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Subtoxic Doses of Cadmium Modulate Inflammatory Properties of Murine RAW 264.7 Macrophages.

Riemschneider S, Herzberg M, Lehmann J - Biomed Res Int (2015)

Bottom Line: Cadmium alone caused a dose-dependent decreased viability of exposed cells.Also production of antibacterial NO was significantly reduced through exposure to 10 µM Cd, maybe explaining better survival of macrophages.Additionally, we could show by analysis via ICP-MS that different effects of Cd in nonactivated and activated macrophages definitely did not result from different Cd uptake rates.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), 04103 Leipzig, Germany.

ABSTRACT
Cadmium (Cd) is a toxic heavy metal that exhibits various adverse effects in the human and animal organism. Its resemblance to essential metals such as calcium, iron, and zinc leads to an unintended uptake in cells after intake through inhalation and ingestion. In this study we investigated the toxicity and the immunomodulatory potential of Cd in nonactivated and activated murine macrophages (i.e., cell line RAW 264.7). Cadmium alone caused a dose-dependent decreased viability of exposed cells. Subtoxic Cd concentrations delayed cell death in macrophages, resulting from cytotoxic storm, producing reactive oxygen species (ROS) and nitric oxide (NO), in response to their stimulation by bacterial antigens via pattern-recognition receptors (PRRs). In addition, production of selected pro- and anti-inflammatory cytokines, the chemokine CXCL1 (KC), and NO was determined. We observed that proinflammatory IL-1β and also CXCL1 were highly upregulated whereas anti-inflammatory or regulatory cytokines IL-6 and IL-10 were suppressed by 10 µM Cd. Also production of antibacterial NO was significantly reduced through exposure to 10 µM Cd, maybe explaining better survival of macrophages. Additionally, we could show by analysis via ICP-MS that different effects of Cd in nonactivated and activated macrophages definitely did not result from different Cd uptake rates.

No MeSH data available.


Related in: MedlinePlus

Influence of Cd on the cell viability of RAW 264.7 macrophages. Cell viability was determined using the WST-1 assay (Roche Diagnostics). 2 × 105 cells/well were seeded in a 96-well microculture plate and subsequently stimulated with various Cd concentrations alone or in the presence of hk S.E. After 24 and 48 h WST-1 Reagent was added for 1 h followed by determination of the optical density in supernatants at 437 nm using a conventional microplate reader (Saphire2, Tecan). Data represent the mean values and are representative of three independent experiments (n = 2 per experiment). ∗ indicates significant (p < 0.05) differences compared to control.
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fig2: Influence of Cd on the cell viability of RAW 264.7 macrophages. Cell viability was determined using the WST-1 assay (Roche Diagnostics). 2 × 105 cells/well were seeded in a 96-well microculture plate and subsequently stimulated with various Cd concentrations alone or in the presence of hk S.E. After 24 and 48 h WST-1 Reagent was added for 1 h followed by determination of the optical density in supernatants at 437 nm using a conventional microplate reader (Saphire2, Tecan). Data represent the mean values and are representative of three independent experiments (n = 2 per experiment). ∗ indicates significant (p < 0.05) differences compared to control.

Mentions: In order to determine the subtoxic dose range of Cd, various Cd concentrations were studied using the impedance-based xCELLigence RTCA system (Figure 1(a)). The CI values show that incubation of RAW 264.7 macrophages with 100 μM Cd caused rapid cell death as early as 5 h of exposure, while 50 μM Cd impaired adherence of RAW 264.7 macrophages after 10 h and 20 μM Cd reduced cell viability after 60 h. However, concentrations between 0.1 μM and 10 μM Cd did not cause changes of the adherence behaviour compared to untreated controls up to 200 h. A simultaneous stimulation of macrophages with hk S.E., a potent trigger of several PRRs, delivered a very similar result for 100 μM and 50 μM Cd (Figure 1(b)). A complete loss of adherence became apparent after 25 h of exposure with 20 μM Cd. The cell viability of Cd-free control started to decrease after 32 h, whereas cell viability in cultures with 0.1 μM and 1 μM Cd showed a moderately prolonged survival since the decrease of CI signal started to decrease 2 h later compared to control. Additionally, 10 μM Cd delayed the loss of adherence by 6 h compared to the control. In parallel, cell viability was also determined using the WST-1 endpoint assay. The results show a decreased cell viability of macrophages following exposure to 100 μM and 50 μM Cd for 24 h and 48 h in nonactivated and activated RAW 264.7 macrophages (Figures 2(a) and 2(b)). Additional stimulation of macrophages with hk S.E. led to WST-1 reduction only after 48 h exposure with 10 μM Cd (Figure 2(b)).


