Particulate matter (PM) exposure is related to pulmonary and cardiovascular diseases, with increased inflammatory status. The release of the proinflammatory interleukin- (IL-) 1
In the last decade great effort has been paid to understand the mechanisms involved in particulate matter (PM) induced adverse health effects. Epidemiological evidence shows an association between exposure to air pollution and the occurrence of respiratory pathologies (chronic bronchitis, COPD) and exacerbation of allergic conditions such as asthma [
PM is a heterogeneous pollutant composed of particles of different chemical composition and different sizes (defined as PM10, PM2.5, and PM0.1 for their aerodynamic diameter). Although the size determines the site of deposition of PM in the respiratory tract [
The fine fraction (PM2.5) is generally composed of primary particles derived from combustion processes, mostly consisting of primary particles with mean diameter lower than 100 nm (PM0.1, ultrafine particles) and secondary aerosol deriving from chemical reaction of free compounds in the atmosphere. The particle composition reflects the sources of emission; indeed fine PM has usually higher content in organic compounds (such as PAHs) and elemental carbon (the soot core of the particles) than the coarse PM.
The coarse fraction (PM10–2.5) is on the contrary dominated by particles derived from abrasion processes, such as the erosion of crustal material, resuspension of deposited particles, and biological components. We have previously shown that the season of PM sampling strongly influences the chemical and biological composition of both coarse and fine PMs [
Moreover, the chemical characterization showed that the PM10 contained crystal silica and other elements which can contribute to its inflammatory potential.
A lot of studies have shown that PM10 exposure promotes inflammation in the lung which is associated with a systemic inflammatory response. Macrophages and lung epithelial cells incubated with PM10 release significantly increased amounts of cytokines and chemokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin IL-1
A critical property of the innate immune system is its ability to discriminate microbes from “self” by the recognition of invariant microbial structure called pathogen-associated recognition patterns (PAMPs) such as lipopolysaccharides (LPS) [
IL-1
The activation of the inflammasome machinery has been related to different mechanisms which have been reviewed in [
It has been shown that particles occurring in ambient PM, such as crystalline silica, as well as different nanoparticles, may induce inflammasome activation [
In the present study it was hypothesized that PM10 due to its chemical and physical nature might induce IL-1
The human monocytes cell line, THP-1, was maintained in Opti-MEM medium supplemented with 10% FBS and 100 U/100 mL Penicillin/Streptomycin at 37°C, 5% CO2. THP-1 cells were differentiated into macrophage-like cells by incubation with phorbol myristate acetate (PMA, 20 nM) (Sigma Aldrich) for 24 h. PMA was then removed and cells were washed and incubated in Opti-MEM (Invitrogen, Italy) medium supplemented with 20% FBS o/n. Cells were treated in 10% FBS medium with summer PM10 at different concentrations (1
Supernatants from control and PM10-exposed cells were collected and stored at −80°C. Supernatants were assayed for IL-1
After 30 min exposure to summer PM10, cells were washed in phosphate-buffered saline 1X (PBS), fixed in paraformaldehyde 4% for 20 min, and washed twice in PBS. Fixed cells were permeabilized with 0.1% Triton X-100 (Sigma Aldrich), 0.1% Tween (Sigma Aldrich), and 2% BSA (Sigma Aldrich) in PBS and incubated o/n with the rabbit anti-human early endosome antibody 1 (EEA1 Antibody, Cell Signaling Technology; dilution 1 : 100). Cells were then washed in PBS and incubated with Alexa fluor-488 (Invitrogen Molecular Probes Srl; dilution 1 : 1000) for 2 h. Samples were mounted on a slide with ProLong mount (Invitrogen Srl) and observed by Axio Observer inverted microscope (Zeiss, Germany).
After exposure to summer PM10 cells were washed in PBS and stored at −80°C. Cells were then lysed in RIPA buffer (50 mM Tris-HCl pH 8; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; Sigma Ladrich Italy) and then sonicated three times for 30 sec on ice. Cell lysates were then separated by 8% SDS-PAGE and transferred on nitrocellulose membranes. Blots were incubated with rabbit polyclonal antibody against human EEA1 (Cell Signaling Technology; dilution 1 : 1000) o/n or anti-actin antibody (Sigma Aldrich, Italy; dilution 1 : 2000). After washes, the membranes were incubated with secondary antibody anti-rabbit IgG (Fab2 fragment-Alkaline Phosphatase, Sigma Aldrich; dilution 1 : 10000) and subsequently incubated with SIGMA FAST BCIP/NBT alkaline phosphatase substrate (Sigma Aldrich) for 10 min for detection. Fold increase data over control, obtained by acquisition of membrane and densitometry analysis with dedicated software (UVP, US), were normalized to the actin content.
THP-1-derived macro-phage untreated and treated with summer PM10 for 24 h at the concentration of 2.5
Samples were prepared for transmission electron microscopy (TEM) using standard procedures. At the end of exposure the cells were fixed in 2.5% glutaraldehyde for 20 min at 4°C and postfixed with 1% osmium tetroxide for 1 h, followed by dehydration using a scale of graded ethanol. Cells were then embedded in Epon resin, and semithin and ultrathin sections were prepared by an ultramicrotome (Ultracut Jung E, Reichert Germany). Ultrathin slides were mounted on copper grids and counterstained by lead citrate and uranyl acetate prior to examination by Jeol JEM 1220 microscope operating at 80 kV and digital images were taken with a Gatan CCD camera.
Results are reported as mean ± standard deviation of at least three independent experiments. Statistical differences were analysed by the software SigmaStat 3.1 performing ANOVA test with post hoc analysis (Dunn’s); if required a parametric statistical analysis was performed.
THP-1-derived macrophages were treated as reported with summer Milan PM10 and with winter PM10 and CB. The experiments showed that IL-1
IL-1
Time-dependent release of IL-1
THP-1-derived macrophages were pre-treated for 1 h with the caspase-1 inhibitor z-YVAD (10
PM10-induced IL-1
Subsequently THP-1-derived macrophages were pretreated with TLR-2 and TLR-4 inhibitors (0.1
Inhibition of PM10-induced IL-1
Treatment with NAC, an inhibitor of oxidative stress, reduced the release of IL-1
Inhibition of PM10-induced IL-1
We focused our study also on the molecular mechanisms involved in summer Milan PM10-induced IL-1
The activity of early endosomes after the exposure to summer PM10 was examined by analysing the expression of the early endosome antigen 1 (EEA1) in THP-1-derived macrophages. The cells were exposed to summer PM10 for 30 min and then assessed by EEA1 immunostaining. We observed that EEA1 expression after 30 min was remarkably increased compared to the control (Figure
PM10-induced overexpression of early endosomes in THP-1-derived macrophages. The cells were treated with summer Milan PM10 at the concentration of 2.5
Early endosome antigen 1 EEA1 protein expression in THP-1-derived macrophages exposed to summer Milan PM10. The cells were treated with summer PM10 (2.5
The interaction between summer Milan PM10 and THP-1-derived macrophages was determined by haematoxylin-eosin staining and electron transmission microscopy (TEM). Haematoxylin-eosin-stained macrophages exposed to summer PM10 (for 6 h) showed a high number of particles attached to the cells (Figures
PM10 interaction with THP-1-derived macrophages. The cells were exposed to summer PM10 at the concentration of 2.5
Recently PM10 was demonstrated to induce IL-1
Most of the in vitro studies of particle-induced IL-1
Also, in the study of Hirota [
Oxidative stress seems to be crucial for the PM10-induced IL-1
Since IL-1
The authors do not have any conflict of interests.
R. Bengalli, E. Molteni, and E. Longhin contributed equally to the in vitro experimental part. M. Gualtieri and R. Magne planned the in vitro experiments and contributed to the revision of the paper. M. Camatini and M. Gualtieri supervised the research activities during the Tosca Project.
M. Camatini and M. Gualtieri greatly thank Cariplo Foundation for financing the Tosca Project during which this research was performed.