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Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC - Part Fibre Toxicol (2011)

Bottom Line: Attention should primarily be focussed on SNP effects on biological barriers.This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation.Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Medical Centre, Institute of Pathology, Mainz, Germany.

ABSTRACT

Background: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.

Methods: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.

Results: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.

Conclusion: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

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The release of inflammatory mediators (sICAM-1, IL-6 and IL-8) is shown after aSNP (Ludox TM-40: light grey, NexSil20: dark grey) incubation on apical/basolateral differentiated cocultures of H441 and ISO-HAS-1, as well as on their appropriate monocultures grown on HTS 24-Transwell® filters. Cells were exposed to aSNPs from the apical side of the filter membrane to mimic an inhalative exposure to aSNPs. After 4 h serum-free incubation, aSNPs were removed and the cells were cultivated for further 20 h. Subsequently, medium supernatant of both compartments (apical: upper well, basolateral: lower well) was examined. Data are depicted as means ± S.D. of one representative (of three independent) experiments with n = 3 samples for each treatment. All independent experiments showed a comparable reaction following aSNP treatment. Exclusively in coculture, an apical exposure of 6 and 60 μg/ml aSNP caused an increased IL-6 and IL-8 release into the lower well (basolateral/endothelial side). For both monocultures increased amounts of sICAM-1, IL-6 and IL-8 in the lower well were not detected below concentrations of 600 μg/ml aSNP. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control
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Figure 4: The release of inflammatory mediators (sICAM-1, IL-6 and IL-8) is shown after aSNP (Ludox TM-40: light grey, NexSil20: dark grey) incubation on apical/basolateral differentiated cocultures of H441 and ISO-HAS-1, as well as on their appropriate monocultures grown on HTS 24-Transwell® filters. Cells were exposed to aSNPs from the apical side of the filter membrane to mimic an inhalative exposure to aSNPs. After 4 h serum-free incubation, aSNPs were removed and the cells were cultivated for further 20 h. Subsequently, medium supernatant of both compartments (apical: upper well, basolateral: lower well) was examined. Data are depicted as means ± S.D. of one representative (of three independent) experiments with n = 3 samples for each treatment. All independent experiments showed a comparable reaction following aSNP treatment. Exclusively in coculture, an apical exposure of 6 and 60 μg/ml aSNP caused an increased IL-6 and IL-8 release into the lower well (basolateral/endothelial side). For both monocultures increased amounts of sICAM-1, IL-6 and IL-8 in the lower well were not detected below concentrations of 600 μg/ml aSNP. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control

Mentions: Due to the different compartments that are formed by growing barrier-forming cells on transwells not only an apical/basolateral differentiated coculture but also polarized monocultures were grown on transwells (see Figure 4). These transwell-monocultures of H441 and ISO-HAS-1 with the same culture conditions and seeding surface serve as a control for the effects of an upper well aSNP exposure on the individual cell types. The analysis of sICAM-1 of the untreated controls of the three approaches (H441/ISO-HAS-1 (upper/lower surface), H441 (upper surface) and ISO-HAS-1 (lower surface) yielded significantly different basic levels of sICAM-1. The transwell-monoculture of H441 released in total 19fold more sICAM-1 compared to the coculture in total. The sICAM-1 response of the cocultures and transwell-monocultures of H441 to aSNPs with a value of 6 μg/ml was most intense (30fold in total) and decreased with increasing aSNP concentration. At concentrations from 6-600 μg/ml aSNPs there was no significant release of sICAM-1 into the lower compartment. With 6000 μg/ml aSNP sICAM-1 increased significantly in the lower well for the cocultures and the transwell-monocultures of H441. sICAM-1 response of the cocultures behaved in similar fashion to the H441 monoculture. Monocultures of ISO-HAS-1 growing at the lower surface of the transwell filter showed no sICAM release upon upper well exposure.


Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: comparison with conventional monocultures.

Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick JC - Part Fibre Toxicol (2011)

The release of inflammatory mediators (sICAM-1, IL-6 and IL-8) is shown after aSNP (Ludox TM-40: light grey, NexSil20: dark grey) incubation on apical/basolateral differentiated cocultures of H441 and ISO-HAS-1, as well as on their appropriate monocultures grown on HTS 24-Transwell® filters. Cells were exposed to aSNPs from the apical side of the filter membrane to mimic an inhalative exposure to aSNPs. After 4 h serum-free incubation, aSNPs were removed and the cells were cultivated for further 20 h. Subsequently, medium supernatant of both compartments (apical: upper well, basolateral: lower well) was examined. Data are depicted as means ± S.D. of one representative (of three independent) experiments with n = 3 samples for each treatment. All independent experiments showed a comparable reaction following aSNP treatment. Exclusively in coculture, an apical exposure of 6 and 60 μg/ml aSNP caused an increased IL-6 and IL-8 release into the lower well (basolateral/endothelial side). For both monocultures increased amounts of sICAM-1, IL-6 and IL-8 in the lower well were not detected below concentrations of 600 μg/ml aSNP. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040689&req=5

Figure 4: The release of inflammatory mediators (sICAM-1, IL-6 and IL-8) is shown after aSNP (Ludox TM-40: light grey, NexSil20: dark grey) incubation on apical/basolateral differentiated cocultures of H441 and ISO-HAS-1, as well as on their appropriate monocultures grown on HTS 24-Transwell® filters. Cells were exposed to aSNPs from the apical side of the filter membrane to mimic an inhalative exposure to aSNPs. After 4 h serum-free incubation, aSNPs were removed and the cells were cultivated for further 20 h. Subsequently, medium supernatant of both compartments (apical: upper well, basolateral: lower well) was examined. Data are depicted as means ± S.D. of one representative (of three independent) experiments with n = 3 samples for each treatment. All independent experiments showed a comparable reaction following aSNP treatment. Exclusively in coculture, an apical exposure of 6 and 60 μg/ml aSNP caused an increased IL-6 and IL-8 release into the lower well (basolateral/endothelial side). For both monocultures increased amounts of sICAM-1, IL-6 and IL-8 in the lower well were not detected below concentrations of 600 μg/ml aSNP. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared to the untreated control
Mentions: Due to the different compartments that are formed by growing barrier-forming cells on transwells not only an apical/basolateral differentiated coculture but also polarized monocultures were grown on transwells (see Figure 4). These transwell-monocultures of H441 and ISO-HAS-1 with the same culture conditions and seeding surface serve as a control for the effects of an upper well aSNP exposure on the individual cell types. The analysis of sICAM-1 of the untreated controls of the three approaches (H441/ISO-HAS-1 (upper/lower surface), H441 (upper surface) and ISO-HAS-1 (lower surface) yielded significantly different basic levels of sICAM-1. The transwell-monoculture of H441 released in total 19fold more sICAM-1 compared to the coculture in total. The sICAM-1 response of the cocultures and transwell-monocultures of H441 to aSNPs with a value of 6 μg/ml was most intense (30fold in total) and decreased with increasing aSNP concentration. At concentrations from 6-600 μg/ml aSNPs there was no significant release of sICAM-1 into the lower compartment. With 6000 μg/ml aSNP sICAM-1 increased significantly in the lower well for the cocultures and the transwell-monocultures of H441. sICAM-1 response of the cocultures behaved in similar fashion to the H441 monoculture. Monocultures of ISO-HAS-1 growing at the lower surface of the transwell filter showed no sICAM release upon upper well exposure.

Bottom Line: Attention should primarily be focussed on SNP effects on biological barriers.This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation.Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: University Medical Centre, Institute of Pathology, Mainz, Germany.

ABSTRACT

Background: To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.

Methods: Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.

Results: Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.

Conclusion: We present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

Show MeSH
Related in: MedlinePlus