Subtoxic Doses of Cadmium Modulate Inflammatory Properties of Murine RAW 264.7 Macrophages.

Riemschneider S, Herzberg M, Lehmann J - Biomed Res Int (2015)

Influence of Cd on the cell viability of RAW 264.7 macrophages. Cell viability was determined using the WST-1 assay (Roche Diagnostics). 2 × 105 cells/well were seeded in a 96-well microculture plate and subsequently stimulated with various Cd concentrations alone or in the presence of hk S.E. After 24 and 48 h WST-1 Reagent was added for 1 h followed by determination of the optical density in supernatants at 437 nm using a conventional microplate reader (Saphire2, Tecan). Data represent the mean values and are representative of three independent experiments (n = 2 per experiment). ∗ indicates significant (p < 0.05) differences compared to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4538338&req=5

fig2: Influence of Cd on the cell viability of RAW 264.7 macrophages. Cell viability was determined using the WST-1 assay (Roche Diagnostics). 2 × 105 cells/well were seeded in a 96-well microculture plate and subsequently stimulated with various Cd concentrations alone or in the presence of hk S.E. After 24 and 48 h WST-1 Reagent was added for 1 h followed by determination of the optical density in supernatants at 437 nm using a conventional microplate reader (Saphire2, Tecan). Data represent the mean values and are representative of three independent experiments (n = 2 per experiment). ∗ indicates significant (p < 0.05) differences compared to control.
Mentions: In order to determine the subtoxic dose range of Cd, various Cd concentrations were studied using the impedance-based xCELLigence RTCA system (Figure 1(a)). The CI values show that incubation of RAW 264.7 macrophages with 100 μM Cd caused rapid cell death as early as 5 h of exposure, while 50 μM Cd impaired adherence of RAW 264.7 macrophages after 10 h and 20 μM Cd reduced cell viability after 60 h. However, concentrations between 0.1 μM and 10 μM Cd did not cause changes of the adherence behaviour compared to untreated controls up to 200 h. A simultaneous stimulation of macrophages with hk S.E., a potent trigger of several PRRs, delivered a very similar result for 100 μM and 50 μM Cd (Figure 1(b)). A complete loss of adherence became apparent after 25 h of exposure with 20 μM Cd. The cell viability of Cd-free control started to decrease after 32 h, whereas cell viability in cultures with 0.1 μM and 1 μM Cd showed a moderately prolonged survival since the decrease of CI signal started to decrease 2 h later compared to control. Additionally, 10 μM Cd delayed the loss of adherence by 6 h compared to the control. In parallel, cell viability was also determined using the WST-1 endpoint assay. The results show a decreased cell viability of macrophages following exposure to 100 μM and 50 μM Cd for 24 h and 48 h in nonactivated and activated RAW 264.7 macrophages (Figures 2(a) and 2(b)). Additional stimulation of macrophages with hk S.E. led to WST-1 reduction only after 48 h exposure with 10 μM Cd (Figure 2(b)).

Bottom Line: Cadmium alone caused a dose-dependent decreased viability of exposed cells.Also production of antibacterial NO was significantly reduced through exposure to 10 µM Cd, maybe explaining better survival of macrophages.Additionally, we could show by analysis via ICP-MS that different effects of Cd in nonactivated and activated macrophages definitely did not result from different Cd uptake rates.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Cell Therapy and Immunology (IZI), 04103 Leipzig, Germany.

ABSTRACT
Cadmium (Cd) is a toxic heavy metal that exhibits various adverse effects in the human and animal organism. Its resemblance to essential metals such as calcium, iron, and zinc leads to an unintended uptake in cells after intake through inhalation and ingestion. In this study we investigated the toxicity and the immunomodulatory potential of Cd in nonactivated and activated murine macrophages (i.e., cell line RAW 264.7). Cadmium alone caused a dose-dependent decreased viability of exposed cells. Subtoxic Cd concentrations delayed cell death in macrophages, resulting from cytotoxic storm, producing reactive oxygen species (ROS) and nitric oxide (NO), in response to their stimulation by bacterial antigens via pattern-recognition receptors (PRRs). In addition, production of selected pro- and anti-inflammatory cytokines, the chemokine CXCL1 (KC), and NO was determined. We observed that proinflammatory IL-1β and also CXCL1 were highly upregulated whereas anti-inflammatory or regulatory cytokines IL-6 and IL-10 were suppressed by 10 µM Cd. Also production of antibacterial NO was significantly reduced through exposure to 10 µM Cd, maybe explaining better survival of macrophages. Additionally, we could show by analysis via ICP-MS that different effects of Cd in nonactivated and activated macrophages definitely did not result from different Cd uptake rates.

No MeSH data available.


Related in: MedlinePlus