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Original Research

Ex vivo stimulation of whole blood as a means to determine glucocorticoid sensitivity

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Pages 89-97 | Published online: 22 Aug 2012

Figures & data

Table 1 Demographics of normal human subjects (n = 12)

Table 2 Real-time PCR primers

Figure 1 Whole blood was stimulated with 100 nM dexamethasone (A) or 1 ng/mL LPS with and without 100 nM dexamethasone (B) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine expression of the GR upregulated genes GILZ, FKBP51, FLAP, and MKP-1 (A) and the GR downregulated genes IL-6, TNFα, and IFNγ (B). The expression of these genes was normalized to the housekeeping genes GAPDH and CAP-1. ΔΔ Ct was calculated for GILZ, FKBP51, FLAP, and MKP-1 as treatment (dexamethasone) against no treatment (A) and for IL-6, TNFα, and IFNγ as treatment (dexamethasone + LPS) against LPS only.

Note: Box plots with whiskers identifying outliers 1.5 times the IQR either above the 3rd quartile or below the 1st quartile are shown from 12 individuals.
Abbreviations: LPS, lipopolysaccharide; PCR, polymerase chain reaction; GR, glucocorticoid receptor; GILZ, glucocorticoid inducible leucine zipper; FKBP51, FK506 binding protein 51; FLAP, arachidonate 5-lipoxygenase-activating protein; MKP- 1, mitogen activated protein kinase phosphatase 1; IL-6, interleukin 6; TNFα, tumor necrosis factor α; IFNγ, interferon γ; IQR, interquartile range; CAP-1, cyclic AMPaccessory protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 1 Whole blood was stimulated with 100 nM dexamethasone (A) or 1 ng/mL LPS with and without 100 nM dexamethasone (B) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine expression of the GR upregulated genes GILZ, FKBP51, FLAP, and MKP-1 (A) and the GR downregulated genes IL-6, TNFα, and IFNγ (B). The expression of these genes was normalized to the housekeeping genes GAPDH and CAP-1. ΔΔ Ct was calculated for GILZ, FKBP51, FLAP, and MKP-1 as treatment (dexamethasone) against no treatment (A) and for IL-6, TNFα, and IFNγ as treatment (dexamethasone + LPS) against LPS only.

Figure 2 Whole blood was stimulated with increasing amounts of dexamethasone (A) or 1 ng/mL LPS with and without increasing concentrations of dexamethasone (B) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine expression of the GR upregulated genes GILZ and FKBP51 (A) and the GR downregulated genes IL-6 and TNFα (B). The expression of these genes was normalized to the housekeeping genes GAPDH and CAP-1. Dose response curves were drawn and the EC50 (A) and IC50 (B) values were calculated.

Note: Box plots with whiskers identifying outliers 1.5 times the IQR either above the 3rd quartile or below the 1st quartile are shown from 9 individuals.
Abbreviations: LPS, lipopolysaccharide; PCR, polymerase chain reaction; GR, glucocorticoid receptor; GILZ, glucocorticoid inducible leucine zipper; IL-6, interleukin 6; TNFα, tumor necrosis factor α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAP-1, cyclic AMP-accessory protein; EC50, half maximal effective concentration; IC50, half maximal inhibitory concentration; IQR, interquartile range.
Figure 2 Whole blood was stimulated with increasing amounts of dexamethasone (A) or 1 ng/mL LPS with and without increasing concentrations of dexamethasone (B) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine expression of the GR upregulated genes GILZ and FKBP51 (A) and the GR downregulated genes IL-6 and TNFα (B). The expression of these genes was normalized to the housekeeping genes GAPDH and CAP-1. Dose response curves were drawn and the EC50 (A) and IC50 (B) values were calculated.

Figure 3 Whole blood was drawn from 10 individuals at 8:30 am, 12:30 pm, 4:30 pm, and 8:30 pm and was stimulated with 100 nM dexamethasone (A and B) or 1 ng/mL LPS with and without 100 nM dexamethasone (C and D) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine expression of the GR upregulated genes GILZ (A) and FKBP51 (B) and the GR downregulated genes IL-6 (C) and TNFα (D).

