Abstract
Purpose::
To investigate whether urban particulate matter of ≤ 2.5µm (PM2.5) induce on proliferation, citotoxicity, nitric oxide, NF-kB, inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) signaling pathways, in a normal human conjunctival epithelial cell line (IOBA-NHC).
Methods::
PM2.5 samples were collected in polycarbonate filters, outdoors, at an intersection with heavy traffic in downtown São Paulo. Particles were extracted from the filters using sonication in distilled water. Suspensions with concentrations of 10, 25, 50 and 100 µg/ml of PM2.5 were prepared. Cells of a normal human conjunctival cell line (IOBA-NHC) were grown on: a) multiwell plates and b) cover slips for 3 days, washed with PBS and switched to a serum-free medium for 24 hours before treatment. Cells were cultured for an additional 1, 2, 4 and 24 hours with 200 µl of 0,10, 25,50, 100 µg/ml of PM2.5 suspension, after that the supernatants were discarded and the cells were washed with PBS and switched to a serum-free medium for 24 hours.. The supernatants were then collected and stored at -80°C. In vitro cell viability, NF-kB p65 nuclear translocation and phosphorylated MAPKs (JNK1/2, ERK1/2) was evaluated by using MTS, immunohistochemistry and western blot respectively. IL-6 and Nitric oxide (NO) were evaluated by using ELISA and Griess reagent respectively.
Results::
Proliferation of IOBA-NHC cells was observed after incubation with PM2.5: at 2 hours , taking 100% as control, 172, 159, 148 and 139 for concentrations of 10, 25, 50 and 100 µg/ml; and at 4 hours, 112, 112, 102, 87 for concentrations of 10, 25, 50 and 100 µg/ml respectively. Citoxicity (87) was observed after 4 hours of incubation with 100 µg/ml. This cell line presented NF-kB nuclear translocation with 1 and 2 hours of incubation at concentrations of 50 and 100 µg/ml. With 2 hours of incubation at concentrations of 25, 50 and 100 µg/ml MAPKs (JNK 1/2, ERK 1/2) activation was induced. At 24 hours of incubation at 50 and 100 µg/ml IL-6 production was induced.
Conclusions::
These data suggest that PM2.5 induces cell proliferation and nuclear NF-kB translocation in human conjunctival epithelial cells with production of pro-inflammatory cytokines. Lower concentrations and shorter time of exposure induced greater proliferation rate in this cell line.
Keywords: conjunctiva • inflammation • cytokines/chemokines