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The immunomodulatory effects of albumin in vitro and in vivo - PubMed

The immunomodulatory effects of albumin in vitro and in vivo

Derek S Wheeler et al. Adv Pharmacol Sci. 2011.

Abstract

Albumin appears to have proinflammatory effects in vitro. We hypothesized that albumin would induce a state of tolerance to subsequent administration of lipopolysaccharide (LPS) in vitro and in vivo. RAW264.7 and primary peritoneal macrophages were treated with increasing doses of bovine serum albumin (BSA) and harvested for NF-κB luciferase reporter assay or TNF-α ELISA. In separate experiments, RAW264.7 cells were preconditioned with 1 mg/mL BSA for 18 h prior to LPS (10 μg/mL) treatment and harvested for NF-κB luciferase reporter assay or TNF-α ELISA. Finally, C57Bl/6 mice were preconditioned with albumin via intraperitoneal administration 18 h prior to a lethal dose of LPS (60 mg/kg body wt). Blood was collected at 6 h after LPS administration for TNF-α ELISA. Albumin produced a dose-dependent and TLR-4-dependent increase in NF-κB activation and TNF-α gene expression in vitro. Albumin preconditioning abrogated the LPS-mediated increase in NF-κB activation and TNF-α gene expression in vitro and in vivo. The clinical significance of these findings remains to be elucidated.

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Figures

Figure 1
Figure 1

Treatment with fraction V bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, Mo) dose-dependently increases TNF-α gene expression in murine peritoneal macrophages in an NF-κB-dependent manner. (a) RAW264.7 peritoneal macrophages were transiently transfected with a 3x NF-κB/luc reporter plasmid prior to treatment with increasing doses of BSA for 24 h (doses in mg/mL shown on the x-axis). BSA treatment resulted in increased NF-κB activation in a dose-dependent manner. As a comparison, LPS treatment (10 μg/mL) resulted in a 3.9-fold increase in relative luciferase activity (data not shown). All experiments were performed in triplicate with 3 wells per condition (*P < .05 compared to control). (b) RAW264.7 peritoneal macrophages were treated with increasing doses of BSA for 24 h (doses in mg/mL shown on the x-axis). BSA treatment resulted in a significant increase in TNF-α, as measured by ELISA. As a comparison, LPS treatment (10 μg/mL) resulted in a much greater increase in TNF-α compared to BSA (TNF-α 7000 pg/mL) (data not shown). All experiments were performed in triplicate with 3 wells per condition (*P < .05 compared to control).

Figure 2
Figure 2

BSA increases TNF-α gene expression in murine peritoneal macrophages in a TLR-4-dependent manner. Primary peritoneal macrophages were isolated from C3H/HeJ (TLR-4 mutant) and C3H/HeOuJ (wild-type) and were treated with BSA for 24 h. As expected, LPS treatment (10 μg/mL) resulted in a significant difference in TNF-α induction between macrophages isolated from C3H/HeJ mice and their wild-type counterparts. BSA treatment resulted in a dose-dependent increase in TNF-α in macrophages isolated from C3H/HeOuJ mice, but not in macrophages isolated from C3H/HeJ mice. All experiments were performed in triplicate with 3 wells per condition.

Figure 3
Figure 3

Albumin preconditioning abrogates LPS-mediated TNF-α gene expression in RAW264.7 macrophages. RAW264.7 peritoneal macrophages were preconditioned with BSA (1 mg/mL) for 18 h prior to a subsequent treatment with LPS (10 μg/mL). LPS treatment resulted in a significant increase in TNF-α expression, as measured by ELISA. Albumin preconditioning, however, significantly abrogated LPS-mediated TNF-α expression. All experiments were performed in triplicate with 3 wells per condition (*P < .05 compared to control; # P < .05 compared to LPS alone).

Figure 4
Figure 4

Albumin preconditioning attenuates LPS-mediated NF-κB activation. (a) NF-κB luciferase experiments. RAW264.7 peritoneal macrophages were transiently transfected with a 3x NF -κB/luc reporter plasmid. Cells were allowed to recover overnight and were then preconditioned with BSA (1 mg/mL) for 18 h prior to a subsequent treatment with LPS (10 μg/mL). LPS treatment resulted in a significant increase in NF-κB promoter activation, which was significantly inhibited by albumin preconditioning. All experiments were performed in triplicate with 3 wells per condition (*P < .05 compared to control; # P < .05 compared to LPS alone). (b) EMSA. RAW264.7 peritoneal macrophages were preconditioned with BSA (1 mg/mL) for 18 h prior to a subsequent treatment with LPS (10 μg/mL). Nuclear protein was harvested at 30 min after LPS and EMSA were performed. LPS treatment resulted in a significant increase in NF-κB binding (Lane 2) compared to either control (Lane 1) or albumin preconditioning alone (Lane 3). Albumin preconditioning abrogated NF-κB binding (Lane 4) compared to LPS alone. Lane 5 (LPS treatment, cold competitor), Lane 6 (LPS treatment, p65 supershift), Lane 7 (LPS treatment, p50 supershift), Lane 8 (Alb + LPS, cold competitor), Lane 9 (Alb + LPS, p65 supershift), and Lane 10 (Alb + LPS, p50 supershift) were performed as additional controls to demonstrate specificity and the nature of the NF-κB. The EMSA shown is representative of 3 separate experiments, all with similar results.

Figure 5
Figure 5

Albumin preconditioning abrogates LPS-mediated TNF-α expression in vivo. C57Bl/6 mice, 20–25 g body wt, were preconditioned with BSA (10 mg/kg body wt, i.p.) or vehicle 18 h prior to subsequent treatment with a lethal dose of LPS (60 mg/kg body wt, i.p.). Plasma was harvested at 6 h, and TNF-α was measured via ELISA. Albumin preconditioning significantly inhibited LPS-mediated plasma TNF-α expression. Experiments were performed in triplicate with 5 mice per experimental group (*P < .05 compared to control; # P < .05 compared to LPS alone).

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