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Immunostimulatory potential of β-lactoglobulin preparations

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Presentation on theme: "Immunostimulatory potential of β-lactoglobulin preparations"— Presentation transcript:

1 Immunostimulatory potential of β-lactoglobulin preparations
Susanne Brix, MS, Lionel Bovetto, PhD, Rodolphe Fritsché, PhD, Vibeke Barkholt, PhD, Hanne Frøkiaer, PhD  Journal of Allergy and Clinical Immunology  Volume 112, Issue 6, Pages (December 2003) DOI: /j.jaci

2 FIG 1 Induction of proliferation and intracellular thiol increase in cow's-milk–naive immune cells after in vitro exposure to β-LG. Splenocytes and MLNs from BALB/c mice were cultured with various concentrations of the different milk proteins and OVA. A, Proliferation of spleen cells.B, Proliferation of MLNs.C, Intracellular thiol level in splenocytes cultured with 1 mg/mL protein. Data are mean ± SD for proliferation, n = 6. Differences in cell proliferation toward the proteins were analyzed for significance using 1-way ANOVA. ∗∗∗P < .001 by Tukey's multiple-comparisons test. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )

3 FIG 2 Purification of β-LG from raw milk using non-denaturating conditions. A, Anionic ion-exchange chromatography of whey proteins. B, Size-exclusion chromatography (Superdex 75 pg column) of the β-LG variant A+B fraction from A. C, SDS-PAGE (4%-12% NuPAGE) after silver staining: molecular weight marker (lane 1), unfractionated whey proteins (lane 2), the purity of the β-LG preparation before (lane 3), and after size-exclusion chromatography (lane 4). The protein amount applied to the SDS-PAGE was ∼2.5 μg. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )

4 FIG 3 No immunomodulatory effects of β-LG from raw milk. Splenocytes and MLNs from cow's-milk–naive BALB/c mice were cultured with an optimal dose of 1 mg/mL protein. A, Proliferation of splenocytes and MLNs. Data (mean ± SD, n = 6) are stimulation index calculated as counts per minute (cpm) of antigen-stimulated cultures divided by that of unstimulated cultures. The cpm of unstimulated cultures was 332 ± 88 for spleen cells and 342 ± 38 for MLNs. B, Cytokine production by spleen cells (mean ± SD, n = 6). C, Increase in cellular thiol in spleen cells. All data are representative of 2 independent experiments. Differences in cell proliferation and cytokine production between proteins were tested using 1-way ANOVA. ∗∗P < .01. ∗∗∗P < .001 by Tukey's multiple-comparisons test. S, Sigma-Aldrich. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )

5 FIG 4 Size-exclusion chromatography of Sigma β-LG separates the immunostimulatory component from β-LG. The Sigma β-LG preparation (20 mg/mL) was fractionated on a Superdex 75 pg column using 150 mmol/L ammonium acetate, pH 6.0. The absorbance was measured at 280 nm (solid black line). The insert presents the whole chromatogram. Proliferation in response to each collected fraction and the unfractionated β-LG was tested on MLNs from cow's-milk–naive BALB/c mice. The proliferation data (mean, n = 6) are shown as stimulation index (black bars). Results are representative of 2 independent experiments. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )

6 FIG 5 The immunostimulatory component of the Sigma β-LG preparation induces cytokine production in murine DC cultures. MBM cells were in vitro expanded by GM-CSF for 8 days and then cultured with 1 mg/mL antigen or 1 μg/mL LPS (E coli O26:B6). After 19 hours, the supernatants were harvested, and the cytokines TNF-α, IL-6, IL-1β, and IL-10 were measured by ELISA. Data (mean ± SD) are derived from 1 experiment using cells from 2 mice and tested in duplicate wells. The F6 and F15 were obtained from size-exclusion chromatography of the Sigma β-LG preparation (Fig 4). S: Sigma-Aldrich; AF: Arla Foods, Denmark. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )

7 FIG 6 The cellular response to Sigma β-LG is reduced in the LPS low-responder strain C3H/HeJ. Proliferation of MLNs from C3H/HeJ and C3H/HeOuJ mice (n = 6) cultured with 1 mg/mL antigen or 10 μg/mL LPS (E coli O55:B5). The F6 and F15 were obtained from size-exclusion chromatography of the Sigma β-LG preparation (Fig 4). ∗∗∗P < .001 by 2-way ANOVA followed by the Bonferroni multiple-comparisons test. NS: Not significant; S: Sigma-Aldrich; AF: Arla Foods, Denmark. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )

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