Lung Hypoxia as Potential Mechanisms for PM-Induced Health Effects
    R827351C011 Urban PM2.5 Surface Chemistry and Interactions with Bronchoalveolar Lavage Fluid (BALF)
    R827351C012 Subchronic PM2.5 Exposure Study at the NYU PM Center
    R827351C013 Long Term Health Effects of Concentrated Ambient PM2.5
    R827351C014 PM Components and NYC Respiratory and Cardiovascular Morbidity
    R827351C015 Development of a Real-Time Monitoring System for Acidity and Soluble Components in Airborne Particulate Matter
    R827351C016 Automated Real-Time Ambient Fine PM Monitoring System