An in vitro alveolar macrophage assay for the assessment of inflammatory cytokine expression induced by atmospheric particulate matter.
Exposures to air pollution in the form of particulate matter (PM) can result in excess production of reactive oxygen species (ROS) in the respiratory system, potentially causing both localized cellular injury and triggering a systemic inflammatory response. PM-induced inflammation in the lung is modulated in large part by alveolar macrophages and their biochemical signaling, including production of inflammatory cytokines, the primary mechanism via which inflammation is initiated and sustained. We developed a robust, relevant, and flexible method employing a rat alveolar macrophage cell line (NR8383) which can be applied to routine samples of PM from air quality monitoring sites to gain insight into the drivers of PM toxicity that lead to oxidative stress and inflammation. Method performance was characterized using extracts of ambient and vehicular engine exhaust PM samples. Our results indicate that the reproducibility and the sensitivity of the method are satisfactory and comparisons between PM samples can be made with good precision. The average relative percent difference for all genes detected during 10 different exposures was 17.1%. Our analysis demonstrated that 71% of genes had an average signal to noise ratio (SNR) ≥ 3. Our time course study suggests that 4 h may be an optimal in vitro exposure time for observing short-term effects of PM and capturing the initial steps of inflammatory signaling. The 4 h exposure resulted in the detection of 57 genes (out of 84 total), of which 86% had altered expression. Similarities and conserved gene signaling regulation among the PM samples were demonstrated through hierarchical clustering and other analyses. Overlying the core congruent patterns were differentially regulated genes that resulted in distinct sample-specific gene expression "fingerprints." Consistent upregulation of Il1f5 and downregulation of Ccr7 was observed across all samples, while TNFα was upregulated in half of the samples and downregulated in the other half. Overall, this PM-induced cytokine expression assay could be effectively integrated into health studies and air quality monitoring programs to better understand relationships between specific PM components, oxidative stress activity and inflammatory signaling potential.
Department of Environmental Chemistry and Technology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.
Zana
Z
Sijan
Department of Environmental Toxicology, Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, Wisconsin, 53718, USA.
Dagmara S
DS
Antkiewicz
Department of Environmental Chemistry and Technology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.
Jongbae
J
Heo
Department of Environmental Toxicology, University of California-Davis, Davis, California, USA.|California Environmental Protection Agency, Air Resources Board, Sacramento, California, USA.
Norman Y
NY
Kado
Department of Environmental Chemistry and Technology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.|Department of Environmental Toxicology, Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, Wisconsin, 53718, USA.
James J
JJ
Schauer
Department of Civil and Environmental Engineering, University of Southern California, Los Angeles, California, 90089, USA.
Constantinos
C
Sioutas
Department of Environmental Chemistry and Technology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.|Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, Wisconsin 53718, USA.
Martin M
MM
Shafer
D000393:Air Pollutants / Q000633:toxicity*; D000818:Animals; D002460:Cell Line; D016000:Cluster Analysis; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D016676:Macrophages, Alveolar / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D052638:Particulate Matter / Q000633:toxicity*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
D000393:Air Pollutants; D016207:Cytokines; D052638:Particulate Matter; D017382:Reactive Oxygen Species
PCR array; ROS; air pollution; cytokines; gene expression; inflammation; macrophages; oxidative stress; particulate matter; toxicity