PMC

Interaction of volcanic ash with the triple cell co-culture. Scanning electron micrographs of the triple cell co-culture membrane exposed to 0.26 ± 0.09 μg/cm² of single exposure to volcanic ash (SEVA), showing direct interaction of VA particles with the different cell types. a and b (inset of (a)) show a representative interaction of the human blood monocyte-derived macrophages (MDM) with VA, which appears to be engulfed by the MDM (b). Images (c) and d (inset of (c)) show a representative interaction of the human monocyte-derived dendritic cells (MDDC) with the VA particles, which are interacting with the pseudopodia of the MDDC (d; indicated with white arrows). Images were collected at 3 kV and 10 mm working distance. Scale bars are 20 μm (a), 2 μm (b), 50 μm (c) and 20 μm (d)

Biochemical response of triple cell co-culture system following exposures to volcanic ash and diesel exhaust particles. a Total reduced glutathione (GSH), b tumour necrosis factor-α (TNF-α) release, c interleukin-8 (IL-8) release and d interleukin-1β (IL-1β) release of the triple cell co-culture model after exposure to 0.26 ± 0.09 μg/cm² of single exposure to volcanic ash (SEVA), 0.89 ± 0.29 μg/cm² of repeated exposure to volcanic ash (REVA), diesel exhaust particles (DEP; 0.02 mg/mL), co-exposure to diesel exhaust particles and 0.26 ± 0.09 μg/cm² of single exposure to volcanic ash (DEP + SEVA), and co-exposure to diesel exhaust particles and 0.89 ± 0.29 μg/cm² of repeated exposure to volcanic ash (DEP + REVA). The respective positive assay controls are tert-Butyl Hydrogen Peroxide (tBHP; 250 μL of 100 mM) and lipopolysaccharide (LPS; 100 μL of 1 μg/mL), added to the apical and basal compartment of the triple cell co-culture, respectively. The negative control was cell culture medium only. Data are presented as the mean ± standard error of the mean. Data shown are related to the following repetitions for each exposure: SEVA n = 4; REVA, DEP, DEP + SEVA and DEP + REVA n = 3; negative and positive controls n = 8. * indicates p < 0.05

Deposition of nebulized respirable fraction of volcanic ash. a Average mass deposition (μg/cm²) of volcanic ash (VA) quantified using a quartz crystal microbalance (QCM), following nebulisation of dry respirable ash (MVO12/7/03) using a dry powder insufflator (DP-4, Penn Century, USA) under the following conditions: single exposure (SEVA) with 4 mg (n = 14), 6 mg (n = 14) or 8 mg (n = 17), as well as repeated exposure (REVA) to 8 mg (nebulised 3× within 15 min; n = 9). Data are presented as the mean ± standard error of the mean. Scanning electron micrographs of nebulized, uncoated ash sample (REVA), show b heterogeneous particle dispersion (WD: 5.53 mm, MAG: 97×) and c an inset of image (b) (WD: 7 mm, MAG: 3.80 k ×). Images were collected at 10 kV. Scale bars are 1 mm (b) and 20 μm (c)

Cell morphology and cytotoxicity of triple cell co-cultures exposed to volcanic ash and diesel exhaust particles. Confocal laser scanning microscopy (LSM) images show the complete triple cell co-culture (i.e. A549 type-II ‘like’ epithelial cell monolayer with human blood monocyte macrophages (MDM) and dendritic cells (MDDC) on the apical and basal sides, respectively) stained for F-actin cytoskeleton (red) and the nuclei (blue). a Control and cultures exposed to b 0.26 ± 0.09 μg/cm² of single exposure to volcanic ash (SEVA), c 0.89 ± 0.29 μg/cm², repeated exposure to volcanic ash (REVA), d diesel exhaust particles (DEP; 0.02 mg/mL), e diesel exhaust particles and 0.26 ± 0.09 μg/cm² of single exposure to volcanic ash (DEP + SEVA), and f diesel exhaust particles and 0.89 ± 0.29 μg/cm² of repeated exposure to volcanic ash (DEP + REVA). Yellow arrows indicate cells undergoing cell division. Scale bars are 20 μm (a-b) and 15 μm (c-f). Images were collected at magnification 63×. g Cytotoxicity determined by the release of lactate dehydrogenase (LDH) from the triple cell co-culture following exposure to SEVA, REVA, DEP, DEP + SEVA and DEP + REVA. Data are presented as fold increase relative to the negative control (supplemented cell culture medium only) ± standard error of the mean. Triton X-100 at 0.2% in phosphate buffered saline (PBS) acted as the positive assay control. LDH data shown are related to the following repetitions for each exposure: SEVA n = 4; REVA, DEP, DEP + SEVA and DEP + REVA n = 3; negative and positive controls n = 8