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Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects.

Oostingh GJ, Casals E, Italiani P, Colognato R, Stritzinger R, Ponti J, Pfaller T, Kohl Y, Ooms D, Favilli F, Leppens H, Lucchesi D, Rossi F, Nelissen I, Thielecke H, Puntes VF, Duschl A, Boraschi D - Part Fibre Toxicol (2011)

Bottom Line: To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses.In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses.The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria. geja.oostingh@sbg.ac.at

ABSTRACT

Background: With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated.

Results: In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin.

Conclusion: The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

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Related in: MedlinePlus

NP-induced IL-8 promoter activation in A549 cells. Four different NP powders were used to test their immunomodulatory effect on A549 cells, measured by the induction of the IL-8 promoter in transfected cells. Unstimulated cells (diamond shaped symbols) or 20 ng/ml TNF-α stimulated cells (square symbols) were exposed to increasing concentrations of NPs for 48 h. Mean values ± SD (n = 3) are shown. The dotted lines represent the values in control cells treated with PBS (100%). The shaded areas represent the SD above and below the control values. Untreated cells gave a luminescence value of 2,799 ± 367 RLU, while TNF-α-stimulated cells had a value of 100,366 ± 3,697 RLU. Data were normalised to the PBS control (9.1% v/v PBS; taken as 100% value) to allow a direct comparison between stimulated and unstimulated cells and to enable combining data from multiple experiments. * p < 0.05 vs. control
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Figure 7: NP-induced IL-8 promoter activation in A549 cells. Four different NP powders were used to test their immunomodulatory effect on A549 cells, measured by the induction of the IL-8 promoter in transfected cells. Unstimulated cells (diamond shaped symbols) or 20 ng/ml TNF-α stimulated cells (square symbols) were exposed to increasing concentrations of NPs for 48 h. Mean values ± SD (n = 3) are shown. The dotted lines represent the values in control cells treated with PBS (100%). The shaded areas represent the SD above and below the control values. Untreated cells gave a luminescence value of 2,799 ± 367 RLU, while TNF-α-stimulated cells had a value of 100,366 ± 3,697 RLU. Data were normalised to the PBS control (9.1% v/v PBS; taken as 100% value) to allow a direct comparison between stimulated and unstimulated cells and to enable combining data from multiple experiments. * p < 0.05 vs. control

Mentions: The A549 reporter cell lines containing the IL-6, IL-8 or TNF-α promoter sequence or 3 copies of the NF-κB binding sequence were used to analyse the inflammation-related effects of NPs. The effect of the wet particles on these reporter cell lines was low or absent as described in a previous publication [35]. The reporter cell lines proved useful in testing the immunotoxic and immunomodulatory effects of dry NPs, since aggregation and agglomeration of these particles did not influence the assay (the analysis is performed on cell lysates, and NPs can be eliminated by washing cells before lysis and centrifuging the lysates before analysis). The effects of four different dry NP preparations (Au, CoO, FexOy and CeO2) on the IL-8 promoter transfected A549 cell line are shown in Figure 7. Cells were either unstimulated or exposed to recombinant human TNF-α. CoO particles were found to decrease the IL-8 cytokine promoter induction, this reduction being most likely due to the decreased cell viability found when cells were incubated with CoO particles (data not shown). The other dry NPs tested did not affect the cell viability (Au, FexOy and CeO2, data not shown). Moreover, Au and FexOy particles did not have a major effect on the cytokine promoter induction. However, the CeO2 particles did significantly induce the IL-8 promoter. In unstimulated cells the induction was by 68%, while in cells activated with TNF-α a smaller increase was observed (31%). This is possibly due to the fact that TNF-α already induced a near-maximal activation (35-fold increase in the IL-8 promoter induction), thus largely masking the effect of the CeO2 NPs.


Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects.

Oostingh GJ, Casals E, Italiani P, Colognato R, Stritzinger R, Ponti J, Pfaller T, Kohl Y, Ooms D, Favilli F, Leppens H, Lucchesi D, Rossi F, Nelissen I, Thielecke H, Puntes VF, Duschl A, Boraschi D - Part Fibre Toxicol (2011)

NP-induced IL-8 promoter activation in A549 cells. Four different NP powders were used to test their immunomodulatory effect on A549 cells, measured by the induction of the IL-8 promoter in transfected cells. Unstimulated cells (diamond shaped symbols) or 20 ng/ml TNF-α stimulated cells (square symbols) were exposed to increasing concentrations of NPs for 48 h. Mean values ± SD (n = 3) are shown. The dotted lines represent the values in control cells treated with PBS (100%). The shaded areas represent the SD above and below the control values. Untreated cells gave a luminescence value of 2,799 ± 367 RLU, while TNF-α-stimulated cells had a value of 100,366 ± 3,697 RLU. Data were normalised to the PBS control (9.1% v/v PBS; taken as 100% value) to allow a direct comparison between stimulated and unstimulated cells and to enable combining data from multiple experiments. * p < 0.05 vs. control
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045340&req=5

Figure 7: NP-induced IL-8 promoter activation in A549 cells. Four different NP powders were used to test their immunomodulatory effect on A549 cells, measured by the induction of the IL-8 promoter in transfected cells. Unstimulated cells (diamond shaped symbols) or 20 ng/ml TNF-α stimulated cells (square symbols) were exposed to increasing concentrations of NPs for 48 h. Mean values ± SD (n = 3) are shown. The dotted lines represent the values in control cells treated with PBS (100%). The shaded areas represent the SD above and below the control values. Untreated cells gave a luminescence value of 2,799 ± 367 RLU, while TNF-α-stimulated cells had a value of 100,366 ± 3,697 RLU. Data were normalised to the PBS control (9.1% v/v PBS; taken as 100% value) to allow a direct comparison between stimulated and unstimulated cells and to enable combining data from multiple experiments. * p < 0.05 vs. control
Mentions: The A549 reporter cell lines containing the IL-6, IL-8 or TNF-α promoter sequence or 3 copies of the NF-κB binding sequence were used to analyse the inflammation-related effects of NPs. The effect of the wet particles on these reporter cell lines was low or absent as described in a previous publication [35]. The reporter cell lines proved useful in testing the immunotoxic and immunomodulatory effects of dry NPs, since aggregation and agglomeration of these particles did not influence the assay (the analysis is performed on cell lysates, and NPs can be eliminated by washing cells before lysis and centrifuging the lysates before analysis). The effects of four different dry NP preparations (Au, CoO, FexOy and CeO2) on the IL-8 promoter transfected A549 cell line are shown in Figure 7. Cells were either unstimulated or exposed to recombinant human TNF-α. CoO particles were found to decrease the IL-8 cytokine promoter induction, this reduction being most likely due to the decreased cell viability found when cells were incubated with CoO particles (data not shown). The other dry NPs tested did not affect the cell viability (Au, FexOy and CeO2, data not shown). Moreover, Au and FexOy particles did not have a major effect on the cytokine promoter induction. However, the CeO2 particles did significantly induce the IL-8 promoter. In unstimulated cells the induction was by 68%, while in cells activated with TNF-α a smaller increase was observed (31%). This is possibly due to the fact that TNF-α already induced a near-maximal activation (35-fold increase in the IL-8 promoter induction), thus largely masking the effect of the CeO2 NPs.

Bottom Line: To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses.In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses.The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria. geja.oostingh@sbg.ac.at

ABSTRACT

Background: With the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated.

Results: In a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin.

Conclusion: The problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

Show MeSH
Related in: MedlinePlus