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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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After aSNP exposure layer integrity of H441 and ISO-HAS-1 in coculture was determined by immunofluorescent localization of junction-associated proteins. The H441 cells of the coculture were incubated with aSNP (NexSil20: concentration range 0.6 - 6000 μg/ml, c: untreated control) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The H441 were labelled for E-cadherin, the ISO-HAS-1 were counterstained for PECAM-1.
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Figure 6: After aSNP exposure layer integrity of H441 and ISO-HAS-1 in coculture was determined by immunofluorescent localization of junction-associated proteins. The H441 cells of the coculture were incubated with aSNP (NexSil20: concentration range 0.6 - 6000 μg/ml, c: untreated control) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The H441 were labelled for E-cadherin, the ISO-HAS-1 were counterstained for PECAM-1.

Mentions: In conventional monoculture morphological alterations were observed at an aSNP concentration of 600 μg/ml for both cell lines H441 and ISO-HAS-1 (see additional file 2 and 3: Figure S2 and S3). 60 μg/ml did not show any changes as a result of the aSNP treatment. Surprisingly, up to a concentration of 600 μg/ml aSNP the E-Cadherin staining pattern of the H441 in coculture showed no difference compared to the untreated control (Figure 6), even though the TER value decreased significantly at this dose (Figure 5). A concentration of 6000 μg/ml aSNPs, a factor 10 higher than in monoculture, caused visible changes in the assembly of cell-cell junctions and first signs of cell detachment (Figure 6). These findings explain the complete breakdown of TER resulting in apically released cytokines passing through the barrier and being basolaterally detectable. The indirectly exposed ISO-HAS-1 (PECAM-1 counterstaining) did not show any morphological alterations even at a concentration of 6000 μg/ml.


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)

After aSNP exposure layer integrity of H441 and ISO-HAS-1 in coculture was determined by immunofluorescent localization of junction-associated proteins. The H441 cells of the coculture were incubated with aSNP (NexSil20: concentration range 0.6 - 6000 μg/ml, c: untreated control) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The H441 were labelled for E-cadherin, the ISO-HAS-1 were counterstained for PECAM-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: After aSNP exposure layer integrity of H441 and ISO-HAS-1 in coculture was determined by immunofluorescent localization of junction-associated proteins. The H441 cells of the coculture were incubated with aSNP (NexSil20: concentration range 0.6 - 6000 μg/ml, c: untreated control) for 4 h in serum-free medium. aSNPs were then removed and cells were cultivated for further 20 h. The H441 were labelled for E-cadherin, the ISO-HAS-1 were counterstained for PECAM-1.
Mentions: In conventional monoculture morphological alterations were observed at an aSNP concentration of 600 μg/ml for both cell lines H441 and ISO-HAS-1 (see additional file 2 and 3: Figure S2 and S3). 60 μg/ml did not show any changes as a result of the aSNP treatment. Surprisingly, up to a concentration of 600 μg/ml aSNP the E-Cadherin staining pattern of the H441 in coculture showed no difference compared to the untreated control (Figure 6), even though the TER value decreased significantly at this dose (Figure 5). A concentration of 6000 μg/ml aSNPs, a factor 10 higher than in monoculture, caused visible changes in the assembly of cell-cell junctions and first signs of cell detachment (Figure 6). These findings explain the complete breakdown of TER resulting in apically released cytokines passing through the barrier and being basolaterally detectable. The indirectly exposed ISO-HAS-1 (PECAM-1 counterstaining) did not show any morphological alterations even at a concentration of 6000 μg/ml.

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