Notes: The expression of these genes was normalized to the housekeeping genes GAPDH and CAP-1. ΔΔCt was calculated for GILZ and FKBP51 as treatment (dexamethasone) against no treatment and for IL-6 and TNFα as treatment (dexamethasone + LPS) against LPS only. The slope of the ΔΔCt value was calculated and tested for significance using a random-effects linear regression analysis.
Abbreviations: LPS, lipopolysaccharide; PCR, polymerase chain reaction; GR, glucocorticoid receptor; GILZ, glucocorticoid inducible leucine zipper; FKBP51, FK506 binding protein 51; IL-6, interleukin 6; TNFα, tumor necrosis factor α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAP-1, cyclic AMP-accessory protein; ΔΔCt, comparative cycle threshold.
Figure 3 Whole blood was drawn from 10 individuals at 8:30 am, 12:30 pm, 4:30 pm, and 8:30 pm and was stimulated with 100 nM dexamethasone (A and B) or 1 ng/mL LPS with and without 100 nM dexamethasone (C and D) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine expression of the GR upregulated genes GILZ (A) and FKBP51 (B) and the GR downregulated genes IL-6 (C) and TNFα (D).

Figure 4 Whole blood from 10 individuals was either treated immediately (closed square) or stored in the EDTA tube at 4°C for 24 hours (open square) prior to stimulation with 100 nM dexamethasone (A) or 1 ng/mL LPS with and without 100 nM dexamethasone (B) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine the expression of the GR upregulated genes GILZ and FKBP51 (A) and the GR downregulated genes IL-6 and TNFα (B).

Notes: The expression of these genes was normalized to the housekeeping genes GAPDH and CAP-1. ΔΔCt was calculated for GILZ and FKBP51 as treatment (dexamethasone) against no treatment and for IL-6 and TNFα as treatment (dexamethasone + LPS) against LPS only.
Abbreviations: LPS, lipopolysaccharide; PCR, polymerase chain reaction; GR, glucocorticoid receptor; GILZ, glucocorticoid inducible leucine zipper; FKBP51, FK506 binding protein 51; IL-6, interleukin 6; TNFα, tumor necrosis factor α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAP-1, cyclic AMP-accessory protein; ΔΔCt, comparative cycle threshold.
Figure 4 Whole blood from 10 individuals was either treated immediately (closed square) or stored in the EDTA tube at 4°C for 24 hours (open square) prior to stimulation with 100 nM dexamethasone (A) or 1 ng/mL LPS with and without 100 nM dexamethasone (B) for 2 hours. RNA was isolated and cDNA transcribed. Real-time PCR was performed to determine the expression of the GR upregulated genes GILZ and FKBP51 (A) and the GR downregulated genes IL-6 and TNFα (B).

Figure 5 Whole blood was drawn at 8:30 am and stimulated with 1 ng/mL LPS with and without 100 nM dexamethasone for 2 hours for real-time PCR or 24 hours for ELISA.

Notes: For real-time PCR, RNA was isolated, cDNA transcribed, and real-time PCR performed to determine expression of IL-6 mRNA. The expression of IL-6 was normalized to the housekeeping genes GAPDH and CAP-1. For EL ISA, serum was separated and IL-6 protein determined using the Human IL-6 EL IZA MAX Set Deluxe Kit (Biolegend). Upon stimulation with 1 ng/mL, 702 ± 922 pg/mL IL-6 was detected. When co-stimulated with dexamethasone 19 ± 22 pg/mL IL-6 was detected. The percentage repression by dexamethasone was calculated by the difference in fold induction by LPS with and without dexamethasone as a percentage of the fold induction by LPS alone. Means and SD are shown from 12 individuals. The two groups were tested for significance using a student’s t-test.
Abbreviations: LPS, lipopolysaccharide; PCR, polymerase chain reaction; EL ISA, enzyme-linked immunosorbent assay; IL-6, interleukin 6; GAPDH, glyceraldehyde- 3-phosphate dehydrogenase; CAP-1, cyclic AMP-accessory protein; SD, standard deviation.
Figure 5 Whole blood was drawn at 8:30 am and stimulated with 1 ng/mL LPS with and without 100 nM dexamethasone for 2 hours for real-time PCR or 24 hours for ELISA.

Table 3 Coefficient of variation over gene

Table 4 Descriptive statistics by figure, gene, and time