Introduction

Diesel exhaust particles (DEP) from traditional diesel engines and fuels have been shown in animal and cell studies to cause a variety of health effects including pulmonary inflammation, increased severity of infections, and allergic lung disease (1,2,3,4,5,6,7). Earlier animal studies also suggested an association between DEP inhalation and elevated risk of thoracic cancer (8); however, more current reassessments of these studies, combined with new research, suggest that the cancers observed in the DEP-exposed rodents could result from a nonspecific particle-overload effect (9,10,11). A significant portion of inhaled DEP is ingested by alveolar macrophages and cleared from the lungs through the mucociliary escalator mechanism (12,13,14). The fate of DEP in lungs and their interactions with lung cells are less well understood. In vitro uptake of DEP by epithelial cells lining the bronchial airways has been reported (15,16), and electron microscopy evidence shows the presence of DEP in lung epithelial cells of guinea pigs exposed to diesel engine exhaust (17). Since human and rodent lung epithelial cells outnumber alveolar macrophages by a factor of 5 to 10 (18), any contribution of epithelial cells to inflammatory processes and particle clearance could be a significant factor in determining the health effects of DEP. However, there is limited quantitative evidence explaining the relative efficacies of macrophages and epithelial cell ingestion of DEP and other airborne particles. Such knowledge would help explain the mechanisms of particle-induced lung diseases, such as fibrosis and cancer, and could be of use in targeting therapies to particular cell types.

DEP are comprised of an elemental carbon core onto which thousands of different organic molecules are adsorbed. The carbon contents of airborne particles derived from the combustion of fossil fuel can therefore be separated into elemental carbon (EC) and organic carbon (OC) fractions. We hypothesized that determination of EC in biological tissues could serve as a measure of uptake of carbonaceous particles, such as DEP, because mammalian cells do not contain any EC per se. Here we describe a technique to isolate and chemically quantify microgram amounts of DEP from cells exposed to known amounts of particles. Using this method, the kinetics and dose-response characteristics of DEP uptake by murine epithelial and macrophages were examined. In addition, the uptake of pure carbon black (CB) versus diesel was compared to determine whether the presence of organics on the DEP influenced this process. Finally, to investigate whether particle ingestion by cells was a result of active cytoskeletal processes, the effect of the phagocytosis inhibitor cytochalasin D on DEP uptake was assessed.

Materials and Methods

Cells and Reagents

LA4, a nontumorogenic murine lung epithelial cell line derived originally from a urethane-induced epithelial adenoma in A/J mice (19), and the MHS cell line, derived from simian virus 40 (SV40) transformation of mouse alveolar macrophages (20), were obtained from ATCC (Manassas, VA, USA). Both cell lines were maintained in RPMI-1640 culture medium supplemented with 2 mM glutamine, 25 nM HEPES buffer, 20 µg/mL gentamycin, and 10% (v/v) fetal bovine serum. DEP (DEP Standard Reference Material 2975, average particle diameter 120 nm, surface area 108 m²/g) were purchased from the National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA). Fine CB particles (CB, Printex 90, average particle diameter 12 nm, surface area 300 m²/g) were a gift from Dr. Vicki Stone, Napier University, Edinburgh, Scotland. Lympholyte-M, a Ficoll suspension used for isolating viable mouse cells by density gradient centrifugation was obtained from Accurate Chemical and Scientific (Westbury, NY, USA).

In Vitro Exposure of Cells to DEP and Ficoll Density Gradient Separation

A DEP/CB stock suspension (5 mg/mL) was prepared in sterile normal saline and sonicated for 1 min using a probe sonicator (Microson Ultrasonic Cell Disrupter; Misonix, Inc., Farmingdale, NY, USA) at maximum amplitude. Cells were seeded in culture at a cell density of 2 × 10⁴/mL in 6-well culture plates (5 mL cell suspension/well), and after 72 h, the sonicated DEP/CB suspension was added at desired concentrations. Control and DEP/CB-treated cells were harvested at the desired time intervals by trypsinization [trypsin-EDTA 1× from Gibco (Invitrogen, Carlsbad, CA, USA); 2 min] and collected by centrifugation at 150× g for 10 min. Cell pellets were resuspended in 5 mL RPMI medium containing 1% fetal calf serum (FCS), layered on top of 2 mL lympholyte-M, and centrifuged at room temperature at 280× g for 20 min. Cells with ingested DEP formed a black band at the Ficoll-RPMI interface, whereas free DEP or CB and any dead cells settled at the bottom of the Ficoll layer. Cells harvested from the interface were washed twice with RPMI medium containing 1% (v/v) FCS and counted in a hemocytometer. Viability of the cells at this stage was >95